METHODS AND COMPOSITIONS FOR REDUCING PERSISTENT ODOR IN CLOTHING AND MITIGATING BIOFILMS

Abstract

Novel methods and compositions for treating textiles and other materials are disclosed in which persistent odor or other symptoms of biofilm presence can be reduced through the use of compositions comprising a biofilm attack agent such as N-acetyl cysteine or certain enzymes, coupled with surfactants and other agents.

Claims

1. A method for treating a textile item having or suspected of having biofilm matter in one or more regions associated with persistent odor, the method comprising applying an enzymatic composition to the one or more regions having biofilm matter, providing suitable time for the enzymatic mixture to attack the 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% 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% to 10% of N-acetyl cysteine.

2. The method of claim 1, wherein 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 on textiles selected from at least one of proteins, carbohydrates, lipids, and carbohydrates, the spores then producing enzymes that attack a portion of said contaminants.

3. The method of claim 2, wherein the concentration of the bacterial spores is between 110.sup.5 and 510.sup.10 colony forming units per ml.

4. The method of claim 1, wherein the enzymatic composition is packaged with indicia instructing a user to wait at least 5 minutes between applying the enzymatic composition and washing the textile, wherein washing comprises washing in water with a laundry detergent.

5. The method of claim 1, wherein the enzymatic composition comprises at least two portions, a first portion comprising an enzyme mixture and a second portion comprising N-acetyl cysteine, further associated with indicia instructing a user to apply both portions to the one or more regions of the textile item associated with persistent odor.

6. The method of claim 5, wherein the first portion of the enzymatic composition is provided as a liquid and the second portion is provided as a liquid or as a powder to be combined with a liquid when applying it to the one or more regions of the textile item associated with persistent odor.

7. The method of claim 5, wherein the second portion of the enzymatic composition comprises from 1% to 90% N-acetyl cysteine, from 1% 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%, that the pH of the resulting aqueous mixture is at least 6.0

8. The method of claim 1, wherein the method is 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.

9. The method of claim 1, further comprising visualizing the presence of suspected biofilm matter using UV light.

10. The method of claim 9, wherein the textile item has malodor and wherein a suitable dye that fluoresces in UV light has been applied to the textile item 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.

11. The method of claim 1 wherein the enzymes of the enzyme mixture comprise less than 25% lipase.

12. The method of claim 1, further comprising treating the textile item with at least 0.01 g of N-acetyl cysteine or with at least 1 gram of a solution having at least 0.1% N-acetyl cysteine.

13. The method of claim 1, wherein the textile item has a malodorous region associated with a portion of the wearer's body, and wherein the wearer is also provided with a deodorant comprising at least 0.1% of an alpha hydroxy acid having a pH from 2.8 to 5.5, wherein the wearer is instructed to apply the deodorant at least once to the underarms or the region on the body associated with malodor detected in the textile item, followed by or preceded by wearing the item of clothing while also using the deodorant applied to the portion of the body associated with a region of malodor in the textile item.

14. The method of claim 1, wherein the item of clothing has a malodorous region associated with a portion of the wearer's body, and wherein the wearer is also provided with a deodorant wipe comprising an aqueous material having at least 0.5% by weight of probiotic material selected from a microbial filtrate, bacteria, or bacterial spores, and wherein the wearer is instructed to apply the wipe at least once to the portion of the wearer's body associated with the malodorous region in the item of clothing such that the probiotic material is present on the skin while wearing the item of clothing after said application of the bioenzymatic material to and washing of the item of clothing.

15. A method for reducing a biofilm in an item of clothing, 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 pantothenic acid, a catechin, and one or more enzymes selected from proteases, amylases, cellulases, DNases, lysozymes, pectinases, and mannanases, 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; and 4) washing the item of clothing.

16. The method of claim 15, wherein step 1 comprises applying a dose of at least 0.01 g of N-acetyl cysteine per 50 cm.sup.2 to the target region.

17. The method of claim 15, further comprising using UV light having a wavelength from 330 nm to 420 nm to identify target regions having biofilm matter, wherein the item of clothing has been previously washed with a laundry detergent comprising an optical brightener that preferentially absorbs onto the biofilm.

18. The method of claim 17, wherein after step 4, UV light is again used to examine the target regions and reveals decreased fluorescence relative to the fluorescence observed when identifying target regions.

19. A composition for reducing persistent odor in a textile garment worn on the body of a user, comprising an aqueous solution of 0.01 to 30% N-acetyl cysteine, at least one surfactant, and at least one enzyme selected from proteases, pectinases, cellulases, amylases, lipases, mannanases, and lysozymes; the composition being associated with indicia instructing a user to apply the composition to a textile garment having persistent odor.

20. The composition of claim 19, wherein the aqueous solution comprises from 0.2 to 5% N-acetyl cysteine and from 0.1% to 5% protease.

21. A two-part composition for reducing odor in a textile garment worn on the body of a user, comprising a first part comprising N-acetyl-cysteine or salt thereof and a second part comprising a cleaning compound selected from one or more of the following: at least 1% of one or more enzymes selected from proteinases, cellulases, amylsases, lipases, pectinases mannanases, and lysozymes, at least 0.01% of bacterial spores capable of producing at least one of proteinases, cellulases, amylsases, lipases, mannanases, and lysozymes in the presence of suitable nutrients, and at least 4% of one or more surfactants, the two parts of the composition being physically separated from each other prior to use and associated with indicia instructing the user to apply both parts of the two-part composition to a region of the textile garment suffering from malodor prior to washing the garment.

22. The two-part composition of claim 21, wherein the indicia specify a recommended dwell time following treatment of the garment with at least one part of the two-part composition, the recommended dwell time ranging from 1 to 60 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0151] FIG. 1 depicts a shirt with UV-fluorescent biofilm-laden zones in the armpits of a shirt being illuminated with UV light.

[0152] FIG. 2 depicts spraying a cleaning composition on a biofilm region.

[0153] FIG. 3 depicts a flowchart showing a process in which biofilm visualization is used to guide treatment of clothing with persistent odor.

[0154] FIG. 4 depicts a flowchart showing steps in a process for treating clothing with persistent odor while also treating odor on the skin of the wearer.

[0155] FIG. 5 depicts one form of a roll-on applicator for applying a biofilm attack solution to portions of clothing or other surfaces to reduce malodor.

[0156] FIG. 6 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.

[0157] FIG. 7 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 regions of clothing or suspected biofilms.

[0158] FIG. 8 depicts a flowchart showing steps in a process for treating clothing with persistent odor.

[0159] FIG. 9 depicts a spray bottle containing a biofilm attack agent inside.

[0160] FIGS. 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 Examples section below.

[0161] FIGS. 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.

[0162] FIGS. 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. FIGS. 13A and 13B show photos of the right and left pits of a triathlon shirt under UV light.

[0163] FIGS. 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.

[0164] FIGS. 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.

[0165] FIGS. 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.

[0166] FIGS. 17A and 17B are before-and-after images of the biofilm region of a pit in the lace dress taken with confocal microscopy with a UV laser and no added dye.

DETAILED DESCRIPTION OF THE DRAWINGS

[0167] Unless otherwise stated, all percentages (%) are percentages by weight.

[0168] 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 comprising optical brighteners or has had an optical brightener solution applied to it followed by rinsing, the preferential absorption of the optical brightener(s) onto the biofilm may cause the biofilm region 24 to fluoresce (not shown), 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.

[0169] 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.

[0170] 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 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.

[0171] FIG. 4 depicts another flowchart 60 beginning with Step 62, wherein a user faced with persistent odor in clothing is provided with a pretreatment product comprising in one or more parts comprising enzymes, surfactants, and a biofilm attack agent for treating clothing with persistent odor. In Step 64, the user may apply UV light or other biofilm visualization tools to verify the possible existence and location of biofilm in items of clothing. In Step 66, the user may also be further with a personal deodorant comprising at least 1% of a carboxylic acid such as an alpha hydroxy carboxylic acid such as mandelic acid and/or lactic acid, having a pH of 2.8 to 5.5 or from 2.8 to 5. The user in Step 68 us instructed to treat clothing items with persistent odor with the pretreatment product prior to washing, and is further instructed in Step 70 to apply the personal deodorant on regions of the body associated with the locations of persistent odor in clothing. In Step 72, after it is washed, the item of clothing may be worn and further monitored for odor or signs of biofilm, repeating any or all of Steps 62 through 70 as needed.

[0172] FIG. 5 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 least 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.

[0173] FIG. 6 depicts a combined assembly 100 for combination in a single package (not shown) of a first container 102 containing a cleaner composition labeled as a laundry pretreatment comprising at least one of NAC, a suspension of bacteria or bacterial spores such as Bacillus subtiliis spores, two or more laundering enzymes, panthenol, at least 5% surfactant such as from 10% to 70% surfactant, and a second container 104 comprising a biofilm attack agent and/or a biofilm modification agent. The contents of the second container 104 are labeled as a biofilm attack powder, but in related embodiments may be a liquid, a slurry, a paste, a powder, individual powder capsules, and the like, and may further 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 (not shown) for use in a wash cycle in which one or more 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.

[0174] 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.

[0175] FIG. 7 depicts an assembly 120 for a two-part system. A first container 122 labeled as 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% such as from 1% to 15% or from 2% to 10% or from 1% 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 labeled biofilm attack powder 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 or the like, may simply be placed in the second container 152 coupled with water, where it will dissolve and allow the biofilm attack powder to become dissolved and able to 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 on a dry basis 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% to 50% panthenol such as from 1% to 35%, 1% to 25%, 1% to 10% or 1% 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 Epsom salt, magnesium citrate, magnesium acetate, magnesium chloride, and the like, sodium chloride, sodium citrate, sodium bicarbonate, sodium carbonate, etc., potassium chloride, potassium hydroxide, wherein the metal salts may comprise from 0.05% to 20% of the biofilm attack composition. In one embodiment, for example, the composition may comprise from 0.05 to 15% each or from 0.1% 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.

[0176] In another embodiment, the biofilm attack composition 144 comprises caffeine, such as from 0.5% to 20%, from 1% to 12%, or from 1% to 5% caffeine. Without wishing to be bound by theory, it is believed that caffeine can have an inhibitory effect on biofilm formation, based on extrapolating studies of caffeine on diverse biofilm situation such as biofilms in dental care and dentures in particular.

[0177] For embodiments in which a solid material such as an NAC-containing mixture is combined with water to make a biofilm attack agent, the packaging system may comprise loose powder in a container (not shown) that is scooped or otherwise metered or delivered to be combined with an amount of water in a container or dispenser. Alternatively, the solid particles may be combined in a solid tablet that can be dissolved in water, including an effervescent tablet that may comprise NAC and a carbon-dioxide releasing material such as sodium carbonate. In yet another embodiment, 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 embodiment, the capsule shells can be discarded rather than dissolved. In particular, sprinkle capsules (not shown) may be used in which relatively large capsules can be readily gripped and twisted to cause them to separate and release their contents. Exemplary sprinkle capsules are described in U.S. patent Ser. No. 10/610,490, Separable capsule for sprinkling applications, issued Apr. 7, 2020 to S. Stegemann et al. Such capsules may be made of insoluble material such as polypropylene (PP), PET, high density poly ethylene material, metal, aluminum, and glass.

[0178] FIG. 8 depicts a flowchart 160 showing steps in a process for treating clothing with persistent odor. In Step 162, a user identifies clothing with apparent perma-odor and then in Step 164, treats the item of clothing with an enzymatic or bioenzymatic cleaning composition. If malodor is eliminated 166, the article can be worn with confidence 168, but if problems remain, in Step 170 the user may visualize potential biofilm areas using UV light or other tools to view fluorescence from absorbed optical whiteners/brighteners (OW), and then in Step 172 treat the biofilm candidate areas with a biofilm attack agent and optionally also with an enzymatic cleaning composition. If there is progress in odor or biofilm reduction 176 (biofilm reduction being known by again visualizing the bofilm area, not shown, for comparison to the previous visualization), then the use of the enzymatic cleaning composition and biofilm attack agent may be continued as needed 178, but if progress is lacking, there may be a need to consider alternate treatments 179 such as physical biofilm removal using a scrub brush, tooth brush, or other form of friction, or a water jet, ultrasonic cleaner, very low of high pH treatments, more intense chemical agents such as antimicrobial agents, high temperature, etc.

[0179] FIG. 9 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.

[0180] The remaining figures are described below in the Examples section.

FURTHER DETAILED DESCRIPTION

Foam Systems

[0181] Foam applicators, which can be used to deliver fabric treatment compositions described herein, can comprise pressurized containers such as spray cans or foam delivery containers similar to popular foam-based shaving cream dispensers and non-pressurized foam delivery systems such as foam generating pumps. Examples of foam generating pumps are described in U.S. Pat. No. 8,267,284, issued Sep. 18, 2012 to E. W. Ray; U.S. Pat. No. 9,718,069, Foam Pump, issued Aug. 1, 2017 to D. M. R. Creaghan et al.; U.S. Pat. No. 8,991,657, Foam soap dispenser with stationary dispensing tube, issued Mar. 31, 2015 to Ciavarella et al.; US Pat. Publication No. 20140203047, Pumps With Container Vents, issued Jul. 24, 2014 to McNulty; US Patent Publication No. 20140054323, Horizontal Pumps, Refill Units and Foam Dispensers with Integral Air Compressors, issued Feb. 27, 2014 to McNulty; U.S. Pat. No. 8,591,207, Pump with side inlet valve for improved functioning in an inverted container, issued Nov. 26, 2013 to Ciavarella; US Patent Publication No. 20130200098, Foaming Liquid Dispenser, issued Aug. 8, 2013 to Li; U.S. Pat. No. 8,579,159, Squeeze action foam pump, issued Jul. 23, 2009 to Nick E. Ciavarella; U.S. Pat. No. 8,336,737, Foam Dispenser, issued Dec. 25, 2012 to E. I. M. van der Heijden; U.S. Pat. No. 6,082,586, Liquid dispenser for dispensing foam, issued Jul. 4, 2007 to Banks; U.S. Pat. No. 5,445,288, Liquid dispenser for dispensing foam, issued Aug. 28, 1995 to Banks; U.S. Pat. No. 5,238,155, Foam generating device, issued Aug. 24, 1993 to Blake.

Synergy with Deodorant Products

[0182] The objective of reducing persistent malodor in clothing can be synergistic with efforts to alter the skin microbiome in regions where malodor occurs, such as the armpits. Synergy can occur when the compositions described herein are applied periodically to the armpits or other regions having malodor, while also taking steps to reduce the growth of microbial malodor sources in the associated regions on the body such as the armpits or groin. Synergy can occur, for example, when using a low pH deodorant such as LUME brand deodorant or LUME deodorant wipes that contain mandelic acid and have a relatively low pH, such as from 2.8 to 5 or from 3.2 to 4.8. Regular use of these mandelic acid-containing deodorant products can have a long-lasting effect on body odor due to the healthy change promoted in the skin microbiome. As the microbiome changes with reduced malodorous bacteria, the clothing being used can also experience reduced addition of malodorous bacteria in use that can strengthen or maintain malodor sources from clothing, particularly when a biofilm exists or is in the process of being established. Meanwhile, reducing or eliminating the biofilm using the bioenzymatic products described herein can help support the objective of reduced body odor on the skin for those using LUME deodorants. Thus, in some embodiments, synergy in odor reduction in clothing and on the body is achieved by providing a user with both a deodorant such as a low pH deodorant comprising mandelic acid and with the bioenzymatic compositions for treating items of clothing described herein.

[0183] In a related embodiment, a user of the bioenzymatic compositions for cleaning items of clothing described herein is also provided with a deodorant such as a cream, stick, spray, or deodorant wipe comprising at least 0.1%, 0.5%, 1%, or 2% of mandelic acid or another carboxylic acid such as an alpha hydroxy acid (in a wipe, this weight percentage is relative to the mass of the applied wetting solution or ointment and not the mass of the wipe itself) and having a pH from 2.8 to 5.5, more specifically from 2.8 to 5 or from 2.8 to 4.5, wherein the user is instructed to use the deodorant regularly such as at least once a week, twice a week, three times a week, daily or at least 3 times within a 10-day period in the underarms or the region on the body associated with malodor detected in clothing and/or a region associated with apparent biofilm, while also being instructed to use the enzymatic compositions described herein to treat a malodorous region of an item of clothing, followed or preceded at least once, alternatively two, three, four, or five times or more, by wearing the item of clothing while also using the deodorant applied to the portion of the body associated with a region of malodor in the item of clothing.

[0184] In one embodiment, the deodorant, particularly the wetting solution of deodorant wipes, may comprise at least 0.5% by weight of probiotic materials such as saccharomyces ferment filtrate, lactobacillus ferment, lactobacillus, or other bacterial ferments or bacteria or bacterial spores, and may have from 1% to 25% probiotic materials, or from 2% to 15%, 1% to 10%, etc. Without wishing to be bound by theory, it is believed that such materials can help improve the skin microbiome and reduce the presence of malodorous bacteria by encouraging healthy bacteria to proliferate. In one embodiment, both an alpha hydroxy acid such as mandelic acid is presence at a concentration of at least 0.3% and one or more of the aforementioned probiotic materials is present at a concentration of at least 0.5%. Also without wishing to be bound by theory, it is believed that such a combination can further enhance the skin microbiome and provide an environment that further supports the objective of overcoming biofilm in clothing by reducing the ability of malodorous bacteria to move from the skin to establish a biofilm in clothing, and by hindering the ability of an existing biofilm in clothing to spread effectively due to the presence of the acidic materials and/or the presence of the probiotic materials that hinder growth of undesirable bacteria.

Sprays

[0185] Aqueous solutions described herein can be applied with a variety of spray, including aerosol sprays driven by a propellant such as butane; pump 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. 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 U.S. Pat. No. 9,248,462, Airless pump system, issued February 2.

Wipes

[0186] Wipes, whether dry, wet, or in other states, can be made from any suitable substrate that provides a flexible surface useful in applying compositions described herein. The wipe may be a porous, flexible wet wipe capable of retaining and applying a liquid solution, or may be flexible dry wipe that can apply a viscous formulation form its surface onto the pudendum. Suitable materials may include nonwoven or woven fabrics, tissue paper, composite or multilayered materials, etc, The wipe may be made from a conventional towelette or wet wipe material or other materials that have been proposed for wipes such as a nonwoven fabric including materials such as spunbond webs, meltblown webs, combinations of polymeric and natural fibers such as spunlace or coform webs, needlepunched webs, hydroentangled or spunlace materials, bonded carded webs, electrospun layers, composites or multilayer fabrics, woven textiles, apertured films, and the like. Polymeric materials used in the production of nonwoven webs, woven webs, and films may include polypropylene, polyethylene, PET, nylons, and the like. Foam pads or layers may be used, including open cell and closed cell foams, such as polyurethane foams, regenerated cellulose foams, and the like.

[0187] Examples of materials that may be used in producing wipes as described herein include those disclosed in any of the following, alone or in combination: U.S. Pat. No. 5,935,880, Dispersible Nonwoven Fabric and Method of Making Same, issued Aug. 10, 1999 to Wang et al.; U.S. Pat. No. 6,315,864, Cloth-Like Base Sheet and Method for Making the Same, issued Nov. 13, 2001 to Anderson et al.; U.S. Pat. No. 6,416,623, Method of Producing an Extensible Paper Having a Three-Dimensional Pattern and a Paper Produced by the Method, issued Jul. 9, 2002 to Hollmark; and U.S. Pat. No. 6,737,068, Wipe Formulation, May 19, 2004.

[0188] Wipes for use with the formulations described herein may also be made, used, or dispensed according to any of the following: U.S. Pat. No. 5,292,581, Wet Wipe, issued Mar. 8, 1994 to Viazmensky et al.; and U.S. Pat. No. 6,537,631, Roll of Wet Wipes, issued Mar. 25, 2003 to Rivera et al. Wipes may be provided from or in association with dispensers that provide a quantity of a formulation as described herein. Such a dispenser can include a combination of wipe dispenser and spray applicator for dispensing active ingredients, as described, for example, in US 20090057331, Wipes Dispenser, published Mar. 5, 2009 by Fryan et al., herein incorporated by reference. Wipes be dispensed from containers such as plastic or metal tubs or cylinders, from flexible pouches such as resealable pouches, form cardboard or other cellulosic containers, and from other known devices for dispensing wipes, including U.S. Pat. No. 6,601,737, Baby Wipe/Rash Cream Dispenser, issued Aug. 5, 2003 to Gartenberg.

Other Dispensers

[0189] A variety of dispenser forms may be used, such as roller ball (roll-on) type bottles, such as those described in U.S. patent Ser. No. 10/206,479, Application head for a product, in particular skin care, comprising an applicator ball held by a magnet, issued to G. Gieux et al., Feb. 19, 2019. Bottles with sponge or perforated tops may be used, such as tops with silicone, rubber, neoprene, or various thermoplastic elastomers or thermoset elastomers, including styrenic block copolymers, thermoplastic polyolefinelastomers, thermoplastic vulcanizates, thermoplastic polyurethanes, thermoplastic copolyesters, thermoplastic polyamides, and various classified thermoplastic 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. An ultraviolet LED or other UV light may also 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 the location of biofilm that may be fluorescent due to absorbing optical brighteners from typical laundry detergents. Examples of related scrubbing units include the elastomeric nubs on top of the OxiClean MaxForce Gel Stick marketed by Church & Dwight Co. (Ewing, N.J.) and the Cosmogen Maxi Squeeze'n Scrub body and face scrubbing product with elastomeric bristles manufactured by Cosmogen (Paris, France), believed to be related to embodiments shown in US Patent Application No. 20100028070, APPLICATOR FOR A FLUID PRODUCT SUCH AS A COSMETIC PRODUCT, published by G. Gieux et al., Feb. 4, 2010. 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, including various forms described in U.S. Pat. No. 8,573,875, Applicator for a fluid product such as a cosmetic product, issued Nov. 5, 2013 to G. Gieux et al.

Other Product Forms

[0190] A variety of other product forms can be considered. The compositions described herein can be applied in the form of a manually pumped spray or aerosol spray, a foam, a wipe, a wetted sponge such as a standalone sponge or a sponge applicator attached to a reservoir of material, a cream, a paste, a solid that can be wiped or rubbed onto clothing such as a laundry soap, a tablet such as a tablet for use in a laundry machine used in addition to laundry detergent, as a component in a laundry detergent or stain spray or fabric softener or bleach product, etc.

[0191] Single-use sponges in individual wraps can be used, for example, with instructions and indicia similar to those for wipe products. The sponge may be polyurethane, regenerated cellulose, and other known sponge materials. The compositions described herein may be impregnated in the sponge or applied shortly prior to use. The sponges may be attached to a wand or gripper element such as a plastic handle. A pre-wetted sponge may be wrapped in foil or plastic prior to use.

[0192] Additional odor-blocking compounds may be considered such as those described in United States Patent Application 20150182436, Deodorant Compositions Having Antibacterial and Odor Blocking Properties and Methods For Using the Same, issued to C. Schmit, Jul. 2, 2015. Such compounds include agrumex (2-tert-butyl cyclohexyl acetate; 2-(1,1-dimethylethyl)-cyclohexanyl acetate); C14 aldehyde (1,4undecanolide; gamma-undecalactone); ambrettolide; anisyl aldehyde; CALONER1951 (7-methyl-3,4-dihydro-2H-1,5-benzodioxepin-3-one); 1-carvone; cedryl methyl ether; citronellol 950; citrylal; CLARITONE (2,4,4,7-tetramethyl-oct-6-en-3-one); -damascone (1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one); -damascone; DYNASCONE 10 (145,5-dimethyl-1-cyclohexen-1-yl)4-penten-1-one); ethyl vanillin; eugenol; evernyl; FARENAL (2,6,10-trimethyl-9-undecenal); floropal (2,4,6-trimethyl-4-phenyl-1,3-dioxane); GLOBALIDE (oxacyclohexadecen-2-one); cyclohexadecenone; methyl dihydro jasmonate; patchouli oil; phenylethyl alcohol; E-isomer of 3-methyl-2-pentenoic acid ethyl ester; E-isomer of 3-methyl-2-octenoic acid ethyl ester; Z-isomer of 3-methyl-2-pentenoic acid ethyl ester; rosaphen (2-methyl-5-phenylpentan-1-ol; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran); rose oxide he (4-methyl-2-(2-methyl-1-propenyl)tetrahydro-2H-pyran); sandel (santalum album); sandolene (2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-01); tetrahydro linalool (3,7-Dimethyloctan-3-ol); timbranol (Isomethyltetrahydroionol); tonalide (7-Acetyl-1,1,3,4,4,6-hexamethyltetralin); and vertocitral (2,4-dimethyl-3-cyclohexene-1-carboxaldehyde), and the like. Also present may be useful bacteria in, for example, pro-biotic formulations. In some embodiments, microencapsulated beneficial microorganisms may be present, including lactic-acid producing microbes.

[0193] For formulations comprising gels, water is usually employed as the dispersion medium for the gel to optimize biocompatibility. Other possible dispersion mediums include non-aqueous solvents, including glycols, such as propylene glycol, butylene glycol, triethylene glycol, hexylene glycol, polyethylene glycols, ethoxydiglycol, and dipropyleneglycol; alcohols, such as ethanol, n-propanol, and isopropanol; triglycerides; ethyl acetate; acetone; triacetin; and combinations thereof. Typically, the medium (e.g., water) constitutes greater than about 75 wt/vol %.

[0194] Any suitable gelling agent, including gellan and other polymers and polysaccharides, may be used, including those described in U.S. Pat. No. 7,619,008, Xylitol for Treatment of Vaginal Infections, issued Nov. 17, 2009 to Yang et al.

[0195] Minerals and inorganic compounds may also be present such as magnesium oxide, Ozokerite, diatomaceous earth, silicon oxide, amorphous sodium silicate (SiO2:Na2O), crystalline layered silicate of formula -Na2Si2O5, zeolites such as Zeolite A (hydrated sodium aluminosilicate), sodium carbonate, sodium bicarbonate, and sodium tripolyphosphate,

[0196] Thus, in one embodiment, a ready-to-use pretreatment or post-treatment composition is contemplated comprising from 0.01% to 3% of at least one of NAC, panthenol, and catechins including EGCG and other catechins; 0.01% to 2% enzymes selected from protease, lipase, cellulose, amylase, laccase and mannnanase; 0.01% to 2% of a mineral comprising a metal oxide (e.g., MgO); 0.01% to 2% a chelant such as EDTA, about 0.1-0.5% essential oil, a surfactant and water.

Chelants

[0197] Complexing or sequestering agents may be employed such as sodium carbonate and sodium bicarbonate, and/or any known chelant or combinations thereof such as Dissolvine GL-47-S(tetrasodium glutamate diacetate, also known as GLDA), a biodegradable chelate based upon L-glutamic acid, manufactured by Akzo Nobel. Other chelants to consider 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), VERSENOL (Dow Chemical, Midland, Mich.), etc. Iron and/or manganese chelating agents may also be considered. Such agents can be selected from the group consisting of amino carboxylates, amino phosphonates, polyfunctionally-substituted aromatic chelating agents and mixtures therein. Other chelating agents are disclosed in U.S. Pat. No. 5,728,671. If utilized, these chelating agents may comprise from about 0.01% to about 5% by weight of the compositions herein.

Rheology Modifying Ingredients

[0198] Many known rheology modifiers can be considered to obtain desired properties, particularly the bioadhesive properties of some embodiments. Gums such as guar gum or xanthan gum or other industrial gums, polyvinyl alcohols, polyacrylates, cellulose-derived polymers such as carboxymethylcellise or hydroxyalkylcellulose polymers, Laponite, clays, carboxomer polymers, starch derivatives including octenyl succinic anhydride (OSA) modified starch and numerous other compounds can be considered. Silicone elastomers can be considered, including those described in U.S. Ser. No. 09/613,266 (P&G),

Skin Benefit Agents

[0199] Non-limiting examples of skin benefit agents that may be considered for use herein are described in The CTFA Cosmetic Ingredient Handbook, 2nd Edition (1992), which includes a wide variety of ingredients commonly used in the skin care industry, and which may be suitable for use in various embodiments of the present invention. Non-limiting examples of skin benefit agents include absorbents, aesthetic components such as fragrances, natural extractives such as witch hazel or aloe vera, colorings/colorants, essential oils, skin sensates, astringents, etc. (e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate), anti-caking agents, antimicrobial agents (e.g., iodopropyl butylcarbamate), antioxidants, colorants, cosmetic astringents, cosmetic biocides, drug astringents, pH adjusters, skin-conditioning and/or moisturizing agents, skin soothing and/or healing agents (e.g., panthenol and derivatives such as ethyl panthenol, pantothenic acid and its derivatives, allantoin, bisabolol, and dipotassium glycyrrhizinate), retinoids, (e.g. retinol palmitate), tocopheryl nicotinate or Vit. E, skin treating agents, vitamins and derivatives thereof. It is to be understood that the actives useful herein can in some instances operate via more than one mode of action.

Foam Embodiments

[0200] In some embodiments, the active ingredients are delivered while in a foam state, such as stable foam, for example, that is produced with or without a propellant. In some versions, a foam is dispensed from a dispenser such as a propellant-free dispenser with pumping action to create the foam from a composition in a foamable carrier, and then applied to a textile material. Propellant-driving foam generators may also be used to deliver the composition in the form of a foam. Examples of foam-based systems are described in U.S. Pat. No. 6,818,204, Stable Foam for Use in Disposable Wipe, issued to Lapidus on Nov. 16, 2004. At least one foam stabilizing agent may also be present in certain foamable embodiments. Suitable foam stabilizing agents may include, without limitation, natural or synthetic gums such as xanthan gum, polyalkylene glycols such as polyethylene glycol, alkylene polyols such as glycerine and propylene glycol and combinations thereof. Typically, the foam stabilizers may be present in an amount from about 0.10% to about 5%.

[0201] The foam may be produced using the F2 Finger Pump Foamer manufactured by AirSpray International (Pompano Beach, Fla.). Such a spring-loaded valve system operates without the use of gas propellants. Upon actuation, precise amounts of air and liquid are mixed, and a foam capable of maintaining its structure for a substantial length of time is dispensed. In addition, the dispenser can deliver a variable amount of foam, thereby reducing waste of the wipe agent contained therein. Details of propellantless foamers are described in U.S. Pat. No. 5,443,569, issued on Aug. 22, 1995, and U.S. Pat. No. 5,813,576, issued Sep. 29, 1998.

Directions for Use

[0202] 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 bacreria and odor on clothing and/or the human body.

[0203] 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.

[0204] 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 Washing Machine Tablet with warm or hot water (if available). Meanwhile, don't forget to use Lume Deodorant for Underarms and Private Parts on your body regularly to reduce bacterial sources of malodor.

EXAMPLES

Enzymatic Sprays

[0205] Several enzymatic solutions were made:

[0206] E1: A first spray, labeled E1, comprised a buffered solution of Novozymes enzymes for laundry detergent in a buffered solution with surfactants and bacterial spores from J-Zyme AB-20X NFC distributed by J Tech Sales (Boca Raton, Fla.), said to employ spores from Nozozymes. The solution comprised about 20% J-Zyme which is said to have about 1.110.sup.9 CFU/ml of bacterial spores. Ingredients: [0207] Sodium citrate [0208] Sodium bicarbonate [0209] 1,3 propanediol from natural feedstock [0210] Alkypolyglucoside from sugar feedstock [0211] Probiotic bacteria blend [0212] Protease [0213] Amylase [0214] Pectate Lyase [0215] Mannanase [0216] 2 types of Cellulase [0217] Lipase [0218] Preservative (0.1% of a blend of methylchloroisothiazolinone and methylisothiazolinone) [0219] Water

[0220] Total enzyme concentration was about 2% by weight. The enzymes here were selected to have optimum activity at a pH of about 7-8.

[0221] E2: Spray E2 was a mix similar to E1 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 naturally derived soyethyl morpholinium ethosulfate.

[0222] The concentration of the quat was about 0.5% and the enzyme concentration was about 2%.

[0223] E3: Spray E3 was 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 10 concentrate comprising Bacillus subtilis spores, a solvent 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.

[0224] E4C: This liquid is a 4:1 concentrate intended upon dilution to give a solution similar to E3, but with slightly reduced surfactant levels to facilitate the concentrate form.

[0225] E4D: Upon 4:1 dilution, the concentrated E4C solution is diluted to normal strength and dubbed E4D.

[0226] 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 with less lipase. This concentrate comprises mix of enzymes, with a total of about 10 to 18% 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 10 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 (the D indicates dilution has occurred).

[0227] 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

[0228] Other enzymes used included: [0229] Pectinase from Phygene Biotechnology Co (Fuzhou, Chna), product PH1561, activity>500 u/mg, CAS 9032-7501. [0230] Pectinase in Kitchen Alchemy Pectinex Ultra SP-L solution from Modernist Pantry, LLC (Eliot, Me.). [0231] Pectinase powder (pectic enzyme), L. D. Carlson Co. (Kent, Ohio). [0232] Alpha-amylase from BOSF (1,4-alpha-D-glucan glucanohydrolase), 10 kU/g, CAS 9000-90-2, EC 232-565-6, powder form. [0233] Amylase powder, BSG (Shakopee, Minn.), product 10019. [0234] Papain, Phygene Biotechnology Co (Fuzhou, Chna), product PH9028, activity>800 u/mg, CAS 9001-73-4. [0235] Papain tablets, Beazyme brand, MCM (Malaysia Chemical Company, Kuala Lumpur), 150,000 USP, purchased in Kuala Lumpur, Malaysia. [0236] Lipase (triacylglycerol acylhydrolase) from Candida rugosa, Ekear Co. (Shanghai, China), product P0114, CAS 9001-62-1 [0237] Cellulase, Phygene Biotechnology Co (Fuzhou, Chna), product PH9018, activity>400 u/mg, CAS 9012-54-8. [0238] Cellulase powder from Heshibi Biotech, China, activity 100,000 u/g. [0239] Cellulase powder, Henan Wan Bang Industrial Co. (Henan Province, China). [0240] Cellulase powder, Zhejiang Yiruo Biotech (Zhejiang Province, China). [0241] Cellulase powder, Shandong Longda Biotech (Shandon Province, China). [0242] Cellulase powder, Yin brand (China). [0243] Lysozyme from egg whites, Bomei Biotech, activity>20,000 u/mg, CAS 12650-88-3. [0244] Lysozyme chloride, Homecare Noflux brand, 90 mg per tablet, purchased in Kuala Lumpur, Malaysia. [0245] E-Zyme Troche lysozyme chloride tablets, 200 mg each, from AV Manufacturing S/B (Malaysia) purchased in Kota Kinabalu, Malaysia. [0246] 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

[0247] AmylaseA: BSG amylase, 1.137 g and BOST amylase, 0.717 g, were stirred unto 28.0 ml of water.

[0248] CellulaseA: 1.1 g of Shandong Longda cellulase powder and 0.45 g of Phygene cellulase powder were mixed into 22 ml of water.

[0249] CellulaseB: 1.3 g of Heshibi cellulase powder was mixed into 29 ml of water.

[0250] 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 was mixed into 53.5 ml of water.

[0251] 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, and then 0.55 g of BOSF amylase powder.

[0252] LysoA: 1 tablet of E-Zyme lysozyme chloride (200 mg) was dissolved into 8.5 ml of water.

[0253] LysoPap: Grind one table of Homecare Noflux lysozyme chloride (90 mg of lysozyme) with one tablet of MCM Beazyme papain and dissolve the powder into 12 ml of water.

[0254] LysoB: Pulverize 2 tablets of E-zyme Troche lysozyme chloride (200 mg each) and dissolve in 30 ml water.

[0255] PAPA: 1.05 g of Phygene papain was combined with 21.7 ml of water.

[0256] PAPB: 1.3 tablets of MCM papain were ground and dissolved into 15 ml of water. Some residual solids remained even after heating. The slurry was then passed through a fine cloth to filter out some solids. 13 g of solution were obtained.

[0257] 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)

[0258] PANNAC: 33 ml of 3.6% NAC at pH 4.9 combined with 0.643 g of panthenol powder.

[0259] 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 NaHCO.sub.3 added to reach a pH of 4.7.

[0260] 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 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

[0261] Pretreatments to attack biofilm were made as follows:

[0262] 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.

NAC Solutions:

[0263] NAC powder was dissolved distilled water to give a 2.1% strength solution, a 1% solution, and a 20% solution.

[0264] NAC solution at 1.4% concentration was adjusted with citric acid and sodium carbonate to achieve a pH of 3.0.

[0265] NAC solution at 2% was made by mixing 3.9 g of NAC in 185 ml of water.

[0266] 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.

[0267] 1 g of NAC in 50 ml of 0.1M KOH solution was prepared with a pH of 9.17.

[0268] 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.

[0269] 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.

[0270] 28.1 ml of NIC (Naturally It's Clean) enzyme solution was combined 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.

[0271] A 2.1% NAC solution at pH 4.0 was made using 1.51 g of Biotal NAC, 0.648 g NaHCO.sub.3, and 71 ml of water.

Pectinase Solutions

[0272] 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.

[0273] 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.

[0274] PNAC3: 0.6 g NAC are 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.

[0275] 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, >10 kU/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, believed to be suitable for fruit pectinases.

[0276] PNAC5: Combine 1.256 g NAC with 1.033 BOSF pectinase (polygalacturonase) into 106 ml g water.

[0277] 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.

[0278] Lysozyme Solutions: Three Malaysian 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.

Herbal Solutions

[0279] BJC1: 500 g of the herbal product Herba patriniae, also known as Bai Jiang Cao or Su Jiang Bai, were obtained from a traditional Chinese medicine outlet in downtown Shanghai, the Beijing Tongren Pharmacy. The material is from the woody roots of the plant and is provided in thin chips and flakes. 58.65 g of the dry material was combined with 600 ml of water an heated in a large pan and stirred for about 30 minutes to form a concentrated extract which was filtered, and 100 ml therefrom was stored in a spray bottle with the label BJC1. The material was relative dark, probably too dark for use in clothing unless further lightened first.

[0280] BJC2: 5 g of Bai Jiang Cao matter were combined with 55 g of 70% ethanol and heated in a pan for about 30 minutes and filtered out, giving 44.5 g of extract. This was combined with 66 g of water to bring the material to a non-flammable range. The light yellow extract was labeled BJC2.

[0281] Steps Toward Perma-Odor

[0282] An effort was made to artificially create persistent odor problems in several shirts, including the following: [0283] Shirt RD1: a red 100% polyester sports shirt under the Voit brand [0284] Shirt RW1: a red and white striped shirt made from 75% cotton and 25% polyester with high stretch. [0285] Shirt Dec1: A 100% polyester men's sports top made by Decathlon of China. [0286] Shirt M1: A maroon shirt made from 47.5% Modal (rayon), 47.5% cotton, and 5% spandex. [0287] Shirt BS1: a black casual short-sleeved shirt made from 100% polyester

[0288] The first four of these shirts were obtained at a second-hand store in Shanghai, while the last was purchased new in Shanghai but had been worn periodically for several years.

[0289] Two malodor sprays were created to add malodor and a biological load in an attempt to infect clothing with malodor sources, Stink Spray 1 and Stink Spray 2.

[0290] To create a more persistent odor problem, a cheese believed to be rich in Micrococcus bacteria was obtained: Fourme D'Ambert, a well known mild surface smeared blue cheese where surface materials from prior blue cheeses are rubbed onto the surface of new cheese being formed, maintaining an ongoing colony of bacteria, molds and years that have been in use for many centuries in the manufacturing of this historic, ancient cheese.

[0291] Shirt BS1: A black-striped polyester shirt was given 6 sprays (about 1 gram) onto each put of Stink Spray 1 and of Stink Spray 2. Then to add additional bacteria, 0.5 g of Fourme D'Ambert cheese, including material from the outer rind, was applied to each pit over an approximately 9 cm9 cm square centered around the underarm stitching for each pit. The cheese was slowly rubbed into the fabric. The two wetted and cheese-smeared pits were folded to be in contact with each other, and then the shirt was rolled up with the pits toward the center of the rolled shirt. About 50 ml of water was sprinkled on the outer surfaces of the rolled shirt to provide moisture to keep the shirt from becoming too dry.

[0292] The shirt was placed in a plastic bag to seal it and it sat at about 25 C. for 36 hours. The smell was revolting. The odorous shirt was then prepared for washing by enzymatic pretreatment of each pit using the enzyme solution, Naturally It's Clean marketed by Enzyme Solutions, Inc. (Garrett, Ind.). Each pit was saturated with the enzyme mix and then washed by hand after about 5 minutes of dwell time. In the hand washing, efforts were made to remove all visible traces of the cheese. The entire shirt was then thoroughly rinsed and then washed in a Siemens front-loading washing machine in a regular wash cycle of 78 minutes using about 100 ml of the Chinese liquid laundry detergent, Blue Bright Moon, a high-performance detergent with enzymes. After this wash, the distinctly malodorous smell in the pits persisted, though it was greatly reduced.

[0293] At this point, about 1 g each of Stink Spray 1 and Stink Spray 2 was again applied to each pit, and about 1 g of Fourme D'Ambert cheese was again smeared onto each pit while still moist from the wash, and again rolled up and stored in a translucent plastic bag. The bag was placed in direct sunlight at an outdoor temperature (in the shade) of 29 C. for 3 hours to seek to further promote brewing of bacteria in the shirt. After 3 hours, the shirt was hand washed in cold water to remove traces of visible cheese and then washed in a washing machine on the fast cycle (38 minutes/cycle). There was no enzymatic pretreatment, but the same laundry detergent was used with approximately the same amount. After the washing, it was noted that the malodor has persisted. The shirt was worn by a male volunteer for one hour to add additional odor. This shirt is BS1.

[0294] Shirt BS1 was later treated with enzymatic spray E2. The left pit was saturated with spray application of 7.0 g of E2, while the right pit was sprayed and saturated with 6 g of BA-2AEG. After about 20 minutes, the shirt was washed in a fast cycle with laundry detergent.

[0295] After washing, the left pit had a pleasant smell, with the right pit still had some malodor, suggesting that the enzymes, bacteria, and soy quat were more effective in this case than the BA-2AEG spray.

Other Shirts

[0296] Shirt Dec1 was treated with 0.7 g of Fourme D'Ambert cheese total smeared on both armpits over a 10 cm5 cm area, and then sprayed with about 1 g of Stink Spray 1 on each pit. Then a total of 4.9 g of Stink Spray 2 was applied on the two pits (about 2.45 g per pit). The moist pits were rolled in contact with each other and the outer layers of the shirt were moistened with about 20 ml of warm water. The shirt was allowed to sit for two hours, at which time the cheese was rinsed off. Then BA-2AEG spray was applied to moisten the left pit. After 10 minutes, the entire shirt was washed with laundry detergent in a fast cycle (38 minutes). After washing, the right pit had malodor, but not the left, suggesting that BA-2AEG spray was effective.

[0297] Another round of odor application followed for shirts RW1, Dec1, M1, and RD1. For each shirt, about 1.2 g of Stink Spray 1 and 1.2 g of Stink Spray 2 were applied to both pits, followed by rubbing in 0.5 g of Fourme D'Ambert cheese to each pit. The moistened pits were placed in contact with each other, rolled up to be within the shirt, and the outer layers were wetted with 50 ml of water sprinkled thereon, then placed in a plastic bag and set into direct sunlight at an outdoor temperature of 29 C. (in the shade) for about 7 hours.

[0298] Shirt Dec 1 after washing had odor in both pits. Shirt M1 was treated with 1.7 g OCACDScl solution on the right pit and 1.56 g of E2 on the left pit, with reduced odor in the left pit. Shirt RW1 was treated with 3.7 g of 2AEG on the right pit and 1.15 g of Oderase suspension on the left pit, with reduced odor in both pits.

[0299] Four of the shirts acquired from a second-hand clothing store in Shanghai were given further treatments, following the attempt to brew perma-odor and following washing. The red and white striped shirt, RW1 (75% cotton, 25% polyester), had retained a slight cheese odor in both pits in spite of washing with an enzyme pretreatment. The red Voit shirt, RD1, 100% polyester, had slight odor (about 1 on a scale of 1 to 5) in both pits. The Dec1 shirt had no detectable odor, and the maroon 47.5% Modal shirt M1 had a sour, stale odor rated at 1 in both pits following washing.

[0300] On RD1, 0.68 g of the exterior matter of Fourme D'Ambert bleu cheese was applied to an 8-cm diameter circle at the right pit, with cheese applied to the inside and outside regions, being smeared in and rubbed. Then 0.73 g of the cheese was applied in the same manner to the left pit. The same was done to shirt Dec1, with 0.63 g of cheese on the right pit and 0.61 g of cheese on the left pit. For these two cheese-treated shirts, the nutrient spray Nut1 was applied, with 4.8 g to the right pit of Dec1 and 4.5 g to the left pit, and 5.8 g each to the right and left pits of RD1.

[0301] Stink spray 1 was then used, which had been refrigerated for over a month. Filtering though linen cloth was again conducted to remove particulates, then 0.9 g each were applied to the right and left pits of RD1, and 1.4 g each applied to the pits of Dec1. Both shirts were then wrapped with the wetted pits within each shirt in contact with each other and at the center of the wrapped shirt. The outer layer were then sprinkled with about 30 ml of water and then placed in a plastic bag to keep the shirts from drying. The sealed bags were then stored at about 70 F. for 7 hours.

[0302] After washing, Dec1 had noticeable odor in the right pit. Another brewing session was conducted, with 2.5 g of Stink Spray 2 applied to each pit and 0.5 g of Stink Spray 1 applied to each pit, followed by 1.15 g of Nut1 spray on both pits. This was placed in a plastic bag and stored on a solid surface above the water level of hot water in a closed cast-iron pot, where the water was kept at about 30-40 C. for about 10 hours. The shirt was then removed and the right pit was treated with 10 ml of NIC enzyme solution on the left pit and then 10 ml of 1.4% NAC in NIC at a pH of 8.08 on the right pit. The treated shirt was allowed to sit for 20 minutes, then was washed in a fast cycle using Bright Blue Moon detergent.

[0303] Later the sheet was treated to brew additional clothing infection. 2.3 g of Nut1 spray was applied to each pit area, followed by 1.86 g of Stink Spray 2 to the left pit and 1.93 g of Stink Spray 1 to the right pit. The shirt was wrapped up into a cylindrical form with the pits in the inside and the outer layers of the cylindrical form were sprinkled with 20 ml of water, then put into a plastic bag overnight. After about 24 hours, the shirt was washed. Both bits manifested malodor after the wash. Now the right pit was sprayed with 1.6 g of 1.8% NAC solution and washed with a standard wash cycle requiring 78 minutes in a Siemens front-loading washer, using Bright Blue Moon laundry detergent. Although the detergent has a fragrance, it was observed that after washing, the left pit appeared to have no malodor nor fragrance, while the right pit manifested fragrance. After drying, the left pit still had no sign of malodor, while the right pit had some odor. It appears that NAC can be effective when used in combination with other agents such as enzymes or catechins.

UK Triathlon Shirt (TR1)

[0304] 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 from 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 NIC enzyme solution alone, with 5.9 g applied. The right pit was treated with 1% NAC solution at pH 3.0, with 1.85 g applied. After two minutes, 6.87 g of EN2 (a mixture of NAC and NIC) at pH 7.00 was also applied to the right pit, 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 purchased in Asia.

[0305] After drying the washed shirt, it was noted that the right pit was substantially free of odor and fragrance, while the left pit manifested fragrance. After an exercise session with the shirt, the right pit had very little odor while the left pit had rapidly developed intense odor, uncharacteristic for the male doing the exercise. It appeared that the left pit suffered from perma-odor in which odor rapidly develops.

[0306] The shirt was washed in a fully 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).

[0307] 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.

[0308] 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.

[0309] 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 NAC-pectinase treatment was successful in reducing a biofilm or the source of the perma-odor, whatever its nature.

Neon Orange Champion Shirt

[0310] A neon orange sports top for women made under the Champion brand, code named CH1, a semi-fitted L/G, 100% polyester shirt was selected. It had been in regular use for exercise over the past 5 years and appeared to have symptoms of perma-odor. Slight odor would still generally be present after washing, and the odor would become strong after a single exercise session.

[0311] As a first attempt at reducing bacteria on the shirt, about 1 g of PuriShield wound healing agent made by FarnAm Co. (Phoenix, Ariz.) was applied to the right pit. This product contains 0.4% of a cationic steroidal antimicrobial that is said to be effective against a wide range of microbes. Treating the pit did not appear to reduce odor. However, after rinsing, the odor appeared to be significantly reduced. A typical exercise session was conducted to apply normal sweat levels to the shirt. No obvious difference in the odor between left and right was apparent.

[0312] Solution EGCD-1 was sprayed onto the left pit of the neon orange shirt after it has been acidified by spraying 1.27 g of citric acid solution (0.9 g citric acid in 100 ml water). 2.84 g of EGCD-1 is applied to the left pit and allowed to sit for 10 minutes. The pit is then rinsed in diluted vinegar (about 20 ml of distilled vinegar at 5% acidity in 500 ml of water).

[0313] The right side of the shirt is first rinsed with warm water and then sprayed with E2, giving 1.55 g in the pit area, saturating the pit, and it was allowed to sit for 5 minutes to allow enzymes to function at about 24 C.

[0314] After hand rinsing the entire shirt thoroughly in warm water and air drying, the left pit that had been treated with EGCD-1 solution smelled noticeably better than the right pit. The shirt was then washed in a standard laundry cycle with Bright Blue Moon (a Chinese brand, Lan Yue Liang) detergent and used in exercise. After exercise, the smell returned in both pits, though after washing the left pit again smelled noticeably better than the right pit when evaluated after two days of air drying. After further cycles of washing and exercise, the right pit had malodor while the left pit had noticeably weaker malodor.

[0315] Several days after the initial trials with the neon orange shirt, further trials were conducted in Borneo, Malaysia. Nova brand N-Acetyl cysteine powder in 300 mg capsules (Nova Laboratories Sdn. Bhd., Sungai Pelek, Sepang, Selangor, Malaysia, product MAL 17046003NZ) 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 forming the edible, water soluble capsules or additional gelatin mixed with the powder.

[0316] 1.97 g of NAC powder removed from the Nova brand capsules was stirred into 52 ml of water to form a solution which was slightly cloudy, likely due to the presence of some gelatin or other polymer in the powder. The resulting solution, approximately 3.2% NAC, 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 shirt side over a roughly circular area about 12 cm in diameter. After two minutes of dwell time, the right pit was then sprayed with the enzymatic laundry pre-treatment spray, Naturally It's Clean from Enzyme Solutions, Inc. (Garrett, Ind.), with 2.33 g applied to the exterior surface and 2.5 g applied to the interior surface.

[0317] The left pit of the neon orange shirt was treated with Naturally It's Clean enzyme 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 of solution applied to the pit. After five minutes of further dwell time, 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.

[0318] 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 saturate it. Then 5.3 ml of solution EGCD-A comprising EGCG, acids, and ethanol was applied to the right pit and allowed to sit for 20 minutes before handwashing and air drying.

[0319] 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.

[0320] 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, N. Dak.) 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 Naturally It's Clean laundry spray (Enzyme Solutions, Inc., Garrett, Ind.). This was then rinsed in cold water and then handwashed with laundry detergent and warm water. After drying, the right pit had no odor, neither malodor nor fragrance from the laundry detergent, while the left pit exhibited both malodor and fragrance.

[0321] 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. Later, after further exercising and washing, the right pit of the neon orange shirt after exercise could still develop odor, but not as intensely as before. After exercise, the left pit had strong odor, even after being treated with fresheners comprising cucurbituril and after treatment with Febreze Free from Procter & Gamble.

[0322] 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 g of Naturally It's Clean Enzyme Spray was applied and allowed to sit for 15 minutes, whereupon the shirt was rinsed by hand and then washed with commercial laundry detergent (Bright Blue Moon).

[0323] 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 cm8 cm around the left pit. This sat for 20 minutes, then 5 g of Naturally It's Clean enzyme spray was applied. After a 5-minute wait, the shirt was placed in a washing machine and washed.

[0324] 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.

[0325] The right pit was then treated with 3.11 g of EGCD-B spray, immediately followed by application of 3.46 g of t1% NAC spray at a PH of 6.4. The left pit was treated with 2% NAC at a pH of 9.17, with 3.6 g applied, followed by treatment with 1% NAC at a pH of 7.8 with 2.68 g applied. Then NIC was applied to the right pit, 2.0 g, and then 1.76 g of NIC was applied 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 referred to herein), it was observed that 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 then treated with EGCD-B spray, about 3 gm. After a 3-hour wait, both the right and left pits were sprayed with 2% NAC solution, 6 g on the left and 7.5 g on the right. After a five minute wait, the shirt was rinsed in warm water and air dried. After two exercise sessions, the pits were at an odor level of about 5. The left pit was treated with NIC, 1.48 g, while the right pit was treated with EN2, 1.44 g, and air dried. The odor level in the right pit was estimated at 3 on a scale of 0 to 5, while the left pit had an odor level of about 3.5. A second evaluator gave scores of about 2 for each pit.

[0326] The pH 3.0 NAC solution was now 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 was then treated with PNAC3 comprising pectinase. 7.3 g of PNAC3 was applied to the pit and surrounding region, saturating the pit area.

[0327] 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 several treatments with enzymes that the shirt has received, though the intensity of the darkened regions has declined.

[0328] After further exercise, with the odor level at 2 in the left pit and 2.5 in the right, solution 1.57 g of Aq14 freshener was applied to the right. After air drying, about 30 minutes later, the right pit odor level was about 1.

[0329] Steps were then taken to reduce the darkened color regions in the pits. The inner right pit was treated with 3.6 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 NIC 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).

[0330] After further exercise sessions, the shirt achieved 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 believed to be associated with biofilm residual matter. The shirt was then washed using Comfort brand detergent.

[0331] 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 NIC spray was applied to the pit and finally 1.4 g 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 additional exercise sessions without washing, the pits now had developed strong malodor with an odor level of about 5. The right put was treated with 2% NAC solution and washed. After further exercise, the odor in the right pit was significantly reduced relative to the left pit.

Additional Shirts: Series K

[0332] 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 applied. It sat for 10 minutes, then the shirt was rinsed and washed. After air drying, the treated pit smelled much better.

[0333] A white shirt, Notations brand, KW1, 100% polyester, had malodor in both pits after exercise, with an odor level at about 5 on a scale of 0 to 5. The right pit was treated with Febreze Free, 1.63 g. The left pit was treated with Oderase, 1.72 g. After drying, both pits had reduced odor, although the odor slowly returned over time and the pit treated with Febreze had lower odor than the other.

[0334] Washing pre-treatments were now applied to the white shirt. Unilever Comfort brand anti-microbial laundry spray, 4.2 g, was applied to the right pit, while EN2 (6.19 g) was applied to the left. After washing, no odor was obvious in either pit.

[0335] A blue Danskin polyester sports shirt for women, BD1, was also tested. After exercise, it had a left pit odor level of about 4 and a right pit odor level of about 2 (scale of 0 to 5). It was treated in the right pit with PNAC4, 4.7 g. After a 3-minute wait, it was washed Comfort laundry detergent.

[0336] A red Danskin sport top, 100 polyester, code name KR1 was also tested.

Additional Shirts, Series AA-AC

[0337] Several shirts from an athletic female volunteer were obtained, including: [0338] 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. [0339] 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. [0340] Shirt AC, a purple Disney shirt made with 50% cotton and 50% polyester.

[0341] 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 (NIC) 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. For shirt AC, 13.8 g of NIC was applied to the right pit and adjacent areas, followed by 3.3 g of E2.

[0342] 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, it is proposed that an additional treatment with pectinase and NAC may be needed to achieve more complete odor reduction in the right pit. Post-exercise data is not available yet for shirt AC.

Perma-Odor Candidates: Series L

[0343] 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.

[0344] 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.

[0345] 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 conventional 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.

[0346] 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 by itself did not appear to have any effect on the odor problems in the shoes.

Treatments with EGCG and NAC

[0347] 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 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 reduced odor once the washed shirt was exposed to further sweating during exercise.

[0348] 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 Naturally It's Clean (NIC) enzyme solution for laundry applied to the right pit. Shirt WL received 6.3 g of NIC 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 then air dried and later 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.

[0349] 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, NIC 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.

[0350] 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 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 (7 g) 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.

[0351] 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 during exercise, more like a new shirt.

Other Shirts: Series XH

[0352] A shirt from SUIS School in Shanghai, male subjects shirt, Sport Bay brand, XL, with perma-odor symptoms. The right pit was treated with 1.8% NAC solution at pH 3.0, with 13 g applied on and around the pit, plus 4 g of EGCD-1 solution was applied. After 6 minutes, it was rinsed, then again 3.8 g of 1.8% NAC was applied to the pit, followed by 7.9 g of N.I.C. enzyme solution after a 3 minute pause, then 2.1 g of EN2 solution. The shirt was then washed with Comfort detergent, air dried, and returned to its owner for more exercise.

[0353] A 100% cotton blue Superman T-shirt, codenamed NS, 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.

[0354] Another shirt, a dark gray 100% polyester basketball shooting shirt with the Decathlon Tarmak mark, assigned code name KV, was provided in an unwashed state with intense malodor at a level of about 5, with notes of ammonia and other malodors in the pits and adjacent zones. The right side of the shirt including the pit and surrounding area was treated with 23 g og 1.8% NAC. The left side was treated with 13 g of PNAC3. It sat for 18 minutes then was washed in a short cycle at 30 C. with Comfort detergent and returned to the user.

[0355] Another shirt, code named CH, a gray partially cotton T-shirt had a right odor level of 4 and 2 in the left pit. The right pit was treated 6.25 g of PNAC4. A soccer shirt from Ghana, an FCP TMN polyester shirt, codename JG, had a right pit odor level of 4-5 and a left pit level of 3-4. The right pit was treated with 4.9 g of PNAC4. A Decathlon polyester 2-layer shirt, codenamed TF, having been heavily used for exercise over the past few months, was treated in the right pit with PNAC4, 6.9 g over a broad region. The left pit was treated with 4.58 g of 2% NAC followed by 3.3 g of NIC solution. After a 30 minute delay, the shirt was washed in a full cycle at 40 C. with Comfort detergent.

[0356] A 60% cotton, 40% polyester T-shirt, code name MD, was received from an athletic male with high odor in both pits. 0.8 g of NIC was applied to each pit. The right pit also received 2.5 g of 2% NAC at pH 9.17.

[0357] A blue Macron sports shirt, 100% polyester, code named BM, was received from an athletic male with an odor level of about 5 in the left pit and 4 in the right pit. The left pit was treated with 6.15 g of 2.15% NAC at pH 6.4 and 3.59 g of E3D, while the right pit received 5.9 g of NIC.

[0358] A black Rodeo shirt, 100% polyester code named KB, was received with both pits at an odor level of 3. The right pit was treated with E3D, 2.92 g, and the left pit was treated with 4.0 g of 2.1% NAC at a pH of 7.8 followed by 3.00 g of E3D.

[0359] Thick soccer socks from an athletic young male were received with high odor, each labeled to indicate right and left. The socks were first wetted with 10 g of water spray, then the foot region was treated with 10.6 g of 2% NAC at pH 7.8 and then 7.1 g of E3D. The right was treated with 6.2 g of E3D, but no NAC.

[0360] A gray polyester woven jacket was received with mild odor in the pits. The right pit was treated with 9 g of PNAC5 and 3.3 g of E3D.

[0361] The shirts, jacket, and socks were put in plastic bags and keps at about 33 C. for 2 hours, then washed in a full cycle at 40 C. using liquid Tide detergent. All seemed odor free after washing. Visualization in UV light revealed no signs of optical brightener accumulation in the pits after washing, in spite of the detergent having strong optical brightener. No obvious fluorescence was visible in the shirts before washing, either, though it is not known if they had been washed recently with a detergent containing optical brighteners.

Rags with Perma-Odor

[0362] Several freshly washed kitchen rags said to have perma-odor were obtained from a family with small children in Wisconsin. Rag 1, a dark green rag about 30 cm square and made from 100% polyster microfiber, had a cheese odor akin to Limburger cheese. Rag 2, a pink doily, had a mildew odor. Rag 3, a cotton rag, had a cheesy odor. Rag 4, apparently cotton, had a musty odor.

[0363] Rag 3 was treated with E4C in one quadrant, labeled quadrant 2, with 5.86 g applied followed by addition of 6 g of water. Rag 2, with a dry mass of 23 g, was sprayed with 5.6 g water and then 5 g of E4C. Rag 1 was wetted with 9.45 g water and then 12.2 g of E4C. The three treated rags were wrapped in plastic and allowed to sit overnight to allow time for bacterial action.

[0364] The rags then went through two wash cycles, first one cycle without added laundry detergent and then a cycle with Persil Pro Clean, a fragrance free detergent. After drying, Rag 2 smelled noticeably better. The treated quadrant of Rag 3 smelled noticeably better while the untreated portions still had a cheesy smell. Unfortunately, Rag 1 still had a cheesy smell. It was hypothesized that the use of the viscous concentrate resulted in poor uniformity in spraying the compound, or that additional agents were needed to better attack a biofilm.

[0365] Four quadrants on the rag were identified. Quadrants 2 and 3 were treated with PNAC4, applying 9.4 g and 10 g, respectively. Herbal solution BJC1, 5.65 g and BJC2, 5.7 g were applied to quadrant 1. ENC was then applied to quadrant 2, 5.4 g, then quadrant 3, 5.8 g, and then quadrant 4, 10.3 g. Quadrant 4 was also given 5 g of water. After sitting for about an hour, Rag 1 was then hand washed in dilute Persil Pro Clean (about 15 g in 2 liters of water) and then machine washed with the same detergent in a regular washing cycle with Persil Pro Clean and dried. After washing, Rag 2 had dramatically reduced odor, essentially odor free on all quadrants.

Shoe Treatments

[0366] 3 pairs of children's shoes were treated. Two pairs were of the Keen band, shoes SL1 (a larger pair) and SL2, and one pair was of the New Feel brand, shoes SL3. For shoes SL1, both the right and the left were treated with 5 g per shoe of 2.1% NAC solution. Shoes SL2 were treated in the right shoe only, first with 10 g of EGCD-1 and then 10.5 g of 2.1% NAC solution. Shoes SL3 were treated in the right shoe with 11.9 g of 2% NAC solution at a pH of 7.8, while the left was treated with 22.5 g of 1% NAC at a pH of 2.18. Following a dwell time of 20 minutes, PuriShield spray was also applied. To shoes SL2, both were given a total of about 10 g of PuriShield. After 3 minutes, 11.5 g of EGCD-1 was applied to the right shoe only. The shoes were then rinsed in warm water, and 1.8 g of PuriShield spray was applied to the right shoe only and allowed to air dry.

[0367] The shoes were hand rinsed in warm water and then air dried. Residual odor was significantly reduced in the treated shoes, but after a few days' of use, odor returned, indicating further treatment was needed.

[0368] Shoes SL3 then had an odor level of about 2 (max of 5) in the left and about 1 in the right shoe. SL1 had an odor level of about 2 in the left and 3 in the right. SL2 had 1 in the left and 2 in the right. Enzymatic treatment was now applied. Shoes SL3 were treated with PNAC3, with 2.7 g sprayed into the right shoe, emphasizing the forward interior region around the toes, and 3.8 g likewise applied to the left shoe. Shoes SL2 were treated with 5.1 g of PNAC3 in the left shoe and 4.8 g of 1.8% NAC at pH 3 was applied to the right shoe, followed by 3.6 g of PNAC3, again emphasizing the regions around the toes but also spraying the complete interior of the shoe. Shoes SL1 were treated with 1.7 g EGCD-1 and 3.9 g of PNAC3 in the right while the left was treated with about 4 g of PNAC1 only. After about 20 minutes, the shoes were rinsed thoroughly in warm water (about 45 C.), then rinsed in a dilute solution of liquid Tide in warm water (about 3%), and then rinsed again.

Fluorescence and Dye Testing

[0369] 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 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.

[0370] 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 operating at 395 nm.

[0371] In one approach, biofilm candidates were stained using Calcofluor White M2R dye, a fluorescent brightener purchased from Phyto Technology Laboratories (Shawnee Mission, Kans.), CAS No. 4404-43-7. A solution was prepared of 0.074% Calcofluor white in water, and given the name CF1.

[0372] Two 100% polyester sports tops, the above-mentioned orange shirt CH1, and a blue top from Danskin, BD1, both with similar design and material, were examined. Both had been used for exercise for several years with persistent odor issues, although CH1 now showed substantial improvement in both pits following the previously described treatments. At this time, the orange top CH1 had been used in multiple exercise sessions since the previously discussed treatments with very little washing to create odor trouble and possible to promote biofilm growth again.

[0373] In examining fluorescence, it was eventually noted that both an SLR camera 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, it was found that better images could be obtained by placing a yellow UV-absorbing lens form UV safety glasses over the eye of the camera. A rubber band was sufficient for holding it in place.

Shirt CH1 (Orange Champion Shirt)

[0374] For shirt CH1, following the previously described treatments, 8.5 g of Tide (Procter and Gamble, Cincinnati, Ohio) liquid laundry detergent in 15 ml of water was applied to the left pit of the wet shirt, while 6.8 g of CF1 was applied to the right pit and arm area and it was then rinsed in warm water for about 1 minute. Tide liquid laundry detergent is believed to contain Calcofluor White, and its fluorescence under UV is consistent with that of Calcofluor White.

[0375] FIG. 10A depicts the left pit of shirt CH1 in visible light after the treatments described previously (as well as the treatment with optical brighter). A dark region 202 is still present that appears to come from within the fabric rather than resting on its surface. It is believed to come from biofilm material such as polysaccharides, proteins, and genetic material, that is capable of adhering to optical brighteners such as CF1 solution. FIG. 10B depicts the same region in UV light using the UVBeast V3 lamp. The dark region 202 of FIG. 10A is now a fluorescent region 204 with the appearance of a purple color, believed to result from Calcofluor White fluorescence couple with the dark matter's blocking of the fluorescence of the shirt material itself. UV light revealed regions in both pits of CH1 where the optical brightener was apparently adhering. The fluorescence in CH1 was bright enough to be detected even with the orange dye of the shirt brightly fluorescing at the same time.

[0376] FIG. 10C offers another view of fluorescence in the left pit. FIG. 10D shows the right pit in visible light, where dark regions 202A and 202B are manifest. FIG. 10E shows the right pit in UV light, where the dark regions 202A and 202B now fluoresce as fluorescent regions 204A and 204B, respectively.

[0377] On the possibility that biofilm was being visualized with the optical brightener, a biofilm treatment was attempted. The left pit of CH1 was treated with 6 g of 2.1% NAC. Then 1.95 g of E3 was applied to the same region. For the right pit, 2.1 g of E3 was applied without NAC. After about 20 minutes, the shirt was washed in a short cycle at 30 C. with Purex detergent. Darkened regions remained, though perhaps slightly lighter. For example, FIG. 10F shows the treated right pit, which still has a darkened region that becomes fluorescent region 204 in UV light.

[0378] The right pit was treated with 7.6 g of CF1 and the left with 7.2 g of CF1. FIG. 10G shows the left pit under the light of the Lightfe UV301D at 365 nm, showing fluorescence remains, though perhaps weaker than initially seen. FIG. 10H shows the fluorescent region 204 of the right pit also with the Lightfe UV301D lamp. After further washing, two exercise sessions, and treatment with 3.44 g of CF1, the right pit of CH1 shows fluorescence, as shown in FIG. 10I, but it seems less than was initially present in FIG. 10D.

[0379] In CH1, one of the dark patches was treated with a needle jet from a Waterpik Water Flosser for dental hygiene, delivering about 1 liter of water to roughly a 1 cm1 cm patch, resulting in significant lightening of the patch, but still with remaining darkened matter that appeared to be deep in the textile material. This is believed to be a remnant of a biofilm that has left material possibly comprising polysachharides and proteins that can adhere to colorants in wash and may form a platform allowing bacteria to more easily attach to the clothing. In general, mechanical means such as a needle jet, brushing, friction, and so forth may be helpful in freeing biofilm remnants from fabric. The treated area was them sprayed with 6.7 g of CF1, rinsed in about 2 liters of warm water for 1 minute, and then examined in UV light showing improvement in the treated spot.

[0380] 3.76 g of spray E3D was applied to CH1's left pit and rubbed into the slightly moist shirt (about 50% moisture). The shirt was put into a plastic bag and rolled up and kept at about 29 C. for over 4 hours, then washed in a fast cycle at 30 C. with Purex detergent. After washing, the shirt was again sprayed with CF1 and viewed in UV light, as shown in FIG. 10J. The fluorescent spots 204 were still present but the intensity of the fluorescence had noticeably but slightly decreased.

Shirt BD1 (Blue Danskin Shirt)

[0381] The 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 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. 11A 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. 11B, 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. 11C shows the fluorescent zone 204 also present in FIG. 11B, 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 FIGS. 11B and 11C on the sleeve about 9 cm from the center of the pit. FIG. 11D 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).

[0382] 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.

[0383] 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 a temperature of 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 again the intensity may have decreased slightly.

[0384] 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 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 detergent 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.

[0385] 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 (about 7 cm wide and 3 cm tall) was substantially reduced in size and intensity.

[0386] 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 PMIX1, 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, while the yellowish zone had been largely removed. The result for the treated left pit is shown in FIG. 11E showing the fluorescent region 204. The shirt was then washed in a full cycle with Tide liquid detergent at 40 C. FIG. 11F shows the result for the treated left pit. These results in FIGS. 11E and 11F still show the fluorescent patch 204, but its intensity to the naked eye was significantly reduced through these treatments.

Fluorescent Testing with a Gray Perma-Odor Sports Shirt

[0387] An athletic female who exercises almost daily reported that her polyester sports top showed symptoms of perma-odor following extensive use. This shirt, code named RA1, was a 100% polyester gray Melange Jersey knit shirt from Academy, Ltd. (Katy, Tex.), made in Kenya. It exhibited strong fluorescence as is in both pits, with no need to treat with Calcofluor. FIG. 12A shows a photo of the right pit under UV light, and FIG. 12B shows the left pit under UV light. After turning the shirt inside out, FIG. 12C 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. 12D for the right pit for comparison, and for the left pit in FIG. 12E. Fluorescence is visible on the inside and outside. A close-up of the left pit, right-side out, is shown in FIG. 12F.

[0388] 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 many other exercise routines that would allow the sweat to be inclined slight 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.

[0389] Shirt RA1 was then treated with E3D with 9.2 g in the right pit region and the left pit first treated 2.1% NAC, 4.54 g, and then 10.7 g of E3D. The initially dry shirt was 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. 12G, 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 in color to the naked eye than in the images converted to black and white. The right pit (not shown) still appeared bright and lacked the visible reduction seen in the left pit, though it may have been slightly reduced in intensity also.

[0390] 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. 12H, also taken without the UV filter in place

[0391] 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. 121, 12J, and 12K, 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% at pH 4.0, followed by individual treatments in the treatment zones.

[0392] In the upper treatment zone shown in FIG. 12I, 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, as confirmed with pH testing paper, followed by dropwise application by pipette of 2.6 g of AmylaseA solution.

[0393] The lower treatment zone shown in FIG. 12K, 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. 12K shows the boundary marker 208 (here a rubber band) marking the upper treatment zone 210 for pectinase solution. Treatment zone 210 contains portions of the fluorescent zone 204 in the left pit.

[0394] In FIG. 12J, 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 then rinsed in fresh water for about 1 minute.

[0395] 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. 12J, 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 was not dramatically different, though it seemed slightly attenuated in both cases. The pit were examined in UV light and photographed with a UV lens in place, with results for the left pit shown in FIG. 12L and the right pit in FIG. 12M.

[0396] Now two treatment zones in the left pit were considered, both circles about 5 cm in diameters, as shown with UV light in FIG. 12N, 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 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. 12O, 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. 12P, taken with the UV filter.

[0397] 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 area and the cuff region on the left sleeve).

[0398] To raise the pH, a solution at pH 9.89 was prepared from 1.56 g sodium carbonate and 1.0 g NaHCO.sub.3 in 91 ml of water. 13 g of this alkaline solution were applied to the treated regions of the shirt and it was then returned to the plastic bag and kept at about 32 C. The shirt was then rinsed and examined. The fluorescence was perhaps slightly less but the dimensions of the fluorescing areas were substantially the same. See FIG. 12Q for the left pit.

[0399] 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 roughly 1% solution of Comfort brand laundry detergent. Residual fluorescence was still visible, but had declined more strongly in the lower half of the pit.

[0400] In a further test, 12 g of CellAmyIA 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. 12R 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 plainly visible in color to the naked eye. The shirt was put in a plastic bag and held at about 33 for 1 hour. Slightly reduced fluorescence was again observed.

[0401] Next the right pit was considered. An upper zone, Section A, in the pit comprising the cuff of the right shirt sleeve having an oval shape about 5 cm wide and 3 cm tall was treated with 3 g of water and then 2.6 g of PAPA. A lower region of similar dimensions was denoted Section B was centered on the seam at the side of the shirt about 8 cm below the seam connecting the sleeve to the shirt. It was treated with 1.1 g of 2% NAC at pH 6.4 and 1.8 g of PAPA. After 20 seconds of mild rubbing, and rinsing, UV light showed further progress in removing the fluorescing material. The same pit was treated again. 3.46 g of 2% NAC solution at pH 9.17 was applied to the upper portion of the pit, while 2.35 g of LysoB was then applied to the upper regions and 2.25 g of LysoB was applied to the lower area. After rinsing, it was observed that fluorescence had been slightly reduced again. See FIG. 12S.

[0402] In another 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.

[0403] 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 reduction in fluorescence. The benefit of the citrate treatment may be most useful for biofilms that have not been treated multiple times already, though it is also believed to have an impact in disrupting living bacteria to reduce their potential to form further biofilms.

[0404] In another test, a strongly 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. *

[0405] In another test, a fluorescent zone under the right pit, on the front of the shirt about 10 cm below the pit along the seam, was treated with both GASTRO-1 and PANNAC. First 0.79 g of PANNAC was applied to a circular area about 2 cm in diameter, and after about 5 minutes 1.0 g of GASTRO-1 was applied to the same spot. After 15 further minutes, 0.75 g of E4D was applied. This was kept at about 22 C. for about 4 hours.

The Triathlon Shirt, TR1

[0406] The triathlon shirt TR1 was examined. Since initial biofilm busting 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.

[0407] 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.

[0408] FIGS. 13A and 13B 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

[0409] 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 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 NaCl, 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, N.Y.) 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 palmitic 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.

[0410] 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 the fluorescence in the pits were difficult to assess, and it is unclear if the trial was successful.

[0411] More visible success came after considering recent research results on the tenacity and heat resistance of some microbes in dry biofilms. It was then 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.

[0412] 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, Wis. that showed fluorescence in both pits under UV light, most strongly in the right pit, a shown in FIG. 14A, 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. Before treating with artificial sweat, both pits were treated with about 3.7 g each of E7D spray, wrapped in plastic, and allowed to site overnight before being washed with TIDE liquid detergent and tumble dried. The fluorescent zone in the right pit appeared slightly weaker at best 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 approximately 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 of added moisture) to reduce wicking and premature drying of the treated areas in the right pit and lower hem. After the first 4 treatments, the shirt was treated again but allowed to air dry without being wrapped in a plastic bag. This, any biofilm would have experienced 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 about 10 minutes in about 3 liters of warm water with 8 g of added TIDE Simply Clean laundry detergent, a detergent highly fluorescent in UV light and believed to contain Calcofluor White. After rinsing and tumble drying, the shirt 200 was examined in UV light, as shown in FIG. 14B and in addition to the initial fluorescent region 204A, was seen to have an new fluorescent region 204B that may represent the growth of new biofilm material capable of absorbing the optical brightener in the laundry detergent. 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

[0413] To further examine potential biofilm zones in various clothing items with a history of perma-odor, work was carried out with confocal and fluorescent 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 in K. VanderWaal, University of Minnesota Nano Center Standard Operating Procedure [for the Nikon C2 Confocal Microscope], 2016; 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 340-380 nm, and then filters the emission band to 435-485 nm). In both cases, no fluorescent dyes were added to the material, but the inherent fluorescence in the clothing, believed to be due to optical brighteners, was relied on.

[0414] FIGS. 15A and 15B 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 (a bioenzymatic mix described above), with added moisture present in that portion of the dress resulting in roughly a 2:1 dilution. For the left pit, first a pretreatment was applied by spraying 2 g of a biofilm attack solution, PANNAC, onto the biofilm region and adjacent fabric. After a 5-minute wait, 1.54 g of E6D was applied to the same region. With some 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. 16A and 16B 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) still fluorescing in the left pit, but essentially nothing visible to the eye remaining in the right pit.

[0415] FIGS. 17A and 17B 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 characteristics of the microscope using the UV laser. FIG. 17A 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. 17B, 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.

Further Observations

[0416] In several shirts with apparent perma-odor, it was observed that after an apparently successful treatment aimed at reducing biofilm, the treated pits when washed in a laundry detergent with fragrance, failed to emit the fragrance that was present elsewhere in the shirt. It was speculated that biofilms or the residual materials left by stains, fabric softeners, and other foreign materials that may be present in clothing, may serve as a scaffolding for absorption and release of both fragrances and odorants beyond what typical synthetic fibers might do on their own. Removing some of the biofilm or other embedded materials on the fibers may take away an odor reservoir for fragrance and malodor alike.

Remarks

[0417] When introducing elements of aspects of the invention or the embodiments thereof, the articles a, an, the, and said are intended to mean that there are one or more of the elements. The terms comprising, including, and having are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0418] All patents and patent publications cited herein may be presumed to be incorporated by reference to the extent it is non-contradictory herewith. When a document is explicitly said to be incorporated by reference, it is also implied that it is incorporated by reference to the extent it is non-contradictory herewith.

[0419] 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 above description shall be interpreted as illustrative and not in a limiting sense.

[0420] While the foregoing makes reference to particular illustrative embodiments, these examples should not be construed as limitations. The inventive system, methods, and products can be adapted for other uses or forms not explicitly listed above, and can be modified in numerous ways within the spirit of the present disclosure. Thus, the present invention is not limited to the disclosed embodiments, but is to be accorded the widest scope consistent with the claims below.

[0421] 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 (NNCIN) under Award Number ECCS-1542202.