FABRIC CARE COMPOSITIONS WITH DOSE-DEPENDENT ANTIMICROBIAL ACTIVITIES THAT REDUCE THE RISK OF INFECTION AND PROMOTE HEALTH AND HYGIENE WHEN USED DISCRETELY OR SYNERGISTICALLY IN A MACHINE WASHING AND DRYING LAUNDRY PROGRAM
20230257682 · 2023-08-17
Assignee
Inventors
Cpc classification
C11D3/48
CHEMISTRY; METALLURGY
C11D1/645
CHEMISTRY; METALLURGY
C11D3/38636
CHEMISTRY; METALLURGY
C11D1/28
CHEMISTRY; METALLURGY
C11D3/38618
CHEMISTRY; METALLURGY
C11D3/30
CHEMISTRY; METALLURGY
C11D1/835
CHEMISTRY; METALLURGY
A01P1/00
HUMAN NECESSITIES
C11D1/94
CHEMISTRY; METALLURGY
International classification
C11D11/00
CHEMISTRY; METALLURGY
A01P1/00
HUMAN NECESSITIES
C11D1/94
CHEMISTRY; METALLURGY
C11D3/00
CHEMISTRY; METALLURGY
C11D3/30
CHEMISTRY; METALLURGY
C11D3/386
CHEMISTRY; METALLURGY
Abstract
This present invention is directed to antimicrobial fabric care compositions that provide detersive benefits such as stain and soil removal and aesthetic benefits like softening, wrinkle-reduction and color-protection and when used discretely or synergistically during the laundry program reduce the risk of infections and promote health and hygiene by providing dose-dependent antimicrobial activities such as sanitizing, disinfecting and removing malodors from fabrics, and imparting 24-48 hour antimicrobial-durability into fabrics, and deactivating biofilms inside the washing machine and preventing biofilm formation inside the washing machine to prevent recontamination of fabrics. The compositions are well-suited for use in domestic, commercial and on-premise-laundry-operations such as healthcare facilities, hotels, cruise ships, and the like. Methods for discrete and synergistic uses to simultaneously treat fabrics and the internal surfaces of the washing machine are also disclosed.
Claims
1. The fabric-treatment method that removes stains and soils from fabrics, and softens fabrics and protects the color of fabrics and reduces the risk of infections and promotes health and hygiene by providing dose-dependent antimicrobial activities such as, removing malodors from fabrics, and sanitizing and disinfecting fabrics, and imparting antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program, and deactivating biofilms inside the WM, and preventing the formation of biofilms in the WM; comprises the process of treating the fabrics in the wash-cycle of the laundry program in which the temperature of the wash-water is ≥16° C. and contains the antimicrobial liquid laundry detergent Composition A which comprises: A) 17-20% by weight of the cationic biocide, Poly (hexamethylene biguanide Hydrochloride; B) 1-4% by weight of the cationic biocide n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride; C) 3-6% by weight of the anionic surfactant blend Sodium Methyl 2-Sulfolaurate and Disodium 2-Sulfolaurate; D) 2-8% by weight of a fatty alcohol ethoxylate C14-15 Pareth 7, with an E.O. content weight % of 56, and a hydroxyl value of 103 mg KOH/g; E) 3-10% by weight of a fatty alcohol ethoxylate with an average number of moles of E.O of 9; F) 3-5% by weight of a Cocoamidopropyl Betaine with an active % from 30-37; G) 0.65-5% by weight of the rheology modifier Poly (oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z, Z); H) 4-6% by weight of an enzyme medley or mixture having equal parts of a lipase, a protease, an amalyse, a mannanase, and a cellulase; I) 4-5.1% by weight 1,3 propanediol; J) with the remaining balance to 100% by weight deionized or distilled water; and/or treating the fabrics in the final-rinse-cycle of the laundry program in which the temperature of the rinse-water is ≥16° C. which contains the antimicrobial liquid fabric softener Composition B comprising: A) 2-5% by weight of the cationic biocide, Poly (hexamethylene biguanide Hydrochloride; B) 2-4% by weight of cationic biocide, n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride; C) 2-6% by weight of Ethanaminium, 2-hydroxy-N, N-bis(2-hydroxyethyl)-Nmethyl-, esters with C16-18 and C18-unsatd. fatty acids, Me sulfates salts; D) 0.8-2% by weight of Wheat, [2-hydroxy-3-[3-(trimethoxysilyl)propoxy] propyl], hydrolyzed; E) 2-2.5% by weight 1,3 propanediol; F) with the remaining balance to 100% deionized or distilled water.
2. The fabric treatment method according to claim 1 that removes stains, soils and malodors from fabrics, and sanitizes or disinfects fabrics, and impart antimicrobial durability into the fabrics wherein the dosage of Composition A, the water-bath and the fabrics are present in the wash-cycle in accordance with Table 01 below, TABLE-US-00031 TABLE 01 Dosage (ounces) To Disinfect To Remove Sanitize Malodor Fabric and Impart Weight Water remove antimicrobial Fabric Type (lbs.) (gals.) Malodors Durability Cotton, Cotton 2.0 0.70 0.50 0.75 blends, 5.0 1.80 1.00 1.50 Synthetic 7.0 2.50 1.60 2.25 fibers 10.0 3.50 2.25 3.25 12.0 4.20 2.55 4.00 15.0 5.30 3.25 5.00 17.0 6.00 3.75 5.50 20.0 7.00 4.50 6.50 25.0 8.70 5.50 8.00
3. The fabric treatment method according to claim 1 that removes stains, soils and malodors from fabrics, and disinfects fabrics, and imparts antimicrobial durability into the fabrics that continues for 24-48 hours and deactivates biofilms inside the WM, and prevents the formation of new biofilms inside the WM wherein the dosage of Composition A, the water-bath and the fabrics are present in the wash-cycle in accordance with Table 02 below, TABLE-US-00032 TABLE 02 Dosage (ounces) Weight Water Composition A Fabric Type (lbs.) (gals.) in the Wash-Cycle Cotton, Cotton 2.0 0.70 1.00 blends, 5.0 1.80 1.75 Synthetic 7.0 2.50 2.50 fibers 10.0 3.50 3.50 12.0 4.20 4.25 15.0 5.30 5.25 17.0 6.00 5.75 20.0 7.00 6.75 25.0 8.70 8.25
4. The fabric treatment method according to claim 1 that softens and protects the color of fabrics and removes malodors from fabrics, and sanitizes fabrics, and imparts antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program wherein the dose-rate of Composition B, the water-bath and the fabrics are present in the final-rinse-cycle in accordance with Table 03 below, TABLE-US-00033 TABLE 03 Dosage (ounces) Weight Water Composition B Fabric Type (lbs.) (gals.) in the Final-Rinse-Cycle Cotton, Cotton 2.0 0.70 0.75 blends, Synthetic 5.0 1.80 1.50 fibers 7.0 2.50 2.25 10.0 3.50 3.25 12.0 4.20 4.00 15.0 5.30 5.00 17.0 6.00 5.50 20.0 7.00 6.50 25.0 8.70 8.00
5. The fabric treatment method according to claim 1 that removes stains soils and malodors from fabrics, and softens and protects the color of fabrics and works synergistically to sanitize fabrics and impart antimicrobial durability into the fabrics that continues for 24-48 hours wherein the dosage of Composition A the water-bath and the fabrics are present in the wash-cycle in accordance with the Table 04 below, and the dosage of Composition B the water-bath and the fabrics are present in the final-rinse-cycle in accordance with Table 04 below, TABLE-US-00034 TABLE 04 Dosage (ounces) Fabric Composition A Composition Weight Water in the B in the Fabric Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 0.50 0.75 Cotton 5.0 1.80 1.00 1.50 blends, 7.0 2.50 1.60 2.25 Synthetic 10.0 3.50 2.25 3.25 fibers 12.0 4.20 2.55 4.00 15.0 5.30 3.25 5.00 17.0 6.00 3.75 5.50 20.0 7.00 4.50 6.50 25.0 8.70 5.50 8.00
6. The fabric treatment method according to claim 1 that removes stains, soils and malodors from fabrics, and softens and protects the color of fabrics and works synergistically to disinfect fabrics and to impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program wherein the dosage of Composition A, the water-bath and the fabrics are present in the wash-cycle in accordance with the Table 05 below and the dosage of Composition B, the water-bath, and the fabrics are present in the final-rinse-cycle in accordance with the Table 05 below, TABLE-US-00035 TABLE 05 Dosage (Ounces) Composition A Composition B Fabric Weight Water in the in the Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 1.00 0.75 Cotton 5.0 1.80 1.75 1.50 blends, 7.0 2.50 2.50 2.25 Synthetic 10.0 3.50 3.50 3.25 fibers 12.0 4.20 4.25 4.00 16.0 5.30 5.25 5.00 17.0 6.00 5.75 5.50 20.0 7.00 6.75 6.50 25.0 8.70 8.25 8.00
7. The fabric treatment method according to claim 1 that removes stains, soils and malodors from fabrics, and softens and protects the color of fabrics and works synergistically to disinfect fabrics and to impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program and deactivates biofilms inside the WM and prevents the formation of biofilms inside the WM wherein the dosage of Composition A, the water-bath and the fabrics are present in the wash-cycle in accordance with the Table 06 below, and the dosage of Composition B, the water-bath, and the fabrics are present in the final-rinse-cycle in accordance with the Table 06 below, TABLE-US-00036 TABLE 06 Dosage (Ounces) Composition A Composition B Fabric Weight Water in the in the Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 1.00 1.00 Cotton 5.0 1.80 1.75 1.75 blends, 7.0 2.50 2.50 2.50 Synthetic 10.0 3.50 3.50 3.50 fibers 12.0 4.20 4.25 4.25 16.0 5.30 5.25 5.25 17.0 6.00 5.75 5.75 20.0 7.00 6.75 6.75 25.0 8.70 8.25 8.25
8. The fabric treatment method according to claim 1 that removes stains soils and malodors from fabrics, and softens and protects the color of fabrics and works synergistically to sanitize fabrics and impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program wherein the dosage of Composition A, the water-bath and the fabrics are present in the wash-cycle in accordance with Table 07 below and the dosage of Composition B, the water-bath, and the fabrics are present in the final-rinse-cycle that follows an intermediate-rinse-cycle, in accordance with Table 07 below; and that removes stains soils and malodors from fabrics and softens and protects the colors of fabrics and works synergistically to disinfect fabrics and impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program wherein the dosage of Composition A the water-bath and the fabrics are present in the wash-cycle in accordance with Table 08 below and wherein the dosage of Composition B the water-bath and the fabrics are present in the final-rinse-cycle that follows an intermediate-rinse-cycle, in accordance with Table 08 below; and that removes stains, soils and malodors from fabrics and softens and protects the colors of fabrics and works synergistically to disinfect fabrics and to impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program and to deactivate biofilms inside the WM and prevents the formation of biofilms inside the WM wherein the dosage of Composition A the water-bath and the fabrics are present in the wash-cycle in accordance with Table 09 below and the dosage of Composition B the water-bath and the fabrics are present in the final-rinse-cycle that follows an intermediate-rinse-cycle, in accordance with Table 09 below, TABLE-US-00037 TABLE 07 Dosage (ounces) Fabric Composition Composition B in Weight Water A in the the Fabric Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 1.00 0.75 Cotton 5.0 1.80 1.60 1.50 blends, 7.0 2.50 2.25 2.25 Synthetic 10.0 3.50 2.55 3.25 fibers 12.0 4.20 3.25 4.00 15.0 5.30 3.75 5.00 17.0 6.00 4.50 5.50 20.0 7.00 5.50 6.50 25.0 8.70 6.75 8.00 TABLE-US-00038 TABLE 08 Dosage (ounces) Fabric Composition Composition B in Weight Water A in the the Fabric Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 1.45 1.00 Cotton 5.0 1.80 2.20 1.75 blends, 7.0 2.50 2.95 2.50 Synthetic 10.0 3.50 3.25 3.50 fibers 12.0 4.20 3.75 4.25 16.0 5.30 4.25 5.25 17.0 6.00 4.50 5.75 20.0 7.00 4.75 6.75 25.0 8.70 5.95 8.25 TABLE-US-00039 TABLE 09 Dosage (ounces) Fabric Composition Composition B in Weight Water A in the the Fabric Type (lbs.) (gals.) Wash-Cycle Final-Rinse-Cycle Cotton, 2.0 0.70 1.70 1.00 Cotton 5.0 1.80 2.45 1.75 blends, 7.0 2.50 3.20 2.50 Synthetic 10.0 3.50 3.45 3.50 fibers 12.0 4.20 4.00 4.25 16.0 5.30 4.50 5.25 17.0 6.00 4.75 5.75 20.0 7.00 5.00 6.75 25.0 8.70 5.20 8.25
9. The method to determine the antimicrobial durability imparted into a fabric during the wash-cycle and/or the rinse-cycle of the laundry program comprises the process of: boiling a test-fabric (50% cotton/50% polyester weighing 441-g) for 1-h in 4-liters of distilled or deionized water containing 2.2-g of Naz CO.sub.3 and 2.2-g of a nonionic wetting agent Makon 10 (Stepan Co. Chicago, Ill.) to remove impurities; and rinsing the test-fabric in 4 liters of boiling distilled or deionized water; and rinsing the test-fabric in 4-liters cold distilled or deionized water until all visual traces of the wetting agent are removed; and centrifuging the test-fabric for 15-seconds in the spin-chamber of a portable washing machine to remove excess water; and air-drying the test-fabric for 24-hr at ambient temperature; and cutting the scoured, dried fabric into 3-in×5-in pieces and labeling the fabric pieces as Control fabric1, Control fabric2 and Test fabric1; and treating Control fabric2 and Test fabric1 for 17-minutes in the wash-tub of a portable washing machine with a wash-water-solution having 75-ml of 400 ppm of AOAC hardwater cooled to 16±2° C. and 532.5 ul of Composition A; or treating Control fabric2 or test fabric1 in the wash-tub of a portable washing machines with a rinse-water-solution having 75-ml of 400 ppm of AOAC hardwater and 532.5 ul of Composition B; and centrifuging Control fabric2 and Test fabric1 in the spin-tub of a portable washing machine for 15-seconds; and air-drying Control fabric2 and Test fabric1 for 2-h at room temperature and removing Control fabric 2 into a 950-ml glass tray (Pyrex Life Sciences, Tewksbury, Mass. USA) containing 150 ml of Bromophenol Blue Dye (Biopharm, Inc Hatfield, AR. USA) until Control fabric2 is completely saturated with the dye; and air-drying Control fabric2 until completely dry; and visually/qualitatively determining the presence or absence of the antimicrobial actives from Composition A or Composition B imparted into Control fabric2 by the presence of the Bromophenol Blue Dye which complexes with the antimicrobial actives from Composition A or Composition B to form a stable blue complex; and thermally curing Test fabric1 in a residential dryer at 65±1° C. for 20-minutes; and inoculating Test fabric1 and Control fabric1 with 0.30-ml of a mixed species inoculum (340 ul of P. aeruginosa ATCC 15442, and S. aureus ATCC 6538, and K. pneumoniae ATCC 4328 and 75 μL of Bovine Serum Albumin solution, 200 μL of Bovine Mucin solution, and 70 μL Tryptone solution) covering a 2 in×3 in area of the bottom third of the fabric strip; and air drying Test fabric1 and Control fabric1 for 24 or 48 hours (the contact period) at 20° C.; and removing Test fabric1 and Control fabric1 into separate wide-mouthed 250-ml Erlenmeyer flasks containing 50-ml of PBS; and vortexing the 250-ml Erlenmeyer flasks for 10-s repeating the vortex period 7 times to elute and suspend the surviving challenge microorganisms; and performing a 10-fold serial dilution to 10.sup.−7 of 200 ul of the microorganism suspension from Control fabric1 and Test fabric1 and plating 0.02 ml of each dilution in duplicate on Tryptic Soy Agar; and incubating the test plates at 35±1° C. for 24-hr; and counting the colonies from the spread-plate for Control fabric1 or Test fabric1 of the most appropriate dilution showing counts from 30-300 cfu/plate and recording the data as cfu/plate to determine the number of surviving organisms; and averaging duplicate plate counts and multiplying by the dilution factor to calculate cfu/mL of the mixed species microorganism suspension; and calculating the Log.sub.10 reduction using the following equation: Log.sub.10 Reduction=Log(B/A), Where: B=Number of viable test microorganisms on Control fabric1, and A=Number of viable test microorganisms on Test fabric1.
10. The method to determine the antimicrobial efficacy of Composition A to sanitize or disinfect fabrics when used in the wash-cycle of a laundry-program comprises treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a wash-water-solution comprised of from about 532.5 ul to about 887.5 ul of Composition A and from about 75-ml to about 125-ml of 400 ppm AOAC Hard Water, for a specified period of time (the exposure time) in the wash-tub of a portable washing machine, and centrifuging the treated 25-g fabric bundle for a specified period of time (the spin-time) in the spin-tub of a portable washing machine, and removing the treated 25-g fabric bundle from the spin-tub of the portable washing machine and aseptically removing the fabric carriers from the treated 25-g fabric bundle into separate 50-ml wide-mouthed tubes containing 10-ml neutralizer broth (3% (w/v) Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, 0.05% (w/v) KH.sub.2PO.sub.4, 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS), and using the drain-hose on the portable washing machine to extract 5-ml of the wash-water-solution into a 50-ml wide-mouthed tube containing 40-ml of concentrated neutralizer broth (6% (w/v) Tween 80, 0.45% (w/v) Lecithin, 2.0% (w/v) Sodium thiosulfate, 3% (w/v) K.sub.2HPO.sub.4, 0.10% (w/v) KH.sub.2PO.sub.4, 2% (w/v) Poly-[sodium-4-styrenesulfonate], 0.2% (v/v) Triton®×100 prepared in PBS), and determine the sanitizing efficacy by serially diluting 1.0 mL neutralized wash water to 10.sup.−2 using dilution fluid containing neutralizers as needed and plating all dilutions in duplicate on Tryptic Soy Agar containing neutralizers as needed, and filtering the remaining neutralizer broth/wash water combination and plating the filter on Tryptic Soy Agar+5% sheep's blood containing neutralizers as needed, and incubate the plates at 35±2° C. for 48 to 54 h; and count and record the colonies as CFU/plate, and average the duplicate plates and multiply by the dilution factor to arrive at CFU/mL, and convert the average count into log.sub.10; or determine disinfecting efficacy of Composition A by filtering the entire volume of wash-water or rinse-water containing neutralizing broth and plating the filter on Tryptic Soy agar+5% sheep's blood containing neutralizers as needed, and incubating the plates, and the tubes containing the swatches for 48 t0 54 hrs at 35±2° C.; and record and report the results as growth (+) or no growth (−) for the filter, plates and wide-mouthed tubes; and/or the method to evaluate the antimicrobial efficacy of Composition B when used in the rinse-cycle of the laundry program comprises treating the 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a rinse-water-solution comprised of from about 532.5 ul to about 887.5 ul of Composition B and from about 75-ml to about 125-ml of 400 ppm AOAC Hard Water, for a specified period of time (the exposure-time) in the wash-tub of a portable washing machine and centrifuging the treated 25-g fabric bundle for a specified period of time (the spin-time) in the spin-tub of a portable washing machine, and removing the treated 25-g fabric bundle from the spin-tub of the portable washing machine and aseptically removing the fabric carriers from the 25-g fabric bundle into separate 50-ml wide-mouthed tubes containing 10-ml neutralizer broth (3% (w/v) Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, KH.sub.2PO.sub.4 0.05% (w/v), 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS), and using the drain-hose on the 2.sup.nd portable washing machine to remove 5-ml of the rinse-water-solution into a 50-ml wide-mouthed tube containing 40-ml of concentrated neutralizer broth 6% (w/v) Tween 80, 0.45% (w/v) Lecithin, 2.0% (w/v) Sodium thiosulfate, 3% (w/v) K.sub.2HPO.sub.4, 0.10% (w/v) KH.sub.2PO.sub.4, 2% (w/v) Poly-[sodium-4-styrenesulfonate], 0.2% (v/v) Triton®×100 prepared in PBS) and determine the sanitizing efficacy by serially diluting 1.0 mL neutralized rinse-water to 10.sup.−2 using dilution fluid containing neutralizers as needed and plating all dilutions in duplicate on Tryptic Soy Agar+5% sheep's blood containing neutralizers as needed, and filtering the remaining neutralizer broth/rinse water solution combination and plating the filter on Tryptic Soy Agar containing neutralizers as needed, and incubate the plates at 35±2° C. for 48 to 54 h, and count and record the colonies as CFU/plate, and average the duplicate plates and multiply by the dilution factor to arrive at CFU/mL, and convert the average count into log.sub.10 to; or determine the disinfecting efficacy of Composition B by filtering the entire volume of rinse-water solution containing neutralizing broth and plating the filter on Tryptic Soy agar+5% sheep's blood containing neutralizers as needed, and incubating the plates, and the tubes containing the swatches for 48 t0 54 hrs at 35±2° C.; and record and record the results as growth (+) or no growth (−) for the filter, plates and wide-mouthed tubes.
11. The method to determine the synergistic efficacy of Composition A when used discretely in the wash-cycle of a laundry-program and Composition B when used discretely in the rinse-cycle of the same laundry program to sanitize or disinfect fabrics, comprises treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a wash-water-solution comprised of from about 532.5 ul to about 887.5 ul of Composition A and from about 75-ml to about 125-ml of 400 ppm AOAC Hard Water, for a specified period of time (the 1.sup.st exposure time) in the wash-tub of a 1.sup.st portable washing machine and centrifuging the treated 25-g fabric bundle for a specified period of time (the spin-time) in the spin-tub of a 1.sup.st portable washing machine; and removing the 25-g fabric bundle from the spin-tub of the 1.sup.st portable washing machine into the wash-tub of the 2.sup.nd portable washing machine; and treating the same 25-g fabric bundle with a rinse-water-solution comprised of from about 532.5 ul to about 887.5 of Composition B and from about 75-ml to about 125-ml of 400 ppm AOAC Hard Water, for a specified period of time (the 2.sup.nd exposure time) in the wash-tub of a 2.sup.nd portable washing machine and centrifuging the treated 25-g fabric bundle for a period of time (the 2.sup.nd spin-time) in the spin-tub of a 2.sup.nd portable washing machine, and rinsing the twice treated 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a rinse water test solution containing from about 75-ml to about a 125-ml of 400 ppm AOAC Hard Water, for a specified period of time (the rinsing-time) in the wash-tub of a 3.sup.rd portable washing machine; and centrifuging the treated 25-g fabric bundle for a period of time (the 3.sup.rd spin-time) in the spin-tub of a 3.sup.rd portable washing machine; and removing the treated 25-g fabric bundle from the spin-tub of the portable washing machine and aseptically remove the fabric carriers from the treated 25-g fabric bundle into separate 50-ml wide-mouthed tubes containing 10-ml neutralizer broth (3% (w/v) Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, KH.sub.2PO.sub.4 0.05% (w/v), 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS); and using the drain-hose on the 3.sup.rd portable washing machine to extract 5-ml of the rinse-water-test-solution into a 50-ml wide-mouthed tube containing 40-ml of concentrated neutralizer broth 6% (w/v) Tween 80, 0.45% (w/v) Lecithin, 2.0% (w/v) Sodium thiosulfate, 3% (w/v) K.sub.2HPO.sub.4, 0.10% (w/v) KH.sub.2PO.sub.4, 2% (w/v) Poly-[sodium-4-styrenesulfonate], 0.2% (v/v) Triton®×100 prepared in PBS) and filter the entire volume of rinse-water-test-solution containing neutralizing broth and plating the filter on Tryptic Soy agar+5% sheep's blood containing neutralizers as needed, and incubating the plates, and the tubes containing the swatches for 48 t0 54 hrs at 35±2° C.; and record and report the results as growth (+) or no growth (−) for the filter, plates and wide-mouthed tubes.
12. The method to determine the amount of unused antimicrobial efficacy that is passed-through from one cycle of the laundry program into another cycle of the laundry program comprises the process of treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a wash-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul of Composition A, for a specified period of time (the exposure time) in the wash-tub of a 1.sup.st portable washing machine, and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 1.sup.st portable washing machine, and transferring the treated fabric bundle into the wash-tub of a 2.sup.nd portable washing machine containing about 500-ml of 400 ppm AOAC Hardwater (the ca-test-sample-water); and agitating the treated 25-g fabric bundle for a specified period of time (the agitation-time) to extract the unused antimicrobial efficacy of Composition A; and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 2.sup.nd portable washing machine to more fully extract the unused antimicrobial efficacy of Composition A; and/or treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a rinse-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul Composition B, for a specified period of time (the exposure time) in the wash-tub of a 1.sup.st portable washing machine, and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 1.sup.st portable washing machine, and transferring the treated 25-g fabric bundle into the wash-tub of a 2.sup.nd portable washing machine containing 500-ml of 400 ppm AOAC Hardwater (the cb-test-sample-water); and agitating the treated 25-g fabric bundle for a specified period of time (the agitation-time) to extract the unused antimicrobial efficacy of Composition B; and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 2.sup.nd portable washing machine to more fully extract the unused antimicrobial efficacy of Composition B; the 1.sup.st portable washing machine and the 2.sup.nd portable washing machine each comprising a cabinet, a wash-tub, a spin-tub, a pulsator, a water-inlet, a control panel, a pulsator-timer-switch, a pulsator-and-drain selector switch, a spin-timer-switch and a drain-hose.
13. The method according to claim 12, whereby the amount of unused Polyhexamethylene biguanide hydrochloride in for example 3,175 g or 7 pounds of fabrics, that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program is determined by quantifying the amount of polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting a Composition A ca-test-sample-water-P1 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition A ca-test-sample-water-P1, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the 25-ml graduated sample tube containing the Composition A ca-test-sample-water-P1; and swirling to thoroughly mix the contents of the graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the 25-ml graduated sample-tube and swirling to thoroughly mix the contents and to observe the color of the Composition A ca-test-sample-water-P1 change from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the graduated sample-tube containing the Composition A ca-test-sample-water-P1 while swirling and counting each drop until the Composition A ca-test-sample-water-P1 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in for example, 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through into the rinse-cycle of the laundry program as follows: for Composition A Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPA.sub.7=(a.sub.PR×b.sub.PR×g.sub.ST×q.sub.SW)(f.sub.WT), where: PPA.sub.7=PHMB-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.PR=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition A ca-test-sample-water-P1 from the wash-tub of the 2.sup.nd washing machine, and b.sub.PR=5 (the ppm of PHMB in the Composition A ca-test-sample-water-P1 from the wash-tub of the 2.sup.nd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of ca-test-sample-water-P1 in the graduated sample tube, and q.sub.SW=quantity of sample water from the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and whereby the amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride in 3,175 g or 7 pounds of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program is determined by quantifying the amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine, that passes-through into the wash-tub of a 2.sup.nd portable washing machine by extracting a Composition A ca-test-sample-water-Q1 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample tube (Taylor Technologies #9198BR) to the 25-ml mark with the ca-test-sample-water-Q1 and pipetting 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the 25-ml graduated sample-tube containing the Composition A ca-test-sample-water-Q1 from the wash-tub of the 2.sup.nd portable washing machine and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the ca-test-sample-water-Q1 sample and swirl to thoroughly mix and to observe the ca-test-sample-water-Q1 color change from colorless to light blue; and titrating the QAC Titrating Solution (Taylor Technologies R-0884) dropwise into the ca-test-sample-water-Q1 while swirling and counting after each drop until the ca-test-sample-water-Q1 color changes from light blue to violet pink; and recording the number of drops of the QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in for example, 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through into the rinse-cycle of the laundry program as follows: for Composition A n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QPA.sub.7=(d.sub.QR×f.sub.QR×g.sub.ST×q.sub.SW)(f.sub.WT) where: QPA.sub.7=QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and d.sub.QR=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition A ca-test-sample-water-Q1 sample from wash-tub of the 2.sup.nd portable washing machine, and f.sub.QR=10 (the ppm of QAC in the Composition A rinse-test-water-Q1 sample from wash-tub of the 2.sup.nd portable washing machine, neutralized by each drop of QAC Titration Solution, and g.sub.ST=quantity of ca-test-sample-water-Q1 in the graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
14. The method according to claim 12 whereby the amount of unused Polyhexamethylene biguanide hydrochloride in for example, 3,175 g or 7 pounds of fabrics that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the amount of polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition B in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting the Composition B cb-test-sample-water-P2 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition B cb-test-sample-water-P2 and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the 25-ml graduated sample tube containing the Composition B cb-test-sample-water-P2 and swirling to thoroughly mix the contents of the 25-ml graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the 25-ml graduated sample-tube containing the cb-test-sample-water-P2 and swirling to thoroughly mix the contents and to observe the color change of the Composition B cb-test-sample-water-P2 from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the 25-ml graduated sample-tube containing the Composition B cb-test-sample-water-P2 while swirling and counting each drop until the Composition B cb-test-sample-water-P2 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition B that remains in 3,175-g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: for Composition B Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPA.sub.7=(a.sub.PA×b.sub.PA×g.sub.SR×q.sub.SW)(f.sub.WT), where: PPA.sub.7=PHMB-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.PA=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition B cb-test-sample-water-P2 sample from the wash-tub of the 2.sup.nd portable washing machine, and b.sub.PA=5 (the ppm of PHMB in the Composition B cb-test-sample-water-P2 sample from the wash-tub of 2.sup.nd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of cb-test-sample-water-Q2 in the 25-ml graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and whereby the amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition B in the wash-tub of a 1.sup.st portable washing machine, that passes-through into the wash-tub of a 2.sup.nd portable washing machine by extracting a Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample tube (Taylor Technologies #9198BR) to the 25-ml mark with the cb-test-sample-water-Q2 and pipetting 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the 25-ml graduated sample-tube containing the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd portable washing machine and swirling to thoroughly mix, and adding 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the cb-test-sample-water-Q2 and observing the cb-test-sample-water-Q2 change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the cb-test-sample-water-Q2 sample while swirling and counting after each drop, until color changes from light blue to violet pink and recording the number of drops of the QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition B that remains in 3,175 g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: for Composition B n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QPA.sub.7×(d.sub.QA×f.sub.AR×g.sub.ST×q.sub.SW)(f.sub.WT) where: QPA.sub.7=QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabric, and d.sub.QA=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd washing machine, and f.sub.AR=10 (the ppm of QAC in the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd washing machine neutralized by each drop of QAC Titration Solution, and g.sub.ST=quantity of cb-test-sample-water-Q2 in the graduated sample tube, and q.sub.SW=total sample water from the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
15. The method to determine the combined amount of unused antimicrobial efficacy that passes-through from one cycle of the laundry program into another cycle of the laundry program comprises the process of treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a wash-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul of Composition A, for a specified period of time (the 1.sup.st exposure time) in the wash-tub of a 1.sup.st portable washing machine and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 1.sup.st portable washing machine, the (1.sup.st spin-time); and transferring the treated 25-g fabric bundle into the wash-tub of a 2.sup.nd portable washing machine containing a rinse-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul Composition B for a specified period of time (the 2.sup.nd exposure time); and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 2.sup.nd portable washing machine (the 2.sup.nd spin-time); and transferring the twice-treated 25-g fabric bundle into the wash-tub of a 3.sup.rd portable washing machine containing about 500-ml of 400 ppm AOAC Hardwater (the cab-test-sample-water); and agitating the twice-treated 25-g fabric bundle for a period of time (the agitation time) to extract the unused antimicrobial efficacy of Composition AB; and centrifuging the twice-treated 25-g fabric bundle for 15-seconds in the spin-tub of the 3.sup.rd portable washing machine to more fully extract the unused antimicrobial efficacy of Composition AB; the 1.sup.st portable washing machine and the 2.sup.nd portable washing, and the 3.sup.rd portable washing machine each comprising a cabinet, a wash-tub, a spin-tub, a pulsator, a water-inlet, a control panel, a pulsator-timer-switch, a pulsator-and-drain selector switch, a spin-timer-switch and a drain-hose.
16. The method according to claim 15 whereby the amount of unused Polyhexamethylene biguanide hydrochloride from Composition A in for example 3,175 g or 7 pounds of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused Polyhexamethylene biguanide hydrochloride from Composition B in for example 3,175 g or 7 pounds of fabrics from the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program is determined by quantifying the combined amount of polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the unused polyhexamethylene biguanide hydrochloride remaining in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine by extracting the Composition AB cab-test-sample-water-PP from the wash-tub of a 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition AB cab-test-sample-water-PP and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the graduated sample tube containing the Composition AB cab-test-sample-water-PP and swirling to thoroughly mix the contents of the graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the graduated sample-tube and swirling to thoroughly mix the contents and to observe the color change of the Composition AB cab-test-sample-water-PP from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the graduated sample-tube containing the Composition AB cab-test-sample-water-PP while swirling and counting each drop until the Composition AB cab-test-sample-water-PP color changes from blue to pinkish purple and persists for 5-7 seconds and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in for example 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the Combined amount of Composition AB Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPPA.sub.7=(a.sub.CR×b.sub.CR×g.sub.ST×c.sub.CW)(f.sub.WT) where: PPPA.sub.7=Composition AB PHMB-Pass-through-antimicrobial-efficacy for 3,175 g or 7 lbs of fabrics, and a.sub.CR=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PP from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.CR=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PP from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of the cab-test-sample-water in the graduated sample tube, and c.sub.CW=the quantity of the Composition AB cab-test-sample-water-PP from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics) and whereby the amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A in 3,175 g or 7 pounds of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B in the same 3,175 g or 7 pounds of fabrics that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the combined amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine, by extracting the Composition AB cab-test-sample-water-QQ from the wash-tub of a 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 25-ml mark with the Composition AB cab-test-sample-water-QQ, and dispensing 1-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the Composition AB cab-test-sample-water-QQ in the graduated sample tube and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into to the Composition AB cab-test-sample-water-QQ and swirling to thoroughly mix and observing the Composition AB cab-test-sample-water-QQ change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the Composition AB cab-test-sample-water-QQ, while swirling and counting after each drop, until the color changes from light blue to violet pink and recording the number of drops of QAC Titrating Solution (Taylor Technologies R-0884) used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the Combined amount of Composition AB n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QQPA.sub.7=(d.sub.CS×f.sub.TS×g.sub.ST×d.sub.SW)(f.sub.WT), where: QQPA.sub.7=Composition AB QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and d.sub.CS=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-QQ from the wash-tub of a 3.sup.rd portable washing machine, and f.sub.TS=10 (the ppm of QAC in the Composition AB cab-test-sample-water-QQ from the wash-tub of the 3.sup.rd portable washing machine neutralized by each drop of QAC Titration Solution) and g.sub.ST=quantity of cb-test-sample-water-Q2 in the graduated sample tube, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 lbs of fabrics); and whereby the amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A in 3,175 g or 7 pounds of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B in the same 3,175 g or 7 lbs of fabrics in the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program, is determined by quantifying the combined amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine, that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride that remains in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine by extracting the Composition AB cab-test-sample-water-PQ1 from the wash-tub of the 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated Sample-tube-1 (Taylor Technologies #9198) to the 10-ml mark with the Composition AB cab-test-sample-water-PQ1, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into graduated sample-tube1 containing the Composition AB cab-test-sample-water-PQ1 and swirling to thoroughly mix the contents of the graduated sample-tube-1; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies (R-0977) into the graduated sample-tube-1 and swirling to thoroughly mix the contents and to observe the color change of the Composition AB cab-test-sample-water-PQ1 change from clear to blue and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the 25-ml graduated sample-tube-1 containing the Composition AB cab-test-sample-water-PQ1 while swirling and counting each drop until the Composition AB cab-test-sample-water-PQ1 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program; and by extracting the Composition AB cab-test-sample-water-PQ2 from the wash-tub of the 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated Sample-tube-2 (Taylor Technologies #9198) to the 25-ml mark with the Composition AB cab-test-sample-water-PQ2, and titrating 1-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the Composition AB cab-test-sample-water-PQ2 in the graduated sample-tube-2 and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the Composition AB cab-test-sample-water-PQ2 in the graduated sample-tube-2 and swirling to thoroughly mix and observing the Composition AB cab-test-sample-water-PQ2 change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the Composition AB cab-test-sample-water-PQ2 in graduated sample-tube-2 while swirling and counting after each drop, until the color changes from light blue to violet pink and persists for 5-7 seconds and recording the number of drops of QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 pounds of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 pounds of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the combined amount of Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A Combined with the combined amount of Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B is quantified by performing the following calculation: CQPA.sub.7=[(a.sub.CP×b.sub.PT×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(d.sub.QC×f.sub.QT×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(a.sub.CG×b.sub.PG×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(d.sub.QB×f.sub.QD×g.sub.ST×q.sub.SW)(f.sub.WT)] where: CQPA.sub.7=Combined PHMB and QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.CP=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.PT=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of cab-test-sample-water-PQ1 in the graduated sample-tube-1, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and d.sub.QC=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.QT=10 (the ppm of QAC in the Composition AB rinse-test-water-PQ1 sample-tube-1 from the wash-tub of the 3.sup.rd washing machine neutralized by each drop of QAC Titration Solution), and g.sub.ST=quantity of cab-test-sample-water-PQ1 in the graduated sample-tube-1, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.PG=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and q.sub.SW=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics) and d.sub.QB=the number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.QD=10 (the ppm of QAC in the Composition AB rinse-test-water-PQ2 sample-tube-1 from the wash-tub of the 3.sup.rd washing machine neutralized by each drop of QAC Titration Solution), and g.sub.ST=quantity of cab-test-sample-water-PQ2 in the graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g, the fabric weight in grams equal to 7 pounds of fabrics.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0060]
[0061] In the image: 1—Polyester fabric at 3× resolution; 11—black space depicting a pore or air gap between the yarns.
[0062]
[0063] In the image: 1—cotton yarn at 150× via Scanning Electron Microscope; 11—black space depicting a pore or air gap between the fibers.
[0064]
[0065] In the image: 1 polyester yarn at 150× via scanning electron microscope; 11—black space depicting a pore or air gap between the fibers.
[0066]
[0067] In the drawing: 1 Cross-section of a piece of fabric; 11—pore or air gap between the yarns; 12—pore or air gap between the fibers; 13—depicts ϕ.sub.V liquid flow rate between yarns and N.sub.W mass flux from yarns to main flow between yarns.
[0068]
In the drawing: 1—schematic drawing of a piece of fabric; 11—fabric fiber with an approximate diameter of 0.015 mm; 12—fabric yarn with an approximate diameter of 0.25 mm.
[0069]
[0070]
[0071]
[0072]
[0073]
[0074] In the diagram: 1—1.sup.st portable WM; 11—wash-tub; 111—Composition A wash water solution; 112—25 g fabric bundle containing 3 inoculated swatches; 12—spin tub; 121—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A removed from the wash tub; 13—control panel; 131—wash cover; 132—wash timer switch; 133—speed selector switch; 134—spin timer switch; 135—spin cover; 14—drain hose used to extract 5 ml sample of the wash water solution; 141—three 50 ml wide mouthed tubes each containing 10 ml neutralizer broth to neutralize the actives in each of 3 fabric swatches removed from 25 g fabric bundle to be cultured to determine disinfectant or sanitizer efficacy; 142—50 ml wide mouthed tube with containing 40 ml concentrated neutralizer broth to neutralize the actives in 5 ml wash-water sample to be cultured to determine disinfectant or sanitizer efficacy.
[0075]
[0076] In the diagram: 1—1.sup.st portable WM; 11—rinse tub; 111—Composition B rinse water solution; 112—25 g fabric bundle containing 3 inoculated swatches; 12—spin tub; 121—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition B removed from the rinse tub; 13—control panel; 131—rinse cover; 132—rinse timer switch; 133—speed selector switch; 134—spin timer switch; 135—spin cover; 14—drain hose used to extract 5 ml sample of the rinse water solution into the 50 ml wide mouthed tube; 141—three 50 ml wide mouthed tubes each containing 10 ml neutralizer broth to neutralize the actives in each of 3 fabric swatches removed from 25 g fabric bundle to be cultured to determine disinfectant or sanitizer efficacy; 142—50 ml wide mouthed tube with 40 ml concentrate neutralizer broth to neutralize the actives in the 5 ml wash-water sample to be cultured to determine disinfectant or sanitizer efficacy.
[0077]
[0078] In the diagram: 1—1.sup.st Portable WM; 11—wash-tub; 111—Composition A wash water solution; 112—25 g fabric bundle containing 3 inoculated fabric swatches; 12—spin tub; 121—25 g fabric bundle containing three inoculated fabric swatches treated with Composition A wash water solution removed from the wash tub; 13—Control panel; 131—wash cover; 132—wash timer switch; 133—speed selector switch; 134—spin timer switch; 135—spin cover; 2—2.sup.nd Portable WM; 21—rinse tub; 211—Composition B rinse water solution; 212—25 g fabric bundle containing 3 inoculated swatches treated with Composition A removed from the spin tub of 1.sup.st PWM; 22—spin tub; 221—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A in the wash tub of 1.sup.st PWM and treated with Composition B removed from the rinse tub; 23—control Panel; 231—rinse cover; 232—rinse timer switch; 233—speed selector switch; 234—spin timer switch; 235—spin cover; 3—3.sup.rd PWM; 31—rinse tub; 311—rinse water test solution; 312—25-g fabric bundle containing 3 inoculated fabric swatches treated with composition A in the wash tub of 1.sup.st PWM and treated with Composition B in the wash tub of 2.sup.nd PWM removed from the spin tub of 2.sup.nd PWM; 32—spin tub; 321—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A in 1.sup.st PWM and Composition B in 2.sup.nd PWM removed from the rinse tub; 33—control panel; 331—rinse-cover; 332 rinse timer switch; 333—speed selector switch, 334—spin timer switch; 335—spin cover; 4—drain hose used to extract a 5 ml rinse water test solution sample; 411—three 50 ml wide mouthed tubes each containing 10 ml neutralizer broth to neutralize the actives in the 3 inoculated swatches removed from the 25 g fabric bundle to be cultured to determine disinfectant or sanitizer efficacy; 412—50 ml wide mouthed tube containing 40 ml concentrated neutralizer broth to neutralize the actives in the 5 ml rinse water solution sample to be cultured to determine disinfectant or sanitizer efficacy.
[0079]
[0080] In the diagram: 1—1.sup.st PWM; 11—wash tub; 111—Composition A wash-water-solution; 112—fabric bundle containing 3 inoculated fabric swatches; 12—spin tub; 121—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A removed from the wash tub; 13—panel; 131—wash cover; 132—wash timer switch; 133—speed selector switch; 134—spin timer switch; 135—spin cover; 2—2.sup.nd PWM, 21—rinse tub; 211—500 ml ca-test-sample-water; 212—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A removed from the spin tub of 1.sup.st PWM; 22—spin tub; 221—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A; a23—control panel; 231—rinse-cover; 232—rinse timer switch; 233—speed selector switch; 234—spin timer switch; 235—spin cover; 24—drain hose used to extract the ca-test-sample-water containing PHMB or QAC; 241—25 ml graduated cylinder used to collect 10 ml PHMB ca-test-sample water or to collect 25 ml QAC ca-test-sample water used for the titration procedure to quantify PHMB or QAC.
[0081]
[0082] In the diagram: 1—1.sup.st PWM; 11—rinse tub; 111—Composition B rinse-water-solution; 112—fabric bundle containing 3 inoculated fabric swatches; 12—spin tub; 121—25 g fabric bundle treated containing 3 inoculated fabric swatches treated with Composition B; 13—control panel; 131—rinse cover; 132—rinse timer switch; 133—speed selector switch; 134—spin timer switch; 135—spin cover; 2—2.sup.nd PWM, 21—rinse tub; 211—500 ml cb-test-sample water; 212—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition B; 22—spin tub; 221—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition B; 23—control panel; 231—rinse-cover; 232—rinse timer switch; 233—speed selector switch; 234—spin timer switch; 235—spin cover; 24—drain hose used to extract the cb-test-sample water containing PHMB or QAC; 241—25 ml graduated cylinder used to collect 10 ml PHMB cb-test-sample water or to collect 25 ml QAC cb-test-sample water used for the titration procedure to quantify PHMB or QAC.
[0083]
[0084] In the diagram: 1—1.sup.st PWM; 11—wash-tub; 111—Composition A wash water solution; 112—25 g fabric bundle containing 3 inoculated fabric swatches; 12—spin tub; 121—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A removed from the wash tub; 2—2.sup.nd PWM; 21—rinse tub; 211—Composition B rinse water solution; 212—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A removed from the spin tub of 1.sup.st PWM; 22—spin tub; 221—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A in the wash tub of the 1.sup.st PWM and Composition B in the rinse tub of the 2.sup.nd PWM; 23—control panel; 231—rinse cover; 232—rinse timer switch; 233—speed selector switch; 234—spin timer switch; 235—spin cover; 3—3.sup.rd PWM; 31—rinse tub; 311—500 ml cab-test-sample water; 312—25 g fabric bundle containing 3 inoculated fabric swatches treated with Composition A in the 1.sup.st PWM and treated with Composition B in the 2.sup.nd PWM removed from the spin tub of the 2.sup.nd PWM; 32—spin tub; 321—25 g fabric bundle treated with Composition A in the wash tub of the 1.sup.st PWM and treated with Composition B in the rinse tub of the 2.sup.nd PWM removed from the rinse test water in the rinse tub; 33—control panel; 331 rinse cover; 332 rinse timer switch; 333 speed selector switch; 334—spin timer switch, 335—spin cover; 4—drain hose used to extract the cab-test-sample water containing PHMB or QAC; 411—25 ml graduated sample tube used to collect 10 ml PHMB cab-test-sample to be used in the titration procedure to quantify Combined PHMB from Composition A and Composition B; 412—25 ml graduated sample tube for 25 ml QAC cab-test-sample water to be used in the titration procedure to quantify Combined QAC from Composition A and Composition B.
[0085]
[0086] In the image: 1—A collection of Control Fabric2 images displaying the Bromophenol Blue Dye Complex which demonstrates the presence of the antimicrobial actives; 11—un-treated Control Fabric2; 12—Control Fabric2 after treatment with ALLD TA08.3; 13—Control Fabric2 after treatment with ALLD TA08.5; 14—Control Fabric2 after treatment with ALLD TA09.2; 15—Control Fabric2 after treatment with ALFS TA01.5.
DEFINITIONS
[0087] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
[0088] As used herein, “Aesthetic benefit” means softening, wrinkle reduction, color protection, and fragrances.
[0089] As used herein, “AFSS” is an acronym for Antimicrobial Fabric Softener Sheet.
[0090] As used herein, “ALLD” is an acronym for Antimicrobial Liquid Laundry Detergent.
[0091] As used herein, “ALFS” is an acronym for Antimicrobial Liquid Fabric Softener.
[0092] As used herein, “antimicrobial benefit” is the result or effect of antimicrobial activity on fabrics that reduces the risk of infection, promotes human health, hygiene and well-being.
[0093] As used herein, “Antimicrobial durability” means the antimicrobial activity and duration of a fabric care composition on fabrics after use in a laundry program.
[0094] As used herein, “Antimicrobial Efficacy” means the ability of a fabric care composition to produce antimicrobial actions such as disinfection, sanitization, and deactivation of harmful microorganisms.
[0095] As used herein, “cMOA” means conventional mechanism of action.
[0096] As used herein, “convection” means the process involved in which molecules of a fabric care composition are transported by the larger scale movement of fluid currents to and through the more porous regions (the Inter-yarn pores) of a fabric.
[0097] As used herein, “detersive benefits” refers to cleansing soils and removing stains from fabrics.
[0098] As used herein, “discrete use” means the individually separate or distinct use of a fabric care compositions during a cycle of a laundry program; i.e. detergent in the wash-cycle.
[0099] As use herein. “disinfectant, disinfection, disinfecting” means a chemical or chemical process that kills harmful microorganisms, usually expressed as 99.999 percent reduction or 5 log.sub.10 reduction of the colony forming units.
[0100] As used herein, “dosage” refers to the quantity or frequency of an antimicrobial composition.
[0101] As used herein, “dose-dependent” refers to the relationship between the activity of an antimicrobial fabric care composition and the dosage; when the dosage of the antimicrobial fabric care composition is changed, the antimicrobial activity changes.
[0102] As used herein, “dose-dependent antimicrobial activities” means antimicrobial activity such as sanitizing, disinfecting, biofilm deactivation, biofilm prevention and antimicrobial durability that are determined by the dose-rate of the fabric care composition used in one or more laundry cycles.
[0103] As used herein, “dose-rate” means the quantity, concentration or strength of the antimicrobial fabric care composition used during the treatment or laundering of fabrics in a laundry program.
[0104] As used herein, “extra rinse cycle” refers to an additional rinse-cycle that's is programmed to occur before the final rinse cycle of the laundry program.
[0105] As used herein, “fabrics” refers to textiles and fabrics such as cotton, cotton blends, synthetic fibers and apparel items such as slacks, shirts, underwear, sweatshirts, sweatpants, socks, oven mitts, slippers, bathrobes, gloves, hats, scarves, jackets, bedding, and bath-towels, hand-towels curtains and the like, mops, sponges and cleaning cloths located in households, on-premise laundry operations, healthcare facilities, and commercial facilities such as athletic/recreational facilities, exercise facilities, health clubs, schools, colleges, universities.
[0106] As used herein, “fabric-treatment plan” refers to a plan for laundering and the antimicrobial treatment of fabrics that specifies a dose-rate of the fabric care composition, the duration of the treatment, the weight of the fabrics to be treated, the quantity and temperature of the aqueous bath, and the temperature of the dryer.
[0107] As used herein, use of the terms “having”,” “including,” “containing” and “comprising” in the claims and/or the specification are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.
[0108] As used herein, “hydrodynamics” means a branch of physics that deals with the motion of fluids and the forces acting on solid bodies immersed in fluids and in motion relative to them.
[0109] As used herein, “hygienically clean” means the reduction or elimination of microorganisms including pathogenic microorganisms from fabrics and textile products to levels that pose no threat of human illness.
[0110] As used herein, “nMOA” is an acronym for non-conventional mechanism of action.
[0111] As used herein, “pass-through antimicrobial efficacy” refers to the unused antimicrobial actives or activity of a fabric care composition that is transferred from one cycle of the laundry program into another cycle of the laundry program via the treated fabrics.
[0112] As used herein, “PHMB” is an abbreviation for Polyhexamethylene biguanide hydroxide.
[0113] As used herein, “PWM” is an acronym for portable washing machine.
[0114] As used herein, “QAC” is an acronym for quaternary ammonium compound and refers to all biocides of that class.
[0115] As used herein, “synergistic use” refers to the combined antimicrobial effect of the individually separate or distinct uses of antimicrobial fabric care compositions during a cycle of a laundry program; i.e. ALLD in the wash-cycle and ALFS in the rinse-cycle and AFSS in the drying-cycle.
[0116] As used herein, “thermal curing” refers to the process of using the heat produced during the drying-cycle of the laundry program to affix the pass-through antimicrobial efficacy into the laundered fabrics.
[0117] As used herein, “use site” refers to the site of application of an antibacterial composition.
[0118] As used herein, “WM” is an acronym for washing machine.
DETAILED DESCRIPTION OF THE INVENTION
[0119] Before describing the present invention in detail, it is to be understood that this invention is to be interpreted by way of illustration and not limited to particularly exemplified embodiments, methods or process parameters that may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to limit the scope of the invention in any manner. All publications, patents, and patent applications cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Citation of any reference is not an admission regarding any determination as to its availability as prior art to the claimed invention.
[0120] This present invention has been contrived considering the matters and state of affairs illustrated herein above and encompass antimicrobial fabric care compositions, uses and methods that remove soils and stains, reduces the risks of bacterial infections, skin allergies and skin irritation by inactivating harmful bacteria, malodors and viruses on fabrics and biofilms in the washing machine. Thus, the present invention provides fabric care compositions for discrete or synergistic use in domestic, healthcare, commercial and on-premise laundry programs for laundering, disinfecting and sanitizing fabrics and inactivating biofilms and imparting antimicrobial durability into the fabrics to inhibit bacterial growth and contamination of fabrics for up 24-48 hours after the laundry program and to prevent the adhesion of biofilms on fabrics and abiotic surfaces inside the washing machine, and encompass:
[0121] [1] Composition A being an antimicrobial liquid laundry detergent (ALLD) composition for use on fabrics in domestic, industrial and on-premise machine-washing laundry programs; Composition A may comprise one or more cationic biocides and various combinations of cationic surfactants, anionic surfactants, nonionic surfactants, amphoteric surfactants, and surfactant blends. Composition A of the present invention may further comprise adjunct constituents, many of which are well known in the art, including but are not limited to: detergency builders, chelating agents, pH adjusting agents, buffers, processing aids, viscosity builders, viscosity modifiers, hydrotropes, optical brighteners, coloring agents, fragrances, fillers, enzymes, as well as other adjunct constituents not particularly elucidated here. These adjunct constituents may be added in any effective amount, but generally the total amount of such adjunct constituents should not exceed about 11.4% by weight of the total weight of the antimicrobial liquid laundry detergent compositions being taught herein.
[0122] Examples of cationic biocides useful in the Composition A include, didecyl dimethyl ammonium chloride, dioctyl dimethyl ammonium chloride, benzalkonium chloride, alkyl dimethyl benzyl ammonium chloride, alkyldimethyl ethylbenzyl ammonium chloride, and other quaternary ammonium compounds, Polyhexamethylene guanidine (PHMG), and Polyhexamethylene biguanide Hydrochloride (PHMB), or combinations or mixtures thereof.
[0123] Examples of anionic surfactants useful in the Composition A include salts of higher fatty acids, salts of higher alcohol sulfates, salts of higher alcohol sulfonic acids, alkyl sulfates, sodium sarcosinates, salts of sulfonated fatty acids, salts of higher alcohol sulphates, sodium dodecylether sulfates, alkyl benzene sulphonates, salts of sulfosuccinates, sodium methyl 2-sulfolaurates, disodium 2-sulfolaurate, sodium laureth ether sulfates, sodium lauryl sulfates, sodium lauryl glucosides hydroxypropylsulphonates, sodium N-Acylphenylalanines such as Coconut N-Acylphenylalanines, Palm Fatty N-Acylphenylalanines, Karanja Fatty N-Acylphenylalanines, Sterculia Fatty N-Acylphenylalanines, High Oleic Sunflower Fatty N-Acylphenylalanines or combinations or mixtures thereof.
[0124] Examples of nonionic surfactants useful in the composition include alkyl polyglycosides, linear alcohol ethoxylates, Alkanolamides, Polyoxyethylene oleyl ether, alcohol ethoxylates, amide ethoxylates, Sorbitan Esters and Sorbitan Ester Ethoxylates and the like and combinations and mixtures thereof.
[0125] Examples of amphoteric surfactants useful in the composition include amide oxides, alkyl betaines, cetyl betaines, lauryl betaines, propionates, D-Glucopyranose, oligomeric, C10-16-alkyl decyl octyl glycosides, D-Glucopyranose, oligomeric, C10-16-alkyl glycosides, and the like and combinations and mixtures thereof.
[0126] Examples of functionalized alkylpolyglucosides useful in the compositions include D-Glucopyranose, oligomeric, decyl octyl glycosides, 2-hydroxy-3-sulfopropyl ethers, sodium salts, polymers with 1,3-dichloro-2-propanol, D-Glucopyranose, oligomeric, decyl octyl glycosides, 2,3-dihydroxypropyl ethers, phosphates, sodium salts, polymers with 1,3-dichloro-2-propanol, Sodium Hydroxypropylphosphate Lauryl glucoside Crosspolymer, D-Glucopyranose, oligomeric, C10-16-alkyl decyl octyl glycosides, 3-((carboxymethyl)bis(2-hydroxyethyl)ammonio)-2-hydroxypropyl ethers, inner salts, polymers with 1,3-dichloro-2-propanol, D-Glucopyranose, oligomeric, C10-16-alkyl glycosides, 3-((carboxymethyl)bis(2-hydroxyethyl)ammonio)-2-hydroxypropyl ethers, inner salts, polymers with 1,3-dichloro-2-propanol, D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate, D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate, D-Glucopyranose, oligomeric, decyl glycosides, 3-(dimethyldodecylammonio)-2-hydroxypropyl ethers, chlorides, polymers with 1,3-dichloro-2-propanol, D-Glucopyranose, oligomeric, C10-16-alkyl glycosides, 3-(dodecyldimethylammonio)-2-hydroxypropyl ethers, chlorides, polymers with 1,3-dichloro-2-propanol, D-Glucopyranose, oligomeric, C10-16-alkyl glycosides, 3-(dimethyloctadecylammonio)-2-hydroxypropyl ethers, chlorides, polymers with 1,3-dichloro-2-propanol or combinations or mixtures thereof.
[0127] Examples of surfactant blends useful in the Composition include, Sodium Laurylglucosides hydroxy propyl sulfonate, and D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate; and Benzenesulfonic Acid, Decyl (sulfophenoxy)-, Disodium salt and Isopropanolamine Dodecylbenzenesulfonate, and Poly (oxy-1,2-ethanediyl), .alpha.-undecyl-.omega.-hydroxy, and Benzenesulfonic acid, oxybis (decyl-, disodium salt, and Propylene-1,2 diol.
[0128] Distilled or deionized water forms a constituent of Composition A. The amount of water added is an amount to provide the balance of the compositions to 100% by weight. The water is added, generally in an amount of 40 to 58% weight, to provide the balance of the total composition.
[0129] One embodiment, ALLD TA04, of Composition A, consists of: (A) from about 2-23% preferably about 8-20% by weight of the cationic biocide Poly (hexamethylene biguanide Hydrochloride, and, (B) from about 2-7% preferably 5-10% by weight of the primary anionic surfactant blend Sodium Methyl 2-Sulfolaurate and Disodium 2-Sulfolaurate; from about 3-6% preferably 5-6% of a nonionic surfactant blend comprised of components (i) a C9-11 Pareth-3 having the formula CH.sub.3(CH.sub.2).sub.n—O(CH.sub.2CH.sub.2O).sub.y where n=9-11 and y=2.5 moles of ethoxylation and component (ii) a C12-13 Pareth-3 having the formula CH.sub.3(CH.sub.2).sub.n—O(CH.sub.2CH.sub.2O).sub.yH where n—12-13 and y=3.0 moles of ethoxylation, and (D) from about 1-7% preferably 3-5% by weight of dimethyl dodecyl betaine, and from about 2-8% preferably 4-5% by weight of the 1-Dodecanamine, N,N-Dimethyl-N-Oxide, and (F) a chelating agent, examples of such chelating agents useful in the present invention include ethylene diamine tetraacetic acid, N,N Diacetic Tetrasodium salt, hydroxyethyl-ethylenediaminetriacetic acid, citric acid, maleic acid, polycyclic acid and gluconic acid. Of these citric acid, N,N Diacetic Tetrasodium salt, and ethylene diamine tetraacetic acid are preferred and N,N Diacetic Tetrasodium salt is most preferred. It is preferred that the metal chelating agent is from about 0.5-2%, preferably 0.5-1% by weight, and (G) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 3-6%, preferably 3-5% by weight, and (H) an adjunct constituent being Monosodium ascorbate being a processing aid from about 1-6% by weight, preferably 2-5% by weight and (I) from about 1-5% by weight, preferably 2-3% by weight of the rheology modifier Poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z,Z).
[0130] Another embodiment, ALLD TA05, of Composition A, consists of: (A) from about 2-23% preferably 8-20% by weight of the cationic biocide Polyhexamethylene biguanide Hydrochloride, and (B) from about 5 to 20% by weight preferably 6-7% by weight of the anionic surfactant sodium laurylglucosides hydroxypropylsulfonates, and (C) from about 2-8% by weight preferably 5-7% by weight of the 1-Dodecanamine, N,N-Dimethyl-N-Oxide, and (D) from about 1-15% by weight preferably about 3-5% by weight of 1-decyl-D-glucopyranoside, and (E) from about 1-7% by weight, preferably 2-5% by weight of 1,3 Propanediol, and (F) a chelating agent, examples of such chelating agents useful in the present invention include ethylene diamine tetraacetic acid, N,N Diacetic Tetrasodium salt, hydroxyethyl-ethylenediaminetriacetic acid, citric acid, maleic acid, polycyclic acid and gluconic acid. Of these citric acid, N, N Diacetic Tetrasodium salt, and ethylene diamine tetraacetic acid are preferred and N,N Diacetic Tetrasodium salt is most preferred. It is preferred that the metal chelating agent is from about 0.5-2%, preferably 0.5-1% by weight, and (G) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 3-6%, preferably 3-5% by weight, and (H) and adjunct constituent Monosodium ascorbate being a processing aid from about 1-5%, preferably 2-3% by weight, and (I) from about 1-5%, preferably 2-3% of a rheology modifier Poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z,Z).
[0131] Another embodiment, ALLD TA07, of Composition A, consists of: (A) from about 2-23% preferably 8-20% by weight of the cationic biocide Polyhexamethylene biguanide Hydrochloride, and (B) from about 1-18.5% preferably 2-10% of the primary surfactant blend consisting of Sodium Laurylglucosides hydroxypropylsulfonate and Sorbitan Oleate Decylglucoside Crosspolymer, and (C) from about 4-10% preferably 5-9% by weight of a surfactant blend comprised of components (i) a C9-11 Pareth-3 having 2.5 moles of ethoxylation and component (ii) a C12-13 Pareth-3 having 3.0 moles of ethoxylation, and (D) from about 1-8% preferable 1-4% of hexylhexopyranose, and (E) from about 3-8% preferably 4-8% by weight of dimethyl dodecyl betaine, and (F) from about 2-8% preferably 4-5% by weight of the 1-Dodecanamine, N,N-Dimethyl-N-Oxide, and (G) a chelating agent, examples of such chelating agents useful in the present invention include ethylene diamine tetraacetic acid, N,N Diacetic Tetrasodium salt, hydroxyethyl-ethylenediaminetriacetic acid, citric acid, maleic acid, polycyclic acid and gluconic acid. Of these citric acid, N, N Diacetic Tetrasodium salt, and ethylene diamine tetraacetic acid are preferred and N,N Diacetic Tetrasodium salt is most preferred. It is preferred that the metal chelating agent is from about 0.5-2%, preferably 0.5-1% by weight, and (H) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 2-7%, most preferably 2.5-6% by weight, and (I) from about 1-5%, preferably 2-3% of a rheology modifier Poly (oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z, Z).
[0132] A preferred embodiment, ALLD TA08, of Composition A consists of: (A) from about 2-23% preferably 8-20% of the Cationic Biocide Polyhexamethylene biguanide Hydrochloride, and (B) from about 2-10% preferably 3-6% by weight of the primary anionic surfactant blend Sodium Methyl 2-Sulfolaurate and Disodium 2-Sulfolaurate, and (C) from about 1-13% preferably 2-8% by weight of an alcohol ethoxylate C14-15 with an E.O. content from about 40-60% preferably 49-57% by weight, and a hydroxyl value of 103 mg KOH/g, (D) from about 2-10% preferably 3-10% of an alcohol ethoxylate with an average number of moles of E.O from about 3-10, preferably 7-9, and (E) from about 1-5% preferably 3-5% by weight of a coconut fatty acid with the molecular formula C.sub.19H.sub.21NO.sub.5, where the mass % of the element C is from about 65-68%, preferably 66-67% and the mass % of element H is from about 4-7% preferably 6-7% and where the mass % of N is from about 1-6%, preferably 2-5% and where the mass % of O is from about 12-28%, preferably 18-24%, and the exact mass of 343.1419727762, and (F) from about 1-5%, preferably 2-3% of a rheology modifier Poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z,Z), and (G) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 3-6%, preferably 3-5% by weight.
[0133] Another preferred embodiment, ALLD TA08.5, of Composition A consists of: (A) from about 2-23% preferably 8-20% of the Cationic Biocide Polyhexamethylene biguanide Hydrochloride, and (B) from about 1-7% preferably 1-4% of the Cationic Biocide n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride, and (C) from about 2-10% preferably 3-6% by weight of the primary anionic surfactant blend Sodium Methyl 2-Sulfolaurate and Disodium 2-Sulfolaurate, and (D) from about 1-13% preferably 2-8% by weight of a fatty alcohol ethoxylate C14-15 Pareth 7, with an E.O. content weight % of 56, and a hydroxyl value of 103 mg KOH/g, and (E) from about 2-10% preferably 3-10% of a fatty alcohol ethoxylate with an average number of moles of E.O of 9, and (F) from about 1-6% preferably 3-6% by weight of a, Cocoamidopropyl Betaine with an Actives % from 30-37.3, preferably 30, and (G) from about 0.51-5%, preferably 0.65-3% of a rheology modifier Poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-hydroxy-, ether with methyl D-glucopyranoside 2,6-di-9-octadecenoate (2:1), (Z,Z), and (H) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 3.0-6.0%, preferably 4-6% by weight; and (I) from about 3.1-6.4% preferably 4.0-5.1% by weight of the solvent 1,3 propanediol.
[0134] Another preferred embodiment, ALLD TA09, of Composition A consists of: (A) from about 2-23% preferably 8-20% of the Cationic Biocide Poly(hexamethylene biguanide Hydrochloride, and (B) from about 4-18% preferably 6-10% by weight of a functionalized Alkylpolyglucosides blend consisting of: (1) Sodium Laurylglucosides hydroxy propyl sulfonate, and (2) D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate, and (C) from about 1-8% preferably 2-6% by weight of 1-Dodecanaminium, N-(carboxymethyl)-N,N-dimethyl-, hydroxide, inner salt, and (D) from about 2.8-6.5% preferably 3.7-6.0% by weight of a D-Glucopyranose, oligomeric, C10-16-alkyl decyl octyl glycosides, 3-((carboxymethyl)bis(2-hydroxyethyl) ammonio)-2-hydroxypropyl ethers, inner salts, polymers with 1,3-dichloro-2-propanol, and (E) from about 1.5-5.2% preferably 2.2-4.9% by weight of a Polyoxyethylene glycol oleyl ether having 20 moles E0, and (F) from about 3.1-6.4% preferably 4.0-5.1% by weight of the solvent 1,3 propanediol, and (G) from about 2.7%-5.1% preferably 3.3 to 4.8% by weight of an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is most preferred that the enzyme medley be from about 3-4% by weight, and (H) an optional a chelating agent wherein no particular limitation is imposed on the chelating agent other than its ability to bind to metal ions. Examples of such chelating agents useful in the present invention include ethylene diamine tetraacetic acid, N,N Diacetic Tetrasodium salt, hydroxyethyl-ethylenediaminetriacetic acid, citric acid, maleic acid, polycyclic acid and gluconic acid. Of these citric acid, N,N Diacetic Tetrasodium salt, and ethylene diamine tetraacetic acid are preferred and N,N Diacetic Tetrasodium salt is most preferred. It is preferred that the metal chelating agent is from about 0.5-2%, preferably 0.5-1% by weight, and (I) from about 3.6-7.0% preferably 5.0-8.3% by weight of 2-Propenoic acid, homopolymer.
[0135] Another preferred embodiment, ALLD TA09.2, of Composition A consists of: (A) from about 2-23% preferably 8-20% of the Cationic Biocide Poly(hexamethylene biguanide Hydrochloride, and (B) from about 20-70% preferably 26-65% by weight of a cationic, anionic and non-ionic surfactant blend consisting of: (1) from about 20-30% by weight of Decyl phenoxybenzenedisulfonic acid, disodium salt, and (2) from about 10-20% by weight of Isopropanolamine dodecylbenzene sulfonate, and (3) from about 10-20% by weight of Poly(oxy-1,2-ethanediyl), .alpha.-undecyl-.omega.-hydroxy, and (4) from about 10-20% by weight of Benzenesulfonic acid, oxybis (decyl-, disodium salt, and (5) from about 1-3% by weight of Propylene-1,2 diol, and (C) from about 1-5% preferably 2-3.5% by weight of 1,3 propanediol, and (D) an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse a mannanase, and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is preferred that the enzyme medley be from about 3-5.5%, preferably 4-4.5% by weight.
[0136] Another preferred embodiment, ALLD TA10, of Composition A consists of: (A) from about 2-23% preferably 8-20% of the Cationic Biocide Poly(hexamethylene biguanide Hydrochloride, and (B) from about 4-18% preferably 8-15% by weight of an amino acid surfactant blend consists of: (1) 2-12% preferably 3-10% of Coconut N-Acylphenylalanines, and (2) 3-13% preferable 2-12% of Palm Fatty N-Acylphenylalanines, and (C) from about 3-7% preferably 4-6.2% of D-Glucopyranose, oligomeric, C10-16-alkyl glycosides, 3-((carboxymethyl)bis(2-hydroxyethyl)ammonio)-2-hydroxypropyl ethers, inner salts, polymers with 1,3-dichloro-2-propanol, and (D) from about 4-7% preferably 5-6% of a D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate, with an HLB from about 12-14, and (E) from about 1.5-5% preferably 1.6-4% of a D-Glucopyranose, oligomeric, decyl octyl glycosides, polymers with epichlorohydrin and sorbitan monooleate, with an HLB from about 8-10, and (F) from about 2.7%-5.1% preferably 3.3 to 4.8% by weight of an adjunct constituent being an enzyme medley or mixture consisting of a lipase, a protease, an amalyse and a cellulase. No particular limitation is imposed on the individual enzymes present in the mixture. It is most preferred that the enzyme medley be from about 3-4% by weight, and G) from about 3-7% preferably 4-5.6% of a 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, polymer with 2-propenamide, and (H) a chelating agent wherein no particular limitation is imposed on the chelating agent other than its ability to bind to metal ions. Examples of such chelating agents useful in the present invention include ethylene diamine tetraacetic acid, N,N Diacetic Tetrasodium salt, hydroxyethyl-ethylenediaminetriacetic acid, citric acid, maleic acid, polycyclic acid and gluconic acid. Of these citric acid, N,N Diacetic Tetrasodium salt, and ethylene diamine tetraacetic acid are preferred and N,N Diacetic Tetrasodium salt is most preferred. It is preferred that the metal chelating agent is from about 0.5-2%, preferably 0.5-1% by weight; and further encompasses:
[0137] [2] Composition B being an Antimicrobial Liquid Fabric Softener (ALFS) composition for use on fabrics in the rinse-cycle of a machine-washing laundry program; Composition B containing one or more cationic biocides and combinations of quaternary softening compounds, nonionic surfactants, performance additives, rheology modifiers, and adjunct constituents such as colorants, fragrances, as well as others not particularly elucidated here. These adjunct constituents may be added in any effective amount, but generally the total amount of such adjunct constituents should not exceed about 4% by weight of the total weight of the antimicrobial liquid fabric softener compositions being taught herein. One embodiment ALFS TA01 of [2] Composition B, consists of: (A) from about 2-12%% preferably about 8-10% by weight of the cationic biocide Poly(hexamethylene biguanide) Hydrochloride, and (B) about 8-19% preferably 17-19% by weight of Dipalmitoylethyl Hydroxyethylmonium Methosulfate, and (C) from about 0.8-3% preferably 0.9-1.5% by weight of a quaternized hydrolyzed wheat protein silicone co-polymer, and (D) from about 0.04-1% preferably 0.05-0.3% by weight of Ethylene glycol monophenyl ether, and (E) from about 0.03-0.9% preferably 0.05-0.7% of ethyl alcohol, and (F) from about 0.08-3.2% preferably 1.2 to 2.6% of Isopropyl alcohol, and (G) from about 0.3-2% preferably 0.5-1.5% by weight of 2-Propenoic acid, homopolymer, and (H) from about 0.2-1.3% preferably 0.2-0.8% by weight of a crosslinked copolymers of acrylamide and methacrylate methylene bisacrylamide,
[0138] Another embodiment, ALFS TA01.5, of [2] Composition B consists of: (A) from about 2-12% preferably about 2-5% by weight of the cationic biocide Poly(hexamethylene biguanide) Hydrochloride, and (B) from about 1-7% preferably 2-4% of the cationic biocide n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride, and (C) from about 3-8% preferably 4-6% by weight of Ethanaminium, 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-, esters with C16-18 and C18-unsatd. fatty acids, Me sulfates (salts), and (D) from about 0.6-3.5% preferably 0.8-1.7% by weight of Wheat, [2-hydroxy-3-[3-(trimethoxysilyl) propoxy] propyl], hydrolyzed, and (E) from about 1.8-4.0% preferably 2-3% by weight of 1,3 Propanediol.
[0139] Another embodiment, ALFS TA02, of [2] Composition B, consists of: (A) from about 2-12% preferably about 2-5% by weight of the cationic biocide Poly(hexamethylene biguanide) Hydrochloride, and (B) from about 5-15% preferably 9-12% by weight of the fabric conditioner Methyl bis(tallowamido ethyl)-2-hydroxyethyl ammonium methyl sulfate, and (C) from about 3.0-8% preferably 3.7 to 7.5% by weight of Methanaminium, N-coco-alkyl-N,N-bis(hydropoly(2-oxyethyl))-,chlorides, and (D) from about 0.3-2% preferably 0.5-1.8% by weight of 2-Propenoic acid, homopolymer, and (E) from about 0.5-1.8% preferably 0.5-1.5% by weight of a quaternized hydrolyzed wheat protein silicone co-polymer, (F) from about 0.1-6% preferably 0.5-1% by weight of Stearyldimonium hydroxypropyl hydrolysed wheat protein, and (G) from about 0.2-1.3% preferably 0.2-0.8% by weight of a crosslinked copolymers of acrylamide and methacrylate methylene bisacrylamide; and further encompasses:
[0140] [3] Composition C being an Antimicrobial Fabric Softener Sheet (AFSS) composition for use on fabrics in the drying-cycle of the machine-drying stage of the laundry program; Composition C containing a cationic biocide and combinations of quaternary softening compounds, a surfactant system, and adjunct constituents such as colorants, fragrances, as well as others not particularly elucidated here. These adjunct constituents may be added in any effective amount, but generally the total amount of such adjunct constituents should not exceed about 4% by weight of the total weight of the disinfectant liquid fabric softener compositions being taught herein.
[0141] One embodiment (AFSS) of [3] Composition C consists of: (A) rom about 2-12%% preferably about 8-10% by weight of the cationic biocide Poly(hexamethylene biguanide) Hydrochloride, and (B) from about 2-8% preferably 3-6% by weight of Dipalmitoylethyl Hydroxyethylmonium Methosulfate, and (C) from about 18-30% preferably 19-27% by weight of a surfactant system comprised of: (1) palmityl palmitate, and (2) alpha-docosyl-omega-hydroxy-poly-(oxy-2-ethanediyl, and (3) 1,3 dihydroxypropan-2-yl 2-hydroxyoctadecanoate and (4) Glycerides, palm-oilmonoglycerides, diglycerides and triglycerides, hydrogenated. No particular limitation is imposed on the individual components present in the surfactant system. It is preferred that the surfactant system not exceed 27% by weight of the total composition; and (D) from about 0.04-1% preferably 0.05-0.3% by weight of Ethylene glycol monophenyl ether, and (E) from about 0.04-0.55% preferably 0.05-0.3% by weight of a Sodium Hydroxide (50%) Solution
[0142] Distilled or deionized water forms a constituent of Composition A, Composition B and Composition C. The amount of water added is an amount to provide the balance of the compositions to 100% by weight. The water is added, generally in an amount of 20 to 80% weight, to provide the balance of the total compositions. Also, as has been previously noted, up to 13% by weight of the total antimicrobial liquid laundry detergent compositions may be comprised of the one or more adjunct constituents.
[0143] Preparation of Representative Formulations. To illustrate the compositions according to the present invention and their antimicrobial effect, representative formulations of Composition A and Composition B were prepared using commercially available stocks of the various ingredients, and are described in Table 1. The amount of each ingredient in the representative formulations is based upon a weight percentage of the total formulation. Each of the representative formulations were prepared by adding a measured amount of the corresponding ingredients specified for ingredient-group A, B, C or D. Ingredients specified for Ingredient-Group A were dispensed into a 250-ml glass beaker in the order listed, while stirring with a magnetic stir bar at 100-150 rpm and heating to 40-45° C. until smooth and homogenous. Ingredients specified for Ingredient-groups B, C and D were combined in a 500 ml glass beaker in the order listed, while stirring with a magnetic stir bar at 100-150 rpm and heating at 40-45° C. until the formulation was smooth and homogenous. Ingredient group A in the 250-ml beaker was added incrementally into the 500-ml beaker containing the ingredients from Ingredient Groups B, C, and D with continuous stirring at 100-150 rpm and heating to 40-45° C. until the formulation was smooth and homogenous. Stirring continued while the formulation cooled to ambient temperature.
TABLE-US-00001 TABLE 1 Representative Formulations of Composition A and Composition B (values expressed as % by weight) Ingredients TA08.3 TA08.5 TA09.2 TA01.5 Deionized Water 22.20 24.40 32.00 20.00 A Polyhexamethylene biguanide hydrochloride (Carbosynth-Biosynth) 17.46 17.00 8.05 4.20 A 1,3 Propanediol (Zemea, Dupont-Tate and Lyle) 8.68 4.54 2.50 2.40 A Alkyl Dimethyl Benzyl Ammonium Chloride (C12 5%, C14 60%, C16 1.75 3.71 A 30%, C18 5%) and Alkyl Dimethyl Ethylbenzyl Ammonium Chloride (68% C12, 32% C14) (BTC 2125M 80%-Stepan Co). Deionized Water 22.20 25.00 22.95 63.13 B Ethanaminium, 2-hydroxy-N, N-bis(2-hydroxyethyl)-Nmethyl-, esters 5.60 B with C16-18 and C18-unsatd. fatty acids, Me sulfates salts Wheat, [2-hydroxy-3-[3-(trimethoxysilyl)propoxy] propyl], hydrolyzed 0.96 B and Ethyl alcohol (Coltide Radiance-LQ-WD) Alcohol C14-15, Ethoxylated (Biosoft N45-7, Stepan Co.) 5.45 5.32 B Alcohols (C12-15 Ln. Saturated) Ethoxylated (Biosoft E-678, Stepan 8.25 5.31 B Co.) N-Cocamidopropyl-N, N-dimethylglycine, hydroxide, inner salt 5.89 4.90 B Monoisopropanolamine, Benzenesulfonic acid C10-16-alkyl derivs. 30.00 B Sodium salts Poly(oxy-1,2-ethanediyl) Alpha-undecylomegahydroxy and Propylene Glycol (Amphosol CG, Stepan Co.) Enzyme Medley (Genencor Int'l Inc.) 3.14 5.35 4.50 C Sodium Methyl 2-Sulfolaurate and Disodium 2-Sulfolaurate (Alpha- 3.77 2.03 D Step PC 48, Stepan Co.) Sodium hydroxide (18%) (LD Carlson Company) 2.96 3.09 D Ethoxylated methyl glucoside oleate (Lubrizol, Inc.) 0.64 0.64 D
Evaluation of Antimicrobial Efficacy Using a Time-Kill Procedure
[0144] Representative formulations of Composition A or Composition B were evaluated for antimicrobial efficacy against challenge microorganisms P. aeruginosa (ATCC 15442) or K. pneumoniae (ATCC 4352) or S. aureus (ATCC 6538) at a dose-rate of 35.5 ul of a respective formulation to 5.0 ml test substance volume comprised of 4.5-ml sterilized-deionized water and 0.5-ml of an inoculum at a contact time of 17-minutes.
[0145] Summary of the Test Procedure: Test microorganisms are prepared in liquid culture medium for bacteria; the suspension of test microorganism is standardized, as needed, by dilution in a buffered saline solution; test and control substances are dispensed in identical volumes to sterile vessels; independently, test and Control substances are inoculated with each test microorganism, then mixed and incubated; control substances are immediately harvested and represent the concentration present at the start of the test, or time zero; at the conclusion of the contact time, a volume of the liquid test solution is harvested and chemically neutralized; dilutions of the neutralized test solution are assayed using appropriate growth media to determine the surviving microorganisms at the respective contact times. Reductions of microorganisms are calculated by comparing initial microbial concentrations to final microbial concentrations.
[0146] Criteria for Scientific Defensibility: For the Suspension Time Kill study to be scientifically defensible, the average number of viable bacteria recovered from the time zero samples must be approximately 1×10.sup.6 cells/ml or greater; ordinary consistency between replicates must be observed for the time zero samples; positive/growth controls must demonstrate growth of appropriate test microorganism; negative/purity controls must demonstrate no growth of test microorganism.
[0147] Test Parameter used in the Study: Test Substance Vol. 5.0355-ml, Duplicate Replicates, Control substance vol. 5.0355-ml, Control substance PBS, Culture growth media Tryptic Soy Broth, Culture growth time: 24-hours, Inoculum vol. 0.50 ml, Inoculum concentration >1.0×10.sup.6 CFU/ml, Contact temperature 16±1° C., Contact time 17-minutes, Neutralizer vol. 9.0-ml, Neutralizer broth: Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, 0.05% (w/v) KH.sub.2PO.sub.4, 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS, Plating media Tryptic Soy Agar, Enumeration plate incubation temperature 36±1° C., Enumeration plate incubation time 24-48 hrs.
[0148] Study Controls: Sterility controls were plated for all media used during each day of the study including the Neutralizer Broth. A viability control was plated on Tryptic Soy Agar to confirm both the viability of the culture and to confirm that the media type used was appropriate for growth.
[0149] Control Results: Neutralizer method Verified, Media sterility Confirmed sterile, Growth confirmation Conformed morphology on TSA.
[0150] Calculations: Log.sub.10, Reduction=Log(B/A), Where B=Number of viable test microorganisms in the control substance immediately after inoculation, and A=Number of viable test microorganisms in the test substance after the contact time.
[0151] Results from the standard time-kill procedure with a contact time of 17 minutes, and a Log.sub.10, reduction value ≥3.0 against all microorganisms tested is acceptable for a “passing criteria as a sanitizer”. A Log.sub.10 reduction value ≥5.0 against all microorganisms tested is acceptable for a “passing criteria as a disinfectant”. The results of the evaluation are summarized in Table 2, below.
TABLE-US-00002 TABLE 2 Log.sub.10 Red. Log.sub.10 Red. of Log.sub.10 Red. of Formulation of S. aureus P. aeruginosa K. pneumoniae TA08.3 >5.88 >5.66 >5.42 TA08.5 >6.40 >6.20 >6.67 TA09.2 >4.14 >4.02 >4.37 TA01.5 >3.31 >3.12 >3.58 TA08.3 >5.88 >5.66 >5.42
[0152] The Log.sub.10, results reported in Table 2 clearly indicate the inventive compositions are within applicants' preferred range as a sanitizer or disinfectant and further demonstrate the antimicrobial efficacy discussed in the above specification.
[0153] Evaluation of Antimicrobial Efficacy Using a Synergistic Time-Kill Procedure
[0154] Representative formulations of Composition A and Composition B were evaluated for synergistic antimicrobial efficacy against challenge microorganisms P. aeruginosa (ATCC 15442) or K. pneumoniae (ATCC 4352) or S. aureus (ATCC 6538) a dose-rate of 24.0 ul of a representative formulation Composition A plus 35.5 ul of a representative formulation of Composition B to a 5.0 ml test substance volume comprised of 4.5-ml sterilized-deionized water and 0.5-ml of an inoculum for a 1.sup.st and 2.sup.nd contact time of 17-minutes.
[0155] Summary of the Synergistic Test Procedure: Test microorganisms are prepared in liquid culture medium for bacteria; the suspension of test microorganism is standardized as needed by dilution in a buffered saline solution; for a synergy study the test substance volume, and the control substance volume, are dispensed into sterile vessels; independently, test and control substances are inoculated with the challenge microorganism, then mixed and incubated; control substances are immediately harvested and represent the concentration present at the start of the test, or time zero; at the conclusion of the 1.sup.st contact time of 17-minutes, 35.5 ul of Composition B is dispensed into the test substance vessel and then mixed and incubated for the 2.sup.nd contact time of 17-minutes. At the conclusion of the 2.sup.nd contact time, a volume of the liquid test solution is harvested and chemically neutralized; dilutions of the neutralized test solution are assayed using appropriate growth media to determine the surviving microorganisms at the respective contact times. Reductions of microorganisms are calculated by comparing initial microbial concentrations to final microbial concentrations.
[0156] Criteria for Scientific Defensibility: For the Synergistic Suspension Time Kill study to be scientifically defensible, the average number of viable bacteria recovered from the time zero samples must be approximately 1×10.sup.6 cells/ml or greater; ordinary consistency between replicates must be observed for the time zero samples; positive/growth controls must demonstrate growth of appropriate test microorganism; negative/purity controls must demonstrate no growth of test microorganism.
[0157] Test Parameters Used in the Study: Test Substance Vol. 5.0559-ml, Duplicate Replicates, Control substance vol. 5.0595-ml, Control substance PBS, Culture growth media Tryptic Soy Broth, Culture growth time: 24-hours, Inoculum vol. 0.50 ml, Inoculum concentration >1.0×10.sup.6 CFU/ml, Contact temperature 16±1° C., 1.sup.st Contact time 17-minutes, 2.sup.nd Contact time 17-minutes, Neutralizer vol. 9.0-ml, Neutralizer broth: Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, 0.05% (w/v) KH.sub.2PO.sub.4, 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS, Plating media Tryptic Soy Agar, Enumeration plate incubation temperature 36±1° C., Enumeration plate incubation time 24-48 hrs.
[0158] Study Controls: Sterility controls were plated for all media used during each day of the study including the Neutralizer Broth. A viability control was plated on Tryptic Soy Agar to confirm both the viability of the culture and to confirm that the media type used was appropriate for growth.
[0159] Control Results: Neutralizer method Verified, Media sterility Confirmed sterile, Growth confirmation Conformed morphology on TSA.
[0160] Calculations: Log.sub.10, Reduction=Log(B/A), Where B=Number of viable test microorganisms in the control substance immediately after inoculation, and A=Number of viable test microorganisms in the test substance after the contact time.
[0161] Results from the synergistic time-kill procedure at a 1.sup.st contact time of 17 minutes with a formulation of Composition A, and a 2.sup.nd contact time of 17 minutes with a formulation of Composition B and a Log.sub.10 reduction value of 3.0-4.9 against all microorganisms tested is acceptable for a “passing criteria as sanitization by synergy”; and a Log.sub.10, reduction value ≥5.0 against all microorganisms tested is acceptable for a “passing criteria as disinfection by synergy”. The results of the evaluation are summarized in Table 3 below.
TABLE-US-00003 TABLE 3 Log.sub.10 Red. Log.sub.10 Red. of Log.sub.10 Red. of Formulation of S. aureus P. aeruginosa K. pneumoniae TA08.3/TA01.5 >5.88 >5.66 >5.42 TA08.5/TA01.5 >6.40 >6.20 >6.67 TA09.2/TA01.5 >5.14 >5.02 >5.34
[0162] The Log.sub.10 results reported in Table 3 clearly indicate the inventive compositions are within applicants' preferred range as a “synergistic disinfectant” and further demonstrate the antimicrobial efficacy discussed in the above specification.
[0163] Standard Method to Evaluate Laundry Sanitizers and Disinfectants Using a Portable Washing Machine
[0164] Representative formulations of Composition A and Composition B were evaluated for sanitizing or disinfecting efficacy in accordance with Product Performance Test Guideline OCSPP 810.2400: Disinfectants and Sanitizers for Use on Fabrics and Textiles; and ASTM E2406-16 Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations, the contents of which are herein incorporated by reference.
[0165] Scope: Under actual-in-use laundry conditions and operations, sets of fabric swatches are inoculated with a suspension of a challenge microorganism or a mixed species of microorganisms and are dried and placed between the 6.sup.th and 7.sup.th fold of a fabric bundle. The fabric bundle and inoculated fabric carriers are treated in a low volume of a Representative formulation of Composition A or Composition B. After a specified treatment time, the test carriers and wash-water are individually cultured either quantitatively (sanitizer efficacy) or qualitatively (disinfectant efficacy).
[0166] Preparation of the Challenge Microorganisms: The challenge microorganism (P. aeruginosa ATCC 15442 or S. aureus ATCC 6538 or K. pneumoniae ATCC 4352) was subcultured on Nutrient Agar A slants through at least one daily transfer. The slants were incubated for 24±2 hours at 35±2° C. One day prior to the test, the growth was washed from the slant using a 5-10 mL aliquot of Phosphate Buffer Dilution Water. The growth suspension was aspirated and minimally, 1 mL was transferred to each of four Nutrient Agar B culture bottles. The bottles were Incubated for 18-24 hours at the conditions listed above with the agar side down. On the day of the test, the challenge microorganism was harvested from the Nutrient Agar B bottles by adding a 3 mL aliquot of Phosphate Buffer Dilution Water (PBDW) and sterile glass beads to each bottle, rocking the bottles back and forth. As needed, the culture was further adjusted by dilution in PBDW to yield approximately 10.sup.8 colony forming units (CFU) per mL for S. aureus and approximately 10.sup.9 CFU/mL of K. pneumoniae and P. aeruginosa.
[0167] Preparation of the Inoculum: A 500 μL inoculum of the challenge microorganism was obtained by adding 25 ul of 5% bovine serum albumin and 100 ul 0.4% mucin and 35 ul 5% to 340 ul of the challenge microorganism suspension.
[0168] Fabric Bundle and Carrier Preparation: A dry scoured fabric was prepared according to the method, and cut into strips 3 in. (7.6 cm) wide and weighing 25±0.1 g each. One end of the 25-g test fabric strip was pierced to secure the fabric onto the outer horizontal extension of a stainless-steel spindle. The fabric strip was wrapped around the three horizontal extensions with sufficient tension to obtain 12 but not 13 laps while using the entire 25-g of fabric. Staples were used to secure the fabric strip end. Additional staples were applied to the 6th and 7th folds along one horizontal side of the 25-g fabric bundle to create “pockets” that will secure individual fabric swatches during laundering. The fabric wrapped spindle was steam-sterilized and dried prior to testing. Fabric swatch carriers of approximately 1-inch×1.5-inch were cut from the scoured fabric and a staple was secured to each carrier to aid in removal from the spindle, and placed in a sterile vessel, autoclave sterilized, and cooled/dried at room temperature prior to use.
[0169] Preparation of the Test Substance: For disinfection efficacy, 532.5 ul of Representative Formulation ALLD TA08.3 or ALLD TA08.5 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the wash-water). For sanitization efficacy, 360 ul of Representative Formulation ALLD TA08.3 or ALLD TA98.5, or 532.5 ul of ALLD TA09.2 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the wash-water) and 532.5 ul of ALFS TA01.5 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the rinse-water). The test substance was used within 1-hour or preparation.
[0170] Contamination of the Carriers: The fabric swatch/carriers were inoculated with a 30 μL aliquot of the prepared challenge microorganisms P. aeruginosa ATCC 15442 or S. aureus ATCC 6538 or K. pneumoniae ATCC 4532, or a mixed species of the prepared challenge microorganisms and dried in a 35±2° C. incubator until visibly dry, but not longer than 30 minutes. The fabric carriers were removed from the incubator and placed individually into the spindle pocket (up to 3 carriers per spindle) between the sixth and seventh folds of the wrapped fabric spindle without overlapping.
[0171] Treatment Conditions: Prior to treatment, the spindle wire was aseptically removed from the 25-g fabric bundle, and the 25-g fabric bundle containing the fabric swatch carriers was placed in the sterilized wash-tub of the portable washing machine containing the wash-water-solution (532.5 ul of representative formulations ALLD TA08.3 or ALLD TA08.5 or ALLD TA09.2 of Composition A mixed with 75 ml 400 ppm AOAC Hard Water Solution and cooled 16±2° C.) or the rinse-water solution (532.5 ul of representative formulation ALFS TA01.5 mixed with 75 ml 400 ppm AOAC Hard Water Solution cooled to 16±2° C.) and the wash-cycle or rinse-cycle was initiated and continued for 17-minutes (the exposure-time). After completion of the wash-cycle or rinse-cycle exposure-time and using sterile forceps, the 25-g fabric bundle containing the three (3) inoculated carriers is removed into the sterilized spin-tub of the portable washing machine and the spin-cycle was initiated and continued for 15-seconds.
[0172] Test Recovery: Following completion of the spin-cycle, the 25-g fabric bundle was removed from the spin-tub of the portable washing machine and the fabric carriers were aseptically removed from the fabric bundle into individual 50-ml wide-mouthed-tubes containing 10-ml of the neutralizer broth 3% (w/v) Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, KH.sub.2PO.sub.4 0.05% (w/v), 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS). The wash-water or rinse-water was also removed into a wide-mouth tube containing 40-ml concentrated neutralizer (4.5% (w/v) Tween 80, 0.45% (w/v) Lecithin, 1.5% (w/v) Sodium thiosulfate, 2.25% (w/v) K.sub.2HPO.sub.4, 0.025% (w/v) KH.sub.2PO.sub.4, 1.5% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS.)
[0173] Determination of Disinfectant Efficacy: The entire volume of wash-water or rinse-water containing neutralizing broth was filtered and the filter was plated on Tryptic Soy Agar+5% Sheep's blood containing neutralizers as needed. The plates and the tubes containing carriers were incubated for 48 to 54 h at 35±2° C. Results are reported as growth (+) or no growth (—). Positive results were confirmed by Gram stain and streaking onto an appropriate growth medium for identification.
[0174] Determination of Sanitizing Efficacy: 1.0 mL neutralized wash water or rinse water was serially diluted to 10.sup.−2 using dilution fluid containing neutralizers as needed. All dilutions were plated in duplicate on Tryptic Soy Agar containing neutralizers as needed. The remaining neutralizer broth/wash water combination or neutralizer broth/rinse water combination was filtered and the filter was plated on Tryptic Soy Agar containing neutralizers as needed. Plates were Incubated at 35±2° C. for 48 to 54 h. To determine survivors, the colonies were counted and recorded as CFU/plate. Duplicate plates were averaged and multiplied by the dilution factor to arrive at CFU/mL. This average count was converted into log.sub.10.
[0175] Disinfectant Test Results: Results from the ASTM E2406 Procedure for Formulations TA08.3 and TA08.5 against all microorganisms tested, with <2 Positive Carriers (PC) out of 9 Total Carriers (TC) and 0 Positive Water Samples (PWS) out of 3 Total Water Samples (TWS) is acceptable for a “passing criteria as a disinfecting laundry detergent”. The results of the evaluation are summarized in Table 4, below.
TABLE-US-00004 TABLE 4 Disinfectant Efficacy at 17 minutes and 16 ± 2° C. TAO8.3 TA08.5 Test Microorganisms PC/TC PWS/TWS PC/TC PWS/TWS P. aeruginosa (ATCC 15442) 1/9 0/3 0/9 0/3 S. aureus (ATCC 6538) 0/9 0/3 0/9 0/3 K. pneumoniae (ATCC 4352) 0/9 0/3 0/9 0/3
The results reported in Table 4 clearly demonstrate the inventive compositions are within applicants' preferred range as a “disinfecting laundry detergent” and further demonstrate the antimicrobial efficacy discussed in the above specification.
[0176] Sanitizer Test Results: Results from the ASTM E2406 Procedure for Formulations TA08.3, TA08.5 TA09.2 and TA01.5 with a Log.sub.10, reduction value ≥3.0 and <5.0 against all microorganisms tested in the Wash-Water ((WR) and the Reduction of organisms on the fabric carriers (CR), is acceptable as a passing criterion as a “sanitizing laundry detergent” or “sanitizing fabric softener”. The results of the evaluation are summarized in Table 5 and Table 6 below.
TABLE-US-00005 TABLE 5 Sanitizing Efficacy at 17-minute contact time and 16 ± 2° C. TAO8.3 TA08.5 W-R C-R W-R C-R Test Microorganisms (Log.sub.10) (Log.sub.10) (Log.sub.10) (Log.sub.10) P. aeruginosa (ATCC 15442) 3.292 3.515 3.596 3.297 S. aureus (ATCC 6538) 3.439 3.466 3.454 3.124 K. pneumoniae (ATCC 4352) 3.470 3.475 3.112 3.467
TABLE-US-00006 TABLE 6 Sanitizing Efficacy at 17-minute contact time and 16 ± 2° C. TA09.2 TA01.5 W-R C-R W-R C-R Test Microorganisms (Log.sub.10) (Log.sub.10) (Log.sub.10) (Log.sub.10) P. aeruginosa (ATCC 15442) 3.220 3.148 3.365 3.088 S. aureus (ATCC 6538) 3.133 3.120 3.338 3.100 K. pneumoniae (ATCC 4352) 3.173 3.436 3.150 3.069
The results reported in Table 4 and Table 5 and Table 6 clearly demonstrate the inventive compositions are within applicants' preferred range as a “sanitizing laundry detergent” and further demonstrates the antimicrobial efficacy discussed in the above specification.
[0177] Synergistic Method to Evaluate Laundry Sanitizers and Disinfectants Using a Portable Washing Machine
[0178] Representative formulations of Composition A and Composition B were evaluated for sanitizing or disinfecting efficacy in accordance with Product Performance Test Guideline OCSPP 810.2400: Disinfectants and Sanitizers for Use on Fabrics and Textiles; and ASTM E2406-16 Test Method for Evaluation of Laundry Sanitizers and Disinfectants for Use in High Efficiency Washing Operations, the contents of which are herein incorporated by reference.
[0179] Scope: Under actual-in-use laundry conditions and operations, sets of fabric swatches are inoculated with a suspension of the test organism and are dried and placed between the 6th and 7th fold of a fabric bundle. The fabric bundle and inoculated fabric carriers are exposed to low volumes of a Representative formulation of Composition A during the wash-cycle of the laundry program, and a representative formulation of Composition B during the rinse-cycle of the laundry program. Following a specified exposure time, the test carriers and rinse-water are individually cultured either quantitatively (sanitizer efficacy) or qualitatively (disinfectant efficacy).
[0180] Preparation of the Challenge Microorganisms: The challenge microorganism (P. aeruginosa ATCC 15442 or S. aureus ATCC 6538 or K. pneumoniae ATCC 4352) was subcultured on Nutrient Agar A slants through at least one daily transfer. The slants were incubated for 24±2 hours at 35±2° C. One day prior to the test, the growth was washed from the slant using a 5-10 mL aliquot of Phosphate Buffer Dilution Water. The growth suspension was aspirated and minimally, 1 mL was transferred to each of four Nutrient Agar B culture bottles. The bottles were Incubated for 18-24 hours at the conditions listed above with the agar side down. On the day of the test, the challenge microorganism was harvested from the Nutrient Agar B bottles by adding a 3 mL aliquot of Phosphate Buffer Dilution Water (PBDW) and sterile glass beads to each bottle, rocking the bottles back and forth. As needed, the culture was further adjusted by dilution in PBDW to yield approximately 10.sup.8 colony forming units (CFU) per mL for S. aureus and approximately 10.sup.9 CFU/mL of K. pneumoniae and P. aeruginosa.
[0181] Preparation of the Inoculum: A 500 μL inoculum of the challenge microorganism was obtained by adding 25 ul of 5% bovine serum albumin and 100 ul 0.4% mucin and 35 ul 5% to 340 ul of the challenge microorganism suspension.
[0182] Fabric Bundle and Carrier Preparation: A dry scoured fabric was prepared according to the method, and cut into strips 3 in. (7.6 cm) wide and weighing 25±0.1 g each. One end of the 25-g test fabric strip was pierced to secure the fabric onto the outer horizontal extension of a stainless-steel spindle. The fabric strip was wrapped around the three horizontal extensions with sufficient tension to obtain 12 but not 13 laps while using the entire 25-g of fabric. Staples were used to secure the fabric strip end. Additional staples were applied to the 6th and 7th folds along one horizontal side of the 25-g fabric bundle to create “pockets” that will secure individual fabric swatches during laundering. The fabric wrapped spindle was steam-sterilized and dried prior to testing. Fabric swatch carriers of approximately 1-inch×1.5-inch were cut from the scoured fabric and a staple was secured to each carrier to aid in removal from the spindle, and placed in a sterile vessel, autoclave sterilized, and cooled/dried at room temperature prior to use.
[0183] Preparation of the Test Substance: For disinfection efficacy, 532.5 ul of Representative Formulation ALLD TA08.3 or ALLD TA08.5 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the wash-water). For sanitization efficacy, 360 ul of Representative Formulation ALLD TA08.3 or ALLD TA98.5, or 532.5 ul of ALLD TA09.2 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the wash-water) and 532.5 ul of ALFS TA01.5 was mixed with 75 ml sterilized deionized water containing 400 ppm AOAC Hard Water Solution and cooled to 16±2° C. (the rinse-water). The test substance was used within 1-hour or preparation.
[0184] Contamination of the Carriers: The fabric swatch/carriers were inoculated with a 30 μL aliquot of the prepared challenge microorganisms P. aeruginosa ATCC 15442 or S. aureus ATCC 6538 or K. pneumoniae ATCC 4532, or a mixed species of the prepared challenge microorganisms and dried in a 35±2° C. incubator until visibly dry, but not longer than 30 minutes. The fabric carriers were removed from the incubator and placed individually into the spindle pocket (up to 3 carriers per spindle) between the sixth and seventh folds of the wrapped fabric spindle without overlapping.
[0185] Treatment Conditions: Prior to exposure, the spindle wire was aseptically removed from the 25-g fabric bundle, and the 25-g fabric bundle containing the fabric swatch carriers was placed in the sterilized wash-tub of the 1.sup.st portable washing machine containing the wash-water solution (532.5 ul of representative formulations ALLD TA08.3 or ALLD TA08.5 or ALLD TA09.2) of Composition A gently mixed with 75 ml 400 ppm AOAC Hard Water Solution and cooled 16±2° C.) and the wash-cycle was initiated and continued for 17-minutes (the exposure-time). After completion of the wash-cycle exposure-time and using sterile forceps, the 25-g fabric bundle containing the three (3) inoculated carriers is removed into the sterilized spin-tub of the 1.sup.st portable washing machine and centrifuged for 15-seconds (the spin-time). After completion of the spin-cycle and using sterile forceps, the 25-g fabric bundle containing the three (3) inoculated carriers is removed from the spin-tub of the 1.sup.st portable washing machine into the sterilized wash-tub of the 2.sup.nd portable washing containing the rinse-water-solution (532.5 ul of representative formulation ALFS TA01.5 gently mixed with 75 ml 400 ppm AOAC Hard Water Solution cooled to 16±2° C.) and the rinse-cycle was initiated and continued for 17-minutes. At the conclusion of the rinse-cycle and using sterile forceps, the 25-g fabric bundle containing the three (3) inoculated carriers is removed into the spin-chamber of the 2.sup.nd portable washing machine and centrifuged for 15-seconds, (the 2.sup.nd spin-time).
[0186] Test Recovery: Following completion of the in-use-laundry operation, and at the end of the 2.sup.nd spin-cycle, the 25-g fabric bundle was removed from the spin-tub of the 2.sup.nd portable washing machine and the fabric carriers were aseptically removed into individual wide-mouthed-tubes containing 10-ml of the neutralizer broth 3% (w/v) Tween 80, 0.3% (w/v) Lecithin, 1.0% (w/v) Sodium thiosulfate, 1.5% (w/v) K.sub.2HPO.sub.4, KH.sub.2PO.sub.4 0.05% (w/v), 1% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS). The rinse-water-solution was also removed from the 2.sup.nd portable washing machine and added to concentrated neutralizing broth ((4.5% (w/v) Tween 80, 0.45% (w/v) Lecithin, 1.5% (w/v) Sodium thiosulfate, 2.25% (w/v) K.sub.2HPO.sub.4, 0.025% (w/v) KH.sub.2PO.sub.4, 1.5% (w/v) Poly-[sodium-4-styrenesulfonate], 0.1% (v/v) Triton®×100 prepared in PBS.)
[0187] Determination of Synergistic Disinfectant Efficacy: The entire volume of rinse-water containing neutralizing broth was filtered and the filter was plated on Tryptic Soy Agar+5% Sheep's blood containing neutralizers as needed. The plates containing the filters and tubes containing the fabric carriers were incubated for 48 to 54 h at 35±2° C.
[0188] Synergistic Disinfectant Test Results: Results from the ASTM E2406 Procedure modified for a Synergy Study for representative Formulations TA08.3+TA01.5 and TA08.5+TA01.5 and TA09.2+TA01.5 against all microorganisms tested, and <2 Positive Carriers (PC) out of 9 Total Carriers (TC) and 0 Positive Water Samples (PWS) out of 3 Total Water Samples (TWS) is acceptable for a “passing criteria as “disinfection by synergy.” The results of the evaluation are summarized in Table 7 and Table 8, below.
TABLE-US-00007 TABLE 7 Disinfectant Synergy Results at 17 minutes and 16 ± 2° C. Washing Temperature TA08.3 + TA01.5 TA08.5 + TA01.5 Test Microorganism PC/TC PWS/TWS PC/TC PWS/TWS P. aeruginosa (ATCC 0/9 0/3 0/9 0/3 15442) S. aureus (ATCC 6538) 0/9 0/3 0/9 0/3 K. pneumoniae 0/9 0/3 0/9 0/3 (ATCC 4532)
TABLE-US-00008 TABLE 8 Disinfectant Synergy Results at 17 minutes and 16± 2° C. TA09.2 + TA01.5 Test Microorganism PC/TC PWS/TWS P. aeruginosa (ATCC 15442) 0/9 0/3 S. aureus (ATCC 6538) 0/9 0/3 K. pneumoniae (ATCC 4532) 0/9 0/3
The results reported in Table 7 and Table 8 clearly demonstrate the inventive compositions are within applicants' preferred range as a “synergistic disinfecting laundry detergent” and further demonstrate the antimicrobial efficacy discussed in the above specification.
[0189] Quantitative Evaluation of Pass-Through Antimicrobial Efficacy of Composition a or Composition B
[0190] Scope: Representative formulations TA08.5 of Composition A and representative formulation TA01.5 of Composition B were evaluated against a challenge microorganism or a mixed species of challenge microorganisms to determine the quantity of unused antimicrobial efficacy remaining in 3,175-g or 7 lbs. of fabrics that passes-through from one cycle of the laundry program into another cycle of the laundry program, using one or more portable washing machines and a titration assay.
[0191] Preparation of the Challenge Microorganisms: Microorganisms were subcultured on Nutrient Agar A through three daily transfers, incubating at 35±2° C. On the day prior to testing, the cells were transferred into French square bottles containing 20 mL Nutrient Agar B and incubated 18 to 24 h at 35±2° C., agar side down. Growth was removed from the French square bottles using three-mL dilution fluid and five sterile glass beads to suspend growth. The cultures were standardized to yield approximately 10.sup.8 colony forming units (CFU) per mL of S. aureus and 10.sup.9 CFU/mL of K. pneumoniae and P. aeruginosa.
[0192] Preparation of the Inoculum: A 505 μL inoculum of the mixed species challenge microorganism was obtained by adding 25 μL of Bovine Serum Albumin solution, 100 μL of Bovine Mucin solution, and 35 μL Tryptone solution added to 345 ul of the microbial suspension.
[0193] Method to Evaluate the Pass-through Antimicrobial Efficacy from Composition A: To determine the amount of unused antimicrobial efficacy from Composition A that passes from the wash-cycle into the rinse-cycle of the laundry program, a 25-g fabric bundle containing 3 fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms is placed in the wash-tub of a 1.sup.st portable washing machine and treated with a wash-water-solution (532.5 ul of a representative formulation of Composition A added into 75 ml of 400 ppm AOAC Hard Water, gently mixed and cooled to 16±2° C.) for 17-minutes (the exposure-time). After the exposure-time in the wash-tub of the 1.sup.st portable washing machine, the treated 25-g fabric bundle is removed into the spin-tub of the 1.sup.st portable washing machine and centrifuged for 15-seconds (the spin-time). After conclusion of the spin-time, the treated 25-g fabric bundle is removed into the wash-tub of a 2.sup.nd portable washing machine and agitated in the rinse-test-water (500-ml of 400 ppm AOAC Hard Water) for 17-minutes (the agitation time), to extract the unused antimicrobial efficacy of Composition A; after conclusion of the agitation time, the treated fabric bundle is removed into the spin-chamber of the 2.sup.nd portable washing machine and centrifuged for 15-seconds (the spin-time) to more fully extract the unused antimicrobial efficacy of Composition A.
[0194] Method to Quantify PHMB from Composition A: The amount of unused Polyhexamethylene biguanide hydrochloride in for example, 3,175-g or 7 lbs. of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program is determined by quantifying the amount of Polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of the 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting a Composition A ca-test-sample-water-P1 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition A ca-test-sample-water-P1, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the 25-ml graduated sample tube containing the Composition A ca-test-sample-water-P1; and swirling to thoroughly mix the contents of the graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the 25-ml graduated sample-tube and swirling to thoroughly mix the contents and to observe the color of the Composition A ca-test-sample-water-P1 change from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the graduated sample-tube containing the Composition A ca-test-sample-water-P1 while swirling and counting each drop until the Composition A ca-test-sample-water-P1 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through into the rinse-cycle of the laundry program as follows: for Composition A Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPA.sub.7=(a.sub.PR×b.sub.PR×g.sub.ST×q.sub.SW)(f.sub.WT), where: PPA.sub.7=PHMB Pass-through-antimicrobial-efficacy for 3.175-g of or 7 lbs. of fabrics, and a.sub.PR=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition A ca-test-sample-water-P1 from the wash-tub of the 2.sup.nd washing machine, and b.sub.PR=5 (the ppm of PHMB in the Composition A rinse-test-water-P sample from the wash-tub of the 2.sup.nd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of ca-test-sample-water-P1 in the graduated sample tube, and q.sub.SW=quantity of sample water from the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0195] Method to Quantify QAC from Composition A: The amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride in 3,175-g or 7 lbs. of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program is determined by quantifying the amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of the 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting a Composition A ca-test-sample-water-Q1 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 25-ml mark with the ca-test-sample-water-Q1 and pipetting 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the 25-ml graduated sample-tube containing the Composition A ca-test-sample-water-Q1 from the wash-tub of the 2.sup.nd portable washing machine and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the ca-test-sample-water-Q1 sample and swirl to thoroughly mix and to observe the ca-test-sample-water-Q1 color change from colorless to light blue; and titrating the QAC Titrating Solution (Taylor Technologies R-0884) dropwise into the ca-test-sample-water-Q1 while swirling and counting after each drop until the ca-test-sample-water-Q1 color changes from light blue to violet pink; and recording the number of drops of the QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through into the rinse-cycle of the laundry program as follows: for Composition A n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QPA.sub.7=(d.sub.QR×f.sub.QR×g.sub.ST×q.sub.SW)(f.sub.WT) where: QPA.sub.7=QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and d.sub.QR=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition A ca-test-sample-water-Q1 sample from wash-tub of the 2.sup.nd portable washing machine, and f.sub.QR=10 (the ppm of QAC in the Composition A rinse-test-water-Q1 sample from wash-tub of the 2.sup.nd portable washing machine, neutralized by each drop of QAC Titration Solution, and g.sub.ST=quantity of ca-test-sample-water-Q1 in the graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0196] Method to Evaluate the Pass-through Antimicrobial Efficacy of Composition B: To determine the amount of unused antimicrobial efficacy from Composition B that passes from the wash-cycle into the rinse-cycle of the laundry program, a 25-g fabric bundle containing 3 fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms is placed in the wash-tub of a 1.sup.st portable washing machine and treated with a wash-water-solution (532.5 ul of a representative formulation of Composition B added into 75 ml of 400 ppm AOAC Hard Water, gently mixed and cooled to 16±2° C.) for 17-minutes (the exposure-time). After the exposure-time in the wash-tub of the 1.sup.st portable washing machine, the treated 25-g fabric bundle is removed into the spin-tub of the 1.sup.st portable washing machine and centrifuged for 15-seconds (the spin-time). After conclusion of the spin-time, the treated 25-g fabric bundle is removed into the wash-tub of a 2.sup.nd portable washing machine and agitated in the rinse-test-water (500-ml of 400 ppm AOAC Hard Water) for 17-minutes (the agitation time), to extract the unused antimicrobial efficacy of Composition B; after conclusion of the agitation time, the treated fabric bundle is removed into the spin-chamber of the 2.sup.nd portable washing machine and centrifuged for 15-seconds (the spin-time) to more fully extract the unused antimicrobial efficacy of Composition B.
[0197] Method to Quantify PHMB from Composition B: The amount of unused Polyhexamethylene biguanide hydrochloride in 3,175-g or 7 lbs. of fabrics that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the amount of Polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition B in the wash-tub of the 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting a Composition B cb-test-sample-water-P2 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition B cb-test-sample-water-P2, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the 25-ml graduated sample tube containing the Composition B cb-test-sample-water-P2; and swirling to thoroughly mix the contents of the graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the 25-ml graduated sample-tube and swirling to thoroughly mix the contents and to observe the color of the Composition B cb-test-sample-water-P1 change from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the graduated sample-tube containing the Composition B cb-test-sample-water-P2 while swirling and counting each drop until the Composition B cb-test-sample-water-P2 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition B that remains in 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: for Composition B Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPA.sub.7=(a.sub.PA×b.sub.PA×g.sub.ST×q.sub.SW)(f.sub.WT), where: PPA.sub.7=PHMB-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.PA=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition B cb-test-sample-water-P2 sample from the wash-tub of the 2.sup.nd portable washing machine, and b.sub.PA=5 (the ppm of PHMB in the Composition B cb-test-sample-water-P2 sample from the wash-tub of 2.sup.nd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of cb-test-sample-water-Q2 in the 25-ml graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0198] Method to Quantify QAC from Composition B: The amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride in 3,175-g or 7 lbs. of fabrics that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition B in the wash-tub of the 1.sup.st portable washing machine that passes-through into the wash-tub of a 2.sup.nd portable washing machine, by extracting a Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample tube (Taylor Technologies #9198BR) to the 25-ml mark with the cb-test-sample-water-Q2 and pipetting 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the 25-ml graduated sample-tube containing the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd portable washing machine and swirling to thoroughly mix, and adding 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the cb-test-sample-water-Q2 and observing the cb-test-sample-water-Q2 change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the cb-test-sample-water-Q2 sample while swirling and counting after each drop, until color changes from light blue to violet pink and recording the number of drops of QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition B that remains in 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: for Composition B n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QPA.sub.7=d.sub.QA×f.sub.AR×g.sub.ST×q.sub.SW)(f.sub.WT) where: QPA.sub.7=QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabric, and d.sub.QA=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd washing machine, and f.sub.AR=10 (the ppm of QAC in the Composition B cb-test-sample-water-Q2 from the wash-tub of the 2.sup.nd washing machine neutralized by each drop of QAC Titration Solution, and g.sub.ST=quantity of cb-test-sample-water-Q2 in the graduated sample tube, and q.sub.SW=total sample water from the wash-tub of the 2.sup.nd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0199] Method to Evaluate the Combined Amounts of PHMB from Composition A and Composition B: To determine the combined amount of unused antimicrobial efficacy that passes-through from one cycle of the laundry program into another cycle of the laundry program comprises the process of treating a 25-g fabric bundle containing 1 or more fabric swatches inoculated with a challenge microorganism or a mixed species of challenge microorganisms with a wash-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul of Composition A, for a specified period of time (the 1.sup.st exposure time) in the wash-tub of a 1.sup.st portable washing machine and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 1.sup.st portable washing machine, the (1.sup.st spin-time); and transferring the treated 25-g fabric bundle into the wash-tub of a 2.sup.nd portable washing machine containing a rinse-water solution comprising 75-ml of 400 ppm AOAC hardwater and 532.5 ul Composition B for a specified period of time (the 2.sup.nd exposure time); and centrifuging the treated 25-g fabric bundle for 15-seconds in the spin-tub of the 2.sup.nd portable washing machine (the 2.sup.nd spin-time); and transferring the twice-treated 25-g fabric bundle into the wash-tub of a 3.sup.nd portable washing machine containing about 500-ml of 400 ppm AOAC Hardwater (the cab-test-sample-water); and agitating the twice-treated 25-g fabric bundle for a period of time (the agitation time) to extract the unused antimicrobial efficacy of Composition AB; and centrifuging the twice-treated 25-g fabric bundle for 15-seconds in the spin-tub of the 3.sup.rd portable washing machine to more fully extract the unused antimicrobial efficacy of Composition AB; the 1.sup.st portable washing machine and the 2.sup.nd portable washing, and the 3.sup.rd portable washing machine each comprising a cabinet, a wash-tub, a spin-tub, a pulsator, a water-inlet, a control panel, a pulsator-timer-switch, a pulsator-and-drain selector switch, a spin-timer-switch and a drain-hose.
[0200] Method to Quantify the Combined Amount of PHMB from Composition A and Composition B: The amount of unused Polyhexamethylene biguanide hydrochloride from Composition A in 3,175 g or 7 lbs. of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused Polyhexamethylene biguanide hydrochloride from Composition B in 3,175 g or 7 lbs. of fabrics from the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program is determined by quantifying the combined amount of polyhexamethylene biguanide hydrochloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the unused polyhexamethylene biguanide hydrochloride remaining in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine, by extracting the Composition AB cab-test-sample-water-PP from the wash-tub of a 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 10-ml mark with the Composition AB cab-test-sample-water-PP, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into the graduated sample tube containing the Composition AB cab-test-sample-water-PP and swirling to thoroughly mix the contents of the graduated sample-tube; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies R-0977) into the graduated sample-tube and swirling to thoroughly mix the contents and to observe the color change of the Composition AB cab-test-sample-water-PP from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the graduated sample-tube containing the Composition AB cab-test-sample-water-PP while swirling and counting each drop until the Composition AB cab-test-sample-water-PP color changes from blue to pinkish purple and persists for 5-7 seconds; and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the Combined amount of Composition AB Polyhexamethylene biguanide hydrochloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: PPPA.sub.7=(a.sub.CR×b.sub.CR×g.sub.ST×c.sub.CW)(f.sub.WT) where: PPPA.sub.7=Composition AB PHMB-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.CR=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PP from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.CR=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PP from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of the cab-test-sample-water in the graduated sample tube, and c.sub.CW=the quantity of the Composition AB cab-test-sample-water-PP from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0201] Method to Quantify the Combined Amount of QAC from Composition A and Composition B: The amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A in 3,175 g or 7 lbs. of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B in the same 3,175 g or 7 lbs. of fabrics that passes-through from the rinse-cycle of the laundry program into the drying-cycle of the laundry program is determined by quantifying the combined amount of n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the unused n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine, by extracting the Composition AB cab-test-sample-water-QQ from the wash-tub of a 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated sample-tube (Taylor Technologies #9198) to the 25-ml mark with the Composition AB cab-test-sample-water-QQ, and dispensing 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the Composition AB cab-test-sample-water-QQ in the graduated sample tube and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into to the Composition AB cab-test-sample-water-QQ and swirling to thoroughly mix and observing the Composition AB cab-test-sample-water-QQ change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the Composition AB cab-test-sample-water-QQ, while swirling and counting after each drop, until the color changes from light blue to violet pink and recording the number of drops of QAC Titrating Solution (Taylor Technologies R-0884) used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the Combined amount of Composition AB n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride Pass-through antimicrobial efficacy is quantified by performing the following calculation: QPA.sub.7=(d.sub.CS×f.sub.TS×g.sub.ST×q.sub.SW)(f.sub.WT), where: QQPA.sub.7=Composition AB QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and d.sub.CS=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-QQ from the wash-tub of a 3.sup.rd portable washing machine, and f.sub.TS=10 (the ppm of QAC in the Composition AB cab-test-sample-water-QQ from the wash-tub of the 3.sup.rd portable washing machine neutralized by each drop of QAC Titration Solution), and g.sub.ST=quantity of cb-test-sample-water-Q2 in the graduated sample tube, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0202] Method to Quantify the Combined Amount of PHMB and QAC from Composition A and Composition B: The amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A in 3,175 g or 7 lbs. of fabrics that passes-through from the wash-cycle of the laundry program into the rinse-cycle of the laundry program and combines with the amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B in the same 3,175 g or 7 lbs. of fabrics in the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program, is determined by quantifying the combined amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride remaining in the 25-g fabric bundle treated with Composition A in the wash-tub of a 1.sup.st portable washing machine, that passes-through into the wash-tub of the 2.sup.nd portable washing machine and combines with the amount of unused Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride that remains in the same 25-g fabric bundle treated with Composition B in the wash-tub of a 2.sup.nd portable washing machine by extracting the Composition AB cab-test-sample-water-PQ1 from the wash-tub of the 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated Sample-tube-1 (Taylor Technologies #9198) to the 10-ml mark with the Composition AB cab-test-sample-water-PQ1, and titrating 12 drops of Biguanide Complexing Reagent (Taylor Technologies #R-#0976) into graduated sample-tube1 containing the Composition AB cab-test-sample-water-PQ1 and swirling to thoroughly mix the contents of the graduated sample-tube-1; and titrating 5 drops of Biguanide Indicator Solution (Taylor Technologies (R-0977) into the graduated sample-tube-1 and swirling to thoroughly mix the contents and to observe the color change of the Composition AB cab-test-sample-water-PQ1 change from clear to blue; and titrating Biguanide Titrating Solution (Taylor Technologies R-0978) dropwise into the 25-ml graduated sample-tube-1 containing the Composition AB cab-test-sample-water-PQ1 while swirling and counting each drop until the Composition AB cab-test-sample-water-PQ1 color changes from blue to pinkish purple and persists for 5-7 seconds, and recording the number of drops of Biguanide Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program; and by extracting the Composition AB cab-test-sample-water-PQ2 from the wash-tub of the 3.sup.rd portable washing machine via the drain-hose and filling a previously cleaned/sterilized 25-ml graduated Sample-tube-2 (Taylor Technologies #9198) to the 25-ml mark with the Composition AB cab-test-sample-water-PQ2, and titrating 1.0-ml of QAC Complexing Reagent (Taylor Technologies R0950) into the Composition AB cab-test-sample-water-PQ2 in the graduated sample-tube-2 and swirling to thoroughly mix, and titrating 3 drops of Toluidine Blue O Indicator (Taylor Technologies R-0881) into the Composition AB cab-test-sample-water-PQ2 in the graduated sample-tube-2 and swirling to thoroughly mix and observing the Composition AB cab-test-sample-water-PQ2 change to light blue and titrating the QAC Titrating Solution (Taylor Technologies R-0884) into the Composition AB cab-test-sample-water-PQ2 in graduated sample-tube-2 while swirling and counting after each drop, until the color changes from light blue to violet pink and persists for 5-7 seconds, and recording the number of drops of QAC Titrating Solution used during the titration procedure to calculate the amount of unused antimicrobial efficacy from Composition A that remains in 3,175 g or 7 lbs. of fabrics after the wash-cycle of the laundry program that passes-through and combines with the unused antimicrobial efficacy from Composition B that remains in the same 3,175 g or 7 lbs. of fabrics after the rinse-cycle of the laundry program that passes-through into the drying-cycle of the laundry program as follows: the combined amount of Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition A Combined with the combined amount of Polyhexamethylene biguanide hydrochloride and n-Alkyl Dimethyl Benzyl Ammonium Chloride and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride from Composition B is quantified by performing the following calculation: ng calculationowiCQPA.sub.7=[(a.sub.CP×b.sub.PT×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(d.sub.QC×f.sub.QT×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(a.sub.CS×b.sub.PG×g.sub.ST×q.sub.SW)(f.sub.WT)]+[(d.sub.QB×f.sub.QD×g.sub.ST×q.sub.SW)(f.sub.WT)] where: CQPA.sub.7=Combined PHMB and QAC-Pass-through-antimicrobial-efficacy for 3,175 g or 7 pounds of fabrics, and a.sub.CP=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.PT=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=quantity of cab-test-sample-water-PQ1 in the graduated sample-tube-1, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and d.sub.QC=number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-PQ1 in sample-tube-1 from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.QT=10 (the ppm of QAC in the Composition AB rinse-test-water-PQ1 sample-tube-1 from the wash-tub of the 3.sup.rd washing machine neutralized by each drop of QAC Titration Solution), and g.sub.ST=quantity of cab-test-sample-water-PQ1 in the graduated sample-tube-1, and q.sub.SW=total sample water from the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and a.sub.CG=the number of drops of Biguanide Titrating Solution (R-0978) used during titration of Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of the 3.sup.rd portable washing machine, and b.sub.PG=5 (the ppm of PHMB in the Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of 3.sup.rd portable washing machine, neutralized by each drop of Biguanide Titrating solution), and g.sub.ST=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and q.sub.SW=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics); and d.sub.QB=the number of drops of QAC Titrating Solution (R0884) used during titration of the Composition AB cab-test-sample-water-PQ2 in sample-tube-2 from the wash-tub of the 3.sup.rd portable washing machine, and f.sub.QD=10 (the ppm of QAC in the Composition AB rinse-test-water-PQ2 sample-tube-1 from the wash-tub of the 3.sup.rd washing machine neutralized by each drop of QAC Titration Solution), and g.sub.ST=quantity of cab-test-sample-water-PQ2 in the graduated sample tube, and q.sub.SW=total sample water in the wash-tub of the 3.sup.rd portable washing machine divided by g.sub.ST, and f.sub.WT=3,175 g (the fabric weight in grams equal to 7 pounds of fabrics).
[0203] Quantitative Analysis and Report: For each representative formulation used in the study, a pass-through antimicrobial efficacy from the wash-cycle into the rinse-cycle of 500-1,000 ppm PHMB indicates a “low concentration of pass-through antimicrobial efficacy for PHMB”, a quantity of 1,050 to 1,750 ppm PHMB indicates a “moderate concentration of pass-through antimicrobial efficacy for PHMB”, and a quantity >1,750 ppm of PHMB indicates a “high concentration of pass-through antimicrobial efficacy for PHMB”; For each representative formulation used in the study, a pass-through antimicrobial efficacy from the wash-cycle into the rinse-cycle, a quantity <175 ppm QAC indicates a “low concentration of pass-through antimicrobial efficacy for QAC”; a quantity of 200-325 ppm QAC indicates a “moderate concentration of pass-through antimicrobial efficacy for QAC”, and a quantity >325 ppm QAC indicates a “high concentration of pass-through antimicrobial efficacy for QAC”; For each representative formulation used in the study, a pass-through antimicrobial efficacy from the rinse-cycle into the drying-cycle <400 ppm PHMB indicates a “low concentration of pass-through antimicrobial efficacy for PHMB”, a quantity of 400 to 800 ppm PHMB indicates a “moderate concentration of pass-through antimicrobial efficacy for PHMB”, and a quantity >800 ppm of PHMB indicates a “high concentration of pass-through antimicrobial efficacy for PHMB” For each representative formulation used in the study, a pass-through antimicrobial efficacy from the rinse-cycle into the drying-cycle, <100 ppm QAC indicates a “low concentration of pass-through antimicrobial efficacy for QAC”, a quantity of 100 to 150 ppm QAC indicates a “moderate concentration of pass-through antimicrobial efficacy for QAC”, and a quantity >150 ppm of QAC indicates a “high concentration of pass-through antimicrobial efficacy for QAC” For each representative formulation used in the study, a combined pass-through antimicrobial efficacy from the rinse-cycle into the drying-cycle, <1,200 ppm PHMB indicates a “low concentration of combined pass-through antimicrobial efficacy for PHMB”, a quantity 1,200 to 1,300 ppm PHMB indicates a “moderate concentration of combined pass-through antimicrobial efficacy for PHMB”; For each representative formulation used in the study, a combined pass-through antimicrobial efficacy from the rinse-cycle into the drying-cycle, <80 ppm QAC indicates a “low concentration of combined pass-through antimicrobial efficacy for QAC”, a quantity 80-175 ppm QAC indicates a “moderate concentration of combined pass-through antimicrobial efficacy for QAC”; For each representative formulation used in the study, a combined pass-through antimicrobial efficacy from the rinse-cycle into the drying-cycle, <1,100 ppm PHMB+QAC indicates a “low concentration of combined pass-through antimicrobial efficacy for PHMB+QAC”, a quantity from 1,100 to 1,300 ppm PHMB+QAC indicates a “moderate concentration of combined pass-through antimicrobial efficacy for PHMB+QAC”, and a quantity >1,300 ppm of PHMB+QAC indicates a “high concentration of combined pass-through antimicrobial efficacy for PHMB+QAC.” The results of the evaluation are summarized in Table 9, Table 10, and Table 11 below;
TABLE-US-00009 TABLE 9 Pass-Through Antimicrobial Efficacy of Representative Formulations from the Wash-Cycle into the Rinse-Cycle Representative PHMB QAC PHMB+ Formulation (ppm) (ppm) QAC TA08.3 2,750 2,750 TA08.5 2,800 300 3,100 TA09.2 1,220 1,220
TABLE-US-00010 TABLE 10 Pass-Through Antimicrobial Efficacy of Representative Formulations from the Rinse-Cycle into the Drying Cycle Representative PHMB QAC PHMB+ Formulation (ppm) (ppm) QAC TA01.5 380 40 420 TA08.3 820 820 TA08.5 910 110 1,020 TA09.2 365 365
TABLE-US-00011 TABLE 11 Combined Pass-Through Antimicrobial Efficacy of Representative Formulations from the Rinse-Cycle into the Drying-Cycle Representative PHMB QAC PHMB+ Formulation (ppm) (ppm) QAC TA08.3 + TA01.5 1,200 40 1,240 TA08.5 + TA01.5 1,290 150 1,440 TA09.2 + TA01.5 745 40 785
[0204] The results reported in Table 9, Table 10 and Table 11 clearly demonstrate that the Pass-through Antimicrobial Efficacy of the inventive compositions are within applicants' preferred range of “moderate to high concentration” of Pass-through Antimicrobial Efficacy as discussed in the above specification.
Evaluation of Antimicrobial Durability of Composition a or Composition B
[0205] Scope: To determine the antimicrobial efficacy and antimicrobial duration (Antimicrobial Durability) of Composition A and Composition B on fabrics after the laundry program, test fabrics were treated with Representative formulations TA08.3 or TA08.5 or TA09.2, or TA01.5 in the wash-tub of a portable washing machine (Zeny Model H01-1669A) for 17-minutes and inoculated with a mixed species of challenge microorganisms (P. aeruginosa-ATCC 15442, +S. aureus-ATCC 6538+K. pneumoniae-ATCC 4352) and left to air dry for 24 or 48 hours (the test period). At the end of the test period, bacterial growth on the test fabric was suspended in 250-ml wide-mouthed Erlenmeyer flasks containing 50-ml PBS (7.2 pH), quantified by 10-fold serial dilution to 10.sup.−7 and compared to a control fabric to determine the Log.sub.10, reduction.
[0206] Preparation of the Test Materials: Impurities on the test fabric (50% cotton/50% polyester woven fabric weighing 441-g) were removed by boiling for 1 hour in 4 liters of distilled or deionized water containing 2.2-g sodium carbonate and 2.2-g of a nonionic wetting (Makon 10 Stepan Co. Chicago, Ill.). Following the 1-hour boiling period, the fabric was rinsed first in 4 liters boiling distilled or deionized water and then in 4 liters cold distilled or deionized water until all visual traces of the wetting agent (foaming) are removed. The fabric was centrifuged in the spin-chamber of a portable washing machine (Zeny Model H01-1669A) to remove excess water then air dried for at least 24 hrs at room temperature. The scoured dry fabric was cut into (3×5.5 in) pieces which serve as Control fabric1, Control fabric2 and Test fabric1.
[0207] Antimicrobial Treatment of the Test Fabric: Control Fabric2 and Test fabric1 were treated with a wash-water-solution (532.5 ul of the representative formulation of ALLD TA08.3 or ALLD TA08.5 or ALLD TA09.2 or ALFS TA01.5, added to 400 ppm AOAC Hard Water, gently mixed and cooled to 16±2° C.) in the wash-tub of a portable washing machine and agitated for 17-minutes, (the exposure-time). After the exposure-time, Control fabric2 and Test fabric1 were removed into the spin-tub of the portable washing machine and centrifuged for 15-seconds, (the spin-time).
[0208] Qualitative Determination of Composition A and Composition B on Control Fabric2: At the conclusion of the spin-time, Control fabric2 was air dried for 2-hrs then placed in a 950-ml glass tray (Pyrex Life Sciences, Tewksbury Mass. USA) containing 150 ml of Bromophenol Blue dye (Biopharm, Inc Hatfield, AR. USA) and remained in the dye until completely saturated. After the dyeing period, Control fabric2 was air-dried and the presence of the antimicrobial actives on the fabric was visually determined by the presence of the Bromophenol Blue dye which complexed with the antimicrobial actives and formed a blue stable complex on the Control fabric. This served as a qualitative control visually demonstrating the presence of the antimicrobial actives on the Control Fabric2.
[0209] Thermal Curing of the Test Fabric: At the conclusion of the spin-time and following antimicrobial treatment with the wash-water-solution, Test fabric1 was thermally cured in a residential dryer for 20 minutes at 65+1° C.
[0210] Quantitative Determination of Antimicrobial Durability: 200 ul of the microorganism suspension from Control fabric1 or Test fabric1 was serially diluted 10-fold to 10.sup.−7 and 0.03 ml of each dilution was plated in duplicate on Tryptic Soy Agar and incubated at 35±1° C. for 24-hr. After the 24-hr incubation period, colonies from the plates for Control fabric1 or Test fabric1 were counted and the data was recorded as cfu/plate. To determine the number of surviving organisms, duplicate plate counts were averaged and multiplied by the dilution factor to calculate cfu/mL of the mixed species suspension.
[0211] Calculations: Log.sub.10, reduction was calculated using the following equation, Log.sub.10 Reduction=Log(B/A), where: B=Number of viable test microorganisms on Control fabric1, and A=Number of viable test microorganisms on Test fabric1.
[0212] Quantitative Analysis and Report: For each Representative Formulation used in the study, a Log.sub.10 reduction 1.20 of the mixed species inoculum on the Test fabric1 at 24-hours is acceptable as a passing criterion tor “24-hour antimicrobial durability”; and a Log.sub.10, reduction 0.90 of the mixed species inoculum on Test fabric1 at 48-hours is acceptable as a passing criterion for “48-hour antimicrobial durability”. The results of the Antimicrobial Durability Study are provided in Table 12 below.
TABLE-US-00012 TABLE 12 Antimicrobial Durability Representative Formulations of Composition A or Composition B (Lod.sub.10 Reduction) Representative 24-hours 48-hours Formulation PA + SA + KP PA + SA + KP TA08.3 1.358 1.160 TA08.5 1.433 1.215 TA09.2 1.014 0.934 TA01.5 1.272 1.176
The results reported in Table 12 clearly demonstrate that the Antimicrobial Durability of the inventive compositions on laundered fabrics are within applicants' preferred range for 24-hour and 48-hour Antimicrobial Durability.
Antimicrobial Efficacy of Composition a and Composition B Against Biofilms
[0213] Scope: The purpose of this study is to assess Composition A and Composition B susceptibility of biofilms using the MBEC Biofilm Assay.
[0214] Preparation of the Culture and Inoculum: Using a cryogenic stock (at −70° C.), a subculture P. aeruginosa (ATCC 15442), or S. aureus (ATCC 6538) or K. pneumoniae (ATCC 4352) was streaked on individual Tryptic Soy Agar (TSA) plates. and Incubated at 35±2° C. for 16 to 18 h. An isolated colony from each bacterial culture was aseptically removed from the streak plate to inoculate individual flasks containing 200 mL of sterile bacterial growth broth Tryptic Soy Broth (TSB). Each flask was Incubated at 35±2° C. and 150±10 rpm for 16 to 18 h. to achieve a viable bacterial density of ≥10.sup.8 CFU/mL for each culture and was checked by serial dilution and plating. 10 μL from each incubation flask was pipetted into individual flasks containing 100 mL of Tryptic Soy Broth (TSB) to adjust the inoculum to an approximate cell density of 10.sup.5 CFU/mL. Each diluted sample was vortexed for 10 seconds to achieve a homogeneous distribution of cells and a 10-fold serial dilution of teach inoculum was performed in triplicate. 20 μL of the serial dilutions from 10.sup.0 to 10−.sup.7 were spot plated on TSA plates and the plates incubated at 35±2° C. for 16 to 18 h and enumerated.
[0215] Growth of the Biofilm: 150 μL of the P. aeruginosa inoculum was added in Columns 1 through 8 (Rows A through D) and well D12 of “Growth Plate No. 1”; 150 μL of the K. pneumoniae inoculum was added in Columns 1 through 8 (Rows E through H) and well E12 of Growth Plate No. 1; 150 μL of the S. aureus inoculum was added in Columns 1 through 8 (Rows A through D) and well D12 of Growth Plate No. 2; and 150 ul of the mixed species inoculum (50 μL of the inoculums P. aeruginosa, and 50 ul of the K. pneumoniae inoculum, and 50 ul of the S. aureus inoculum) was added in Columns 1 through 8 (Rows E through H) and well E12 of Growth Plate No. 2. A corresponding peg lid was placed on each of Growth Plate No. 1 and Growth Plate No. 2. The completed MBEC Devices were placed on an orbital shaker set to 110±10 rpm and incubated in a humidified incubator at 35±2° C. for 16 to 18 h.
[0216] Biofilm Growth Check: After the incubation period, flame-sterilized pliers, were used to break off pegs D12 and E12 from the lid of Growth Plate No. 1, and pegs D12 and E12 from the lid of Growth Plate No. 2. Each peg was placed into a separate sterile microfuge tube that contained 1.0 mL of buffered water. Each peg-containing tube was placed in a stainless-steel tray and floated in a sonicator and sonicated on high for 30±5 min. Each tube was serially diluted by transferring 0.1 mL to sterile microfuge tubes containing 0.9 mL buffered water then spot plated on TSA. This served as a biofilm growth check.
[0217] Preparation pf the Challenge Plates: Challenge Plate No. 1: 200 μL of sterile TSB was added to well A12 which served as the device sterility control (SC), and 200 μL of sterile neutralizer was added to column 7 and well B12 which served as the device neutralizer toxicity control (N) and sterility control; and 165 μL of sterile neutralizer was added to column 6, followed by 35 ul of TA08.3 added to wells A6 and E6, and 35 ul of TA08.5 added to wells B6 and F6, and 35 ul of TA09.2 added to wells C6 and G6, and 35 ul of TA01.5 added to wells D6 and H6 all of which served as the neutralizer effectiveness control; and 200 μL of buffered water was added to column 8 and well C12 which served as the untreated control and buffered water sterility control (SC); and 200 ul of TA08.3 was added to Columns 1 through 5 (Row A and Row E); and 200 ul of TA08.5 was added to Columns 1 through 5 (Row B and Row F); and 200 ul of TA09.2 was added to Columns 1 through 5 (Row C and Row G); and 200 ul of TA01.5, was added to Columns 1 through 5 (Row D and Row H); Challenge Plate No. 2: 200 μL of sterile TSB was added to well A12 which served as the device sterility control (SC), and 200 μL of sterile neutralizer was added to column 7 and well B12 which served as the device neutralizer toxicity control (N) and sterility control (SC); and 165 μL of sterile neutralizer was added to column 6, followed by 35 ul of TA08.3 added to wells A6 and E6, and 35 ul of TA08.5 added to wells B6 and F6, and 35 ul of TA09.2 added to wells C6 and G6, and 35 ul of TA01.5 added to wells D6 and H6, all of which served as the neutralizer effectiveness control; and 200 μL of buffered water was added to column 8 and well C12 which served as the untreated control and buffered water sterility control (SC); 200 ul of TA08.3 was added to Columns 1 through 5 (Row A and Row E); and 200 ul of TA08.5 was added to Columns 1 through 5 (Row B and Row F); and 200 ul of TA09.2 was added to Columns 1 through 5 (Row C and Row G); and 200 ul of TA01.5, was added to Columns 1 through 5 (Row D and Row H).
[0218] Preparation of the Rinse-Plates: Rinse Plate No. 1 and Rinse Plate No. 2, were prepared by adding 200 μL of buffered water to each well of the Rinse Plates.
[0219] Preparation of the Recovery Plates: Recovery Plate No. 1 and Recovery Plate No. 2 were prepared by adding 100 μL of neutralizer to each well of the Recovery Plates.
[0220] Removal of Planktonic Cells: Planktonic cells from the biofilm that formed on the lids of Growth Plate No. 1 and Growth Plate No. 2 were removed by setting the lid from each Growth Plate into its corresponding Rinse Plate for 10 seconds.
[0221] Challenge of the Biofilm: After the 10 s rinse period, the lid from Growth Plate No. 1 was transferred from Rinse Plate No. 1 to Challenge Plate No. 1 and the lid from Growth Plate No. 2 was transferred from Rinse Plate No. 2 to Challenge Plate No. 2. After the transfer, each Challenge Plates was incubated on benchtop at room temperature for 17-minutes.
[0222] Disaggregation of the Biofilm: After the 17-minute contact time the lid from each Challenge Plate was transferred to its corresponding Recovery Plate and remained for 10 seconds to be neutralized. Following neutralization, each recovery plate with its MBEC lid was placed in a stainless-steel tray in the sonicator and sonicated on high for 30±5 min to remove and disaggregate the biofilm.
[0223] Qualitative Determination of the MBEC: After the sonication period, 100 μL of sterile TSB was added to each well of each Recovery Plate and each Recovery plate was covered with a new sterile non-pegged lid and placed in a humidified incubator at 35±2° C. for 24 h.
[0224] Qualitative Analysis and Results: Qualitative results were determined following the 24 h incubation of the recovery plates by visual scoring (±growth) based on the presence or absence of turbidity in the wells of the recovery plates. A minimum of 4 clear wells (CW) out of 5 total wells (TW) is acceptable for a passing criteria of a “Strong biofilm deactivator”, a minimum of 3 clear wells (CW) out of 5 total wells (TW) is acceptable for a passing criteria of a “Moderate biofilm deactivator” and a minimum of 2 clear wells (CW) out of 5 total wells (TW) is a passing criteria of a “weak biofilm deactivator.” The results of the evaluation are summarized in Table 13 below.
TABLE-US-00013 TABLE 13 Biofilm Deactivation Results @ 17-minute Contact Time PA SA KP MS Formulations CW/TW CW/TW CW/TW CW/TW TA08.3 3/5 4/5 4/5 3/5 TA08.5 4/5 4/5 5/5 4/5 TA09.2 1/5 35 3/5 1/5 TA01.5 2/5 1/5 2/5 2/5
[0225] The results reported in Table 13 demonstrate that the inventive compositions are within applicants' preferred range of “strong, moderate and weak biofilm deactivator.”
[0226] Biofilm Growth Inhibition Assay
[0227] Scope: The Biofilm Inhibition Assay was performed to determine the efficacy of representative formulations TA08.3 and TA08.5 and TA09.2, and TA01.5 to inhibit the adhesion, formation and the growth of isolated and mixed species biofilms formed by P. aeruginosa, K. pneumoniae, S. aureus and a mixed species. The assay was performed in duplicate at 24 and 48 hours.
[0228] Preparation of the Culture/Inoculum: An overnight culture at 35±2° C. in tryptic soy broth (TSB) was conducted an isolate and mixed species of the challenge microorganisms P. aeruginosa (ATCC 15442), or K. pneumoniae (ATCC 4532) or S. aureus (ATCC 6538), or a mixed species of the challenge microorganisms.
[0229] Preparation of the Test Plates: Using four sterile 96-well flat-bottom polystyrene microtiter plates, 35.5 ul of TA08.3 was dispensed in Columns 1-6 (Row A and Row E) of Test Plate No. 1 through Test Plates No. 4, and 35.5 ul of TA08.5 was dispensed into Columns 1-6 (Row B and Row F) of Test Plate No. 1 through Test Plate No. 4; and 35.5 ul of TA09.2 was dispensed into Columns 1-6 (Row C and Row G) of Test Plate. 1 through Test Plate No. 4; and 35.5 ul of TA01.5 was dispensed into Columns 1-6 (Row D and Row H) of Test Plate No. 1 through Test Plate No. 4. Test Plate No. 1 through Test Plate No. 4 were dried for two hours in an incubator at 35±2° C., to form a residue of the respective Compositions in the test wells. Excess Composition was removed by aspiration.
[0230] Growth of the Biofilm: After the 2-hour drying period, 164.5 ul of the P. aeruginosa cell suspension was inoculated in Column 1-6 (Row A through Row D) of Test Plate No. 1 and Test Plate No. 3, and 164.5 ul of the K. pneumoniae cell suspension was inoculated in Columns 1-6 (Row E through Row H) of Test Plate No. 1 and Test Plate No. 3; Positive Control wells in Column 7-9 (Row A through Row D) of Test Plate No. 1 and Test Plate No. 3 were inoculated with 164.5 ul of the P. aeruginosa cell suspension; and Positive Control wells in Columns 7-9 (Row E through Row H) of Test Plates No. 1 and Test Plate No. 3 were inoculated with 164.5 ul of the K. pneumoniae cell suspension; and Positive Control Wells in Column 7-9 (Rows A through Row D) of Test Plates No. 2 and Test Plate No. 4 were inoculated with 164.5 ul of the S. aureus cell suspension; and Positive Control Wells in Column 7-9 (Row E through Row H) of Test Plates No. 2 and Test Plate No 4 were inoculated with 164.5 ul of the mixed species suspension. Negative Control wells in Columns 10-12 (Row A through Row H) of Test Plate No. 1 through Test Plate No. 4 were inoculated with 164.5 ul of sterilized Tryptic Soy Broth. Test Plates No. 1 and Test Plate No. 3 were incubated at 35±2° C. for 24 hours. Test Plate No. 2 and Test No. 4 were incubated at 35±2° C. for 48 hours.
[0231] Removal of Planktonic Cells: Following the respective incubation periods, the test wells of Test Plate No. 1 through Test Plate No. 4 were gently washed 3 times with 200 ul phosphate buffered solution (PBS) and dried for 10 minutes in an inverted position.
[0232] Determination of Biofilm Adherence: Following the 10-minute drying period, each test well of Test Plate 1 through Test Plate No. 4 was stained with 50 ul of 0.1% crystal violet for 10 minutes at room temperature. Following the 10-minute staining period, each test well of Test Plate 1 through Test Plate No. 4 was gently washed 3 times with 200 ul distilled water and dried in an inverted position for 10 minutes. Following the drying period, 200 ul of 5% Acetic Acid was added to each test well of Test Plate No. 1 through Test Plate No. 4 to solubilize the stained biofilm mass.
[0233] Qualitative Analysis and Report: Qualitative results were determined following the 24 h or 48 h incubation of the test plates, by visual scoring, based on the color of the stain in the test wells. 5 out of 6 test wells for the organisms tested that appear to be ‘Clear (CW)” indicate that the Composition is a “Strong Biofilm Inhibitor”; 4 out of 6 test wells of the organisms tested that appear to have a “clear to light blue hue (CtB)”, indicate that the Composition is a “Moderate Biofilm Inhibitor”, and 3 out of 6 wells for the organisms tested that appear to have a “blue to violet hue (BtV)”, indicate the Composition is a “Weak Biofilm inhibitor”. The results of the evaluation are summarized in Table 14 and Table 15 below.
TABLE-US-00014 TABLE 14 Biofilm Inhibition Results 24-hour Incubation @ 35± 2° C. Formulations PA SA KP MS TA08.3 4/6 CW 5/6 CW 5/6 CW 4/6 CW TA08.5 5/6 CW 6/6 CW 6/6 CW 5/6 CW TA09.2 3/6 CtB 3/6 CtB 3/6 CtB 3/6 CtB TA01.5 4/6 CW 3/6 CW 3/6 CW 3/6 CW
TABLE-US-00015 TABLE 15 Biofilm Inhibition Results 48-hour Incubation @ 35± 2° C. Formulations PA SA KP MS TA08.3 4/6 CW 4/6 CW 5/6 CW 4/6 CW TA08.5 5/6 CW 6/6 CW 6/6 CW 6/6 CW TA09.2 3/6 CtB 3/6 CtB 3/6 CtB 2/6 CtB TA01.5 4/6 CW 3/6 CW 3/6 CW 3/6 CW
[0234] The results reported in Table 14 and in Table 15 clearly demonstrate the inventive compositions are within applicants’ preferred range of “Strong Biofilm Inhibitor to Weak Biofilm Inhibitor” and further demonstrates the antimicrobial efficacy of the present invention as discussed in the above specification.
[0235] Malodor Control Assay
[0236] Scope: Determine the effectiveness of representative compositions TA08.3, TA08.5, TA09.2 and TA01.5 to remove malodors produced by biofilms formed by isolates or mixed species of the challenge microorganisms P. aeruginosa (ATCC 15442) or K. pneumoniae (ATCC 4352) or S. aureus (ATCC 6538).
[0237] Preparation of the Inoculum: Overnight culture at 35±2° C. in trypticase soy broth (TSB) was conducted for an isolate of the challenge microorganism P. aeruginosa, or K. pneumoniae or S. aureus and an isolate of a mixed species of the challenge microorganisms.
[0238] Preparation of the Test Materials: Medium sized paper clip, 3/16 in×1.0 in stainless steel washers, and 4.0-ounce French-square bottles (the Test Bottles) containing 125 ml of Tryptic Soy Broth (TSB) were autoclaved for 15 min @ 121° C. Following the autoclave period, Test Bottles 1-4 were inoculated with 2 ul of P. aeruginosa (ATCC 15442) cell suspension; and Test Bottles 5-8 were inoculated with 2 ul of K. pneumoniae (ATCC 4538) cell suspension; and Test Bottles 9-12 were inoculated with 2 ul of S. aureus (ATCC 6538) cell suspension, and Test Bottles 13-16 were inoculated with 2 ul of the mixed species of the challenge microorganisms (PA+KP+SA) cell suspension and the paper clips were used to suspend the stainless steel washers in the inoculated broth in the test bottles. The test bottles were then incubated at 35±2° C. on an orbital shaker at 150±10 rpm overnight.
[0239] Test Procedure: After the incubation period, a stainless-steel washer was removed from each of Test Bottles 1-4 containing the P. aeruginosa biofilm into 8-ounce mason jars (Control Jars 1-4); and a stainless-steel washer was removed from each of Test Bottles 5-8 containing the K. pneumoniae biofilm into 8-ounce mason jars (Control Jars 5-8); and a stainless-steel washer was removed from each of Test Bottles 9-12 containing the S. aureus biofilm into 8-ounce mason jars (Control Jars 9-12); and a stainless-steel washer was removed from each Test Bottles 13-16 containing the Mixed species biofilm, into 8-ounce mason jars (Control jars 13-16). In like manner, the remaining stainless-steel washers were removed from Test Bottles 1-4 into 8-ounce mason jars (Challenge Jars 1-4) containing a solution of 532.5 ul of Composition TA08.3 and 75 ml of distilled water; and the remaining stainless-steel carriers were removed from Test Jars 5-8 into 8-ounce mason jars (Challenge Jars 5-8) containing a solution of 532.5 ul of Composition TA08.5 and 75 ml of distilled water; and the remaining stainless-steel washers were removed from Test Jars 9-12 into 8-ounce mason jars (Challenge Jars 9-12) containing a solution of 532.5 ul of Composition TA09.2 and 75 ml of distilled water; and the remaining stainless-steel washers were removed from Test Jars 13-16 into 8-ounce mason jars (Challenge Jars 13-16) containing a solution of 532.5 ul of Composition TA01.5 and 75 ml of distilled water. The stainless-steel washers remained in the Challenge Jars with the lids closed overnight at room temperature. Following the overnight incubation period, each of 3 panelists smelled the contents in a Control Jar and after 60 seconds smelled the contents of a corresponding Challenge Jar. There was a 3-minute wait time for each panelist between analyzing the smell in the successive Control and corresponding Challenge Jars. There was a wait time of 30 minutes before the next panelist sampled the Control and Challenge jars. The Panelists rated the smell in the Challenge Jar in comparison to the smell in the Control Jar using the Malodor Evaluation Criteria outlined in Table 16 below.
TABLE-US-00016 TABLE 16 Malodor Evaluation Criteria Rating Intensity Description of Zone 0 no Malodor No detectable malodor similar to the odor in the Control Jar 1 Moderate Malodor detected and similar to the Malodor odor in the Control Jar. 2 Strong Malodor easily detected and similar to Malodor the odor in the Control Jar. 3 Intense Malodor immediately detected and Malodor equal to the odor in the Control Jar.
A score of “0” (no malodor) indicates “Strong Malodor Removal”; a score of “1” (moderate malodor) indicates “Moderate Malodor Removal”; a score of “2” (Strong Malodor) indicates “Mediocre Malodor Removal”; and a score of “3” (intense malodor) indicates “inferior Malodor Removal”. The Results provided in Tables 17 below show the malodor scores for the Representative Formulas as determined by panelist.
TABLE-US-00017 TABLE 17 Panelist #1 TA08.3 TA08.5 TA09.2 TA01.5 Scores Scores Scores Scores P K S M P K S M P K S M P K S M 0 0 0 0 0 1 1 0 1 0 1 0 1 0 2 0 Panelist #2 TA08.3 TA08.5 TA09.2 TA01.5 Scores Scores Scores Scores S K S M P K S M P K S M P K S M 0 0 1 0 0 0 1 0 1 1 2 0 2 1 0 1 Panelist #3 TA08.3 TA08.5 TA09.2 TA01.5 Scores Scores Scores Scores P K S M P K S M P K S M P K S M 1 1 2 0 1 1 1 1 1 0 2 1 2 1 1 0
The results reported in Table 17 clearly demonstrate that 87.5% of the panelists' scores are within applicants' preferred range of a “Strong Malodor Removal to a Moderate Malodor Removal”, and further demonstrates the antimicrobial efficacy of the present invention as discussed in the above specification.
[0240] Minimal Essential Media Elution Assay
[0241] Scope: The MEM Elution Assay was performed to determine the cytotoxicity of composition TA08.3 or TA08.5 or TA09.2 or TA01.5 concentrate and the cytotoxicity of the composition-extraction-water from the fabrics treated with the compositions.
[0242] Assay Preparation: A layer of agar was added over an L929 Mouse Fibroblast cell monolayer to act as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable materials. The representative formulations or the composition-extraction-water from the representative formulations were then placed on top of the agar layer and incubated. The cell monolayers were examined and scored based on the degree of cellular destruction. All test method acceptance criteria were met. Testing was performed in compliance with US FDA good manufacturing practice (GMP) regulations 21 CFR Parts 210, 211 and 820.
[0243] Procedure: Six well cell culture plates were seeded with a verified quantity of industry standard L-929 cells (ATCC CCL-1) and incubated at 35±2° C. with 5±1% CO.sub.2 for no less than 24 hours, until confluency approaches 80-90%. The agar overlay consisted of an equal mixture of 1% noble agar and 2× Minimal Essential Media+10% bovine serum. The test articles were applied to clean or sterile filter discs testing no less than 0.1 mL per well. Positive and negative reference controls were included with each assay. All tests were performed using three test wells per test article. After the addition of the test articles, the cell culture plates were incubated as described above for 24-26 hours. The results from the three wells were averaged to give an average cytotoxicity score.
[0244] Analysis and Report: Following incubation, cells were evaluated microscopically using the evaluation criteria outline in Table 18 below.
TABLE-US-00018 TABLE 18 MEM Elution Valuation Criteria Grade Reactivity Description of Zone 0 None No detectable zone around or under the test article 1 Slight Some malformed or degenerate cells under the test article. 2 Mild Zone limited to area under the test article and less than 0.45 cm beyond the test article 3 Moderate Zone extends 0.45 to 1.0 cm beyond the test article. 4 Severe Zone extends greater than 1 cm beyond the test article.
TABLE-US-00019 TABLE 19 MEM Elution Control Results Scores Amount Identification #1 #2 #3 Avg Tested Negative Control: 0 0 0 0 ≥100 mm.sup.2 Polypropylene pellets per well Positive Control: 4 4 4 4 ≥100 mm.sup.2 Latex Natural Rubber per well
A grade from 0-1 for the “Composition Extraction-water” indicates the Composition has an “Exceptional Cytotoxicity Profile”. A grade of 2-3 for the Concentrated Composition indicates the Compositions have an “Acceptable Cytotoxicity Profile.” The test results for the Assay are summarized in Table 20 below:
TABLE-US-00020 TABLE 20 Scores Amount Formulation #1 #2 #3 Avg Tested TA08.3 1 1 1 1 ≥100 mm.sup.2 Rinse-Water per well TA08.5 1 1 1 1 ≥100 mm.sup.2 Rinse-Water per well TA09.2 1 1 1 1 ≥100 mm.sup.2 Rinse-Water per well TA01.5 1 1 1 1 ≥100 mm.sup.2 Rinse-Water per well TA08.3 3 3 3 3 ≥100 mm.sup.2 Concentrate per well Ta08.5 3 3 3 3 ≥100 mm.sup.2 Concentrate per well TA09.2 3 3 3 3 ≥100 mm.sup.2 Concentrate per well TA01.5 3 3 3 3 ≥100 mm.sup.2 Concentrate per well
[0245] The results reported in Table 20 clearly demonstrate the inventive compositions are well within applicants preferred rang of “Excellent Cytotoxicity Profile” for the composition extraction-water and an “Acceptable Cytotoxicity Profile” for the Concentrated Composition, and further demonstrates the safety credentials of the inventive compositions.
[0246] ASTM D 4265 Standard Guide for Evaluating Stain Removal Performance in Home Laundering
[0247] Scope: Representative formulations TA08.3 and TA08.5 and TA09.2 were evaluated for stain removal on 3″×4″ 100% Cotton fabric swatches having 101×77 threads per sq. in., and 3″×4″ 50/50 polyester-cotton fabric swatches having 89×57 threads per sq. in. The test fabrics were purchased from Scientific Services S/D (Sparrowbush, N.Y.) and were soiled/stained with dust-sebum, or barbecue sauce, or chocolate ice cream, or grape juice, or ground-in-clay, or coffee, or black Ink, or grass.
[0248] Laundry Conditions: The test conditions were fixed within the range of a traditional household laundry program. All washings were performed in a top-loading portable washing machine (Zeny Model H01-1669A). The fabric load was 3.5 lbs. consisting of white cotton T-shirts and the soiled/stained fabric swatches. The washing machine was set on “soft-setting”; the cold-water-wash-cycle time was 17-minutes at a temperature of 16±2° C. followed by a high spin speed, and a cold-water-rinse cycle at a temperature of 16±2° C. Water quantity was 1.25 gallons with 400 ppm Hard water. The concentration of Representative formulations TA08.3 or TA08.5 or TA09.2 was 1.20 ounces.
[0249] Order of Addition to the Wash-Cycle: 4 gal of water were added to the machine, followed by 1.5 ounces of Representative formula TA08.3 or TA08.5 or TA09.2, and the test fabrics; the machine wash-cycle was started.
[0250] Drying the Fabrics: After washing, the fabric load was dried in a standard residential dryer (Amana Model: NED4655EW1) on high heat for 20 minutes. After drying, the fabric-samples sat in ambient conditions, out of direct sunlight, for 24 hours.
[0251] Analysis and Report: After the 24-hour period, the L, a*, b* values for all samples, including the unstained fabric samples were measured using a BELEY Colorimeter and the Stain Removal Index (SRI) was calculated using the following equation: SRI=100−[(L.sub.C−L.sub.W).sup.2+(a.sub.C−a.sub.W).sup.2+(b.sub.C b.sub.W).sup.2].sup.1/2, where: L=Reflectance, a=redness/greenness, b=yellowness/blueness, c=unstained fabric cleaned in treatment conditions, w=stained fabric cleaned in the treatment conditions. The SRI value range is from 0 to 100, with a value of 70-79 indicating “minor soil/stain removal”, and a value of 80-89 indicating “moderate soil/stain removal, and a value of 90-99 indicating “exceptional soil/stain removal”, and a value of 100 indicating “complete soil/stain removal”.
[0252] Results: The results of the Stain removal Assay are reported in Table 21 below.
TABLE-US-00021 TABLE 21 TA08.3 TA08.5 TA09.2 SRI Values Stain Type C P/C C P/C C P/C Dust Sebum 77.82 89.95 78.25 87.69 88.03 94.55 Barbecue Sauce 81.85 89.39 90.69 90.79 88.01 94.83 Chocolate 79.52 81.93 77.47 87.14 90.06 96.33 Ice Cream Grape Juice 74.19 90.97 78.48 86.80 79.03 94.17 Ground- 85.34 90.93 84.92 89.74 81.15 91.35 in-Clay Coffee 73.91 79.55 76.01 82.98 81.80 85.40 Black Ink 76.31 83.99 84.99 82.64 79.09 81.30 Grass 77.25 84.70 78.87 85.79 81.88 94.71
As can be seen from the results reported in Table 21, the reported SRI values indicate a range of detergency from “acceptable soil/stain removal” to “exceptional soil/stain removal” for the Representative Formulations of Composition A of the present invention and are well within the applicant's preferred ranges of detergency.
Treatment Methods for Fabrics and the Abiotic Surfaces in the Washing Machine During the Laundry Program
[0253] The present invention provides treatment methods for using the embodiments of Composition A or Composition B or Composition C discretely or synergistically during the laundry program to sanitize, disinfect and reactively remove malodors from fabrics, and impart antimicrobial durability into the fabrics that continues after the laundry program for 24-48 hours, and to deactivate and prevent the growth of biofilms in the Washing Machine.
General Treatment Methods and Uses of the Antibacterial Fabric Care Compositions in the Laundry Program
[0254] The antimicrobial liquid laundry detergent embodiments of Composition A are preferably applied during the wash-cycle of the laundry program to reactively remove malodors and sanitize or disinfect the fabrics and to deactivate and prevent the formation of biofilms on the abiotic surfaces in the WM and to impart antimicrobial durability into the fabrics that continues for 24-48 after the laundry program,
[0255] The antimicrobial liquid fabric softener embodiments of Composition B are preferably applied during the rinse-cycle of the laundry program to reactively remove malodors and sanitize the fabrics and to remove and to prevent the formation of biofilms on the abiotic surfaces in the WM and to impart antimicrobial durability into the fabrics that continues for 24-48 after the laundry program.
[0256] The antimicrobial fabric softener sheet embodiments of Composition C, are preferably applied during the drying-cycle of the laundry program to reactively remove malodors, and sanitize the fabrics and to impart antimicrobial durability into the fabrics that continues for 24-48 after the laundry program.
Treatment Methods to Sanitize, Disinfect and Impart Antimicrobial Durability into the Fabrics During the Wash-Cycle of the Laundry Program:
[0257] Use of an embodiment of Composition A to treat fabrics during the wash-cycle of the laundry program reactively removes malodors and sanitizes or disinfects the fabrics when the fabrics are treated during the wash-cycle of the laundry program in accordance with the dosage instructions provided in Table 22, below.
TABLE-US-00022 TABLE 22 Formulations TA08.3 and TA08.5 of Composition A Dosage (ounces) To To Sanitize Disinfect and and Weight Water remove Remove Fabric Type (lbs.) (gals.) Malodors Malodors Cotton Fabrics 2.0 0.70 0.50 0.75 and Cotton/ 5.0 1.80 1.00 1.50 Polyester 7.0 2.50 1.60 2.25 Blends 10.0 3.50 2.25 3.25 12.0 4.20 2.55 4.00 15.0 5.30 3.25 5.00 17.0 6.00 3.75 5.50 20.0 7.00 4.50 6.50 25.0 8.70 5.50 8.00
Treatment Methods to Sanitize and to Imparts Antimicrobial Durability into the Fabrics During the Rinse-Cycle of the Laundry Program
[0258] Use of an embodiment of Composition B to treat fabrics during the rinse-cycle of the laundry program reactively removes malodors and sanitizes the fabrics when the fabrics are treated during the rinse-cycle of the laundry program in accordance with the dosage instructions provided in Table 23, below.
TABLE-US-00023 TABLE 23 Representative Formula TA01.5 of Composition B Dosage (ounces) Weight Water To Sanitize and Fabric Type (lbs.) (gals.) Remove Malodors Cotton Fabrics 2.0 0.70 0.75 and Cotton/ 5.0 1.80 1.50 Polyester 7.0 2.50 2.25 Blends 10.0 3.50 3.25 12.0 4.20 4.00 15.0 5.30 5.00 17.0 6.00 5.50 20.0 7.00 6.50 25.0 8.70 8.00
Treatment Methods to Disinfect Fabrics and Deactivate and Prevent Biofilm Formation in the WM and Impart Antimicrobial Durability into the Fabrics During the Wash Cycle of the Laundry Program.
[0259] Use of an embodiment of Composition A to treat fabrics during the wash-cycle of the laundry program reactively removes malodors, disinfects fabrics, and deactivates biofilms and prevents the formation of biofilms in the WM when the fabrics are treated during the wash cycle of the laundry program in accordance with the dosage instructions provided in Table 24, below.
TABLE-US-00024 TABLE 24 Formulations TA08.3 and TA08.5 of Composition A Dosage (ounces) To Disinfect, Remove Weight Water Malodors and Fabric Type (lbs.) (gals.) Deactivate Biofilms Cotton Fabrics 2.0 0.70 1.00 and Cotton/ 5.0 1.80 1.75 Polyester 7.0 2.50 2.50 Blends 10.0 3.50 3.50 12.0 4.20 4.25 15.0 5.30 5.25 17.0 6.00 5.75 20.0 7.00 6.75 25.0 8.70 8.25
General Synergistic Method to Remove Malodors and Sanitize or Disinfect Fabrics and Impart Antimicrobial Durability into the Fabrics During the Laundry Program
[0260] Use of an embodiment of Composition A to treat fabrics during the wash-cycle of the laundry program and use of an embodiment of Composition B to treat fabrics during the rinse-cycle of the same laundry program work synergistically to reactively remove malodors, sanitize, or disinfect the fabrics and to impart antimicrobial durability into the fabrics that continues for 24-48 hours after the laundry program. More specifically, the unused antimicrobial efficacy from an embodiment of Composition A used in the wash-cycle passes-through to the rinse-cycle and synergizes with the unused antimicrobial efficacy from an embodiment of Composition B to reactively remove malodors and sanitize or disinfect the fabrics and impart antimicrobial durability into the fabrics that continues after the laundry program for 24-48 hours.
General Synergistic Methods to Deactivate or Prevent Biofilm Formation in the WM During the Wash-Cycle or Rinse-Cycle and Impart Antimicrobial Durability into the Fabrics
[0261] Use of an embodiment of Composition A to treat fabrics during the wash-cycle and use of an embodiment of Composition B to treat fabrics during the rinse-cycle of the same laundry program works synergistically to reactively remove malodors, disinfect the fabrics, deactivate existing biofilms, prevent the formation of new biofilm, and to impart a 24 to 48-hour Antimicrobial Durability into the fabrics. More specifically, the unused antimicrobial efficacy from an embodiment of Composition A used in the wash-cycle can pass-through to the rinse-cycle and synergize with an embodiment of Composition B to reactively remove malodors and disinfect the fabrics and deactivate existing biofilms, prevent the formation of new biofilms, and impart Antimicrobial Durability into the fabrics that continues after the laundry program for 24-48 hours.
Synergistic Method to Sanitize and Impart an Antimicrobial Durability into the Fabrics During the Laundry Program
[0262] Use of an embodiment of Composition A to treat fabrics during the wash-cycle of the laundry program and use of an embodiment of Composition B to treat fabrics during the rinse-cycle of the same laundry program work synergistically to reactively remove malodors from fabrics, and sanitize fabrics and to impart Antimicrobial Durability into the fabrics that continues after the laundry program for up to 24 hours when the fabrics are treated during the wash-cycle of the laundry program and the fabrics are treated during the rinse cycle of the same laundry program in accordance with the dosage instructions presented in Table 25, below.
TABLE-US-00025 TABLE 25 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.5 of Composition B. Dosage (Ounces) Weight Water Composition Composition Fabric Type (lbs.) (gals.) A B Cotton 2.0 0.70 0.50 0.75 Fabrics and 5.0 1.80 1.00 1.50 Cotton/ 7.0 2.50 1.60 2.25 Polyester 10.0 3.50 2.25 3.25 Blends 12.0 4.20 2.55 4.00 16.0 5.30 3.25 5.00 17.0 6.00 3.75 5.50 20.0 7.00 4.50 6.50 25.0 8.70 5.50 8.00
[0263] When an extra rinse cycle is programmed to occur between the wash-cycle containing an embodiment of Composition A and the final-rinse-cycle containing an embodiment of Composition B, less than an appreciable amount of the unused antimicrobial efficacy of an embodiment of Composition A will pass-through into the final-rinse—cycle. Accordingly, a method to synergistically remove malodors from fabrics, and sanitize fabrics and impart Antimicrobial Durability into the fabrics that continue after the laundry program for up to 24 hours consists of treating the fabrics during the wash-cycle with an embodiment of Composition A and treating the fabrics during the final-rinse-cycle of the same laundry program with an embodiment of Composition B in accordance with the dosage instructions presented in Table 26 below.
TABLE-US-00026 TABLE 26 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.3 of Composition B. Dosage (Ounces) Fabric Weight Water Composition Composition Type (lbs. (gal.) A B Cotton 2.0 0.70 0.75 0.75 Fabrics 5.0 1.80 1.50 1.50 and 7.0 2.50 2.25 2.25 Cotton/ 10.0 3.50 3.25 3.25 Polyester 12.0 4.20 4.00 4.00 Blends 16.0 5.30 5.00 5.00 17.0 6.00 5.50 5.50 20.0 7.00 6.50 6.50 25.0 8.70 8.00 8.00
Synergistic Method to Disinfect and Impart Antimicrobial Durability into the Fabrics During the Laundry Program
[0264] Use of an embodiment of Composition A during the wash-cycle of the laundry program and use of an embodiment of Composition B during the rinse-cycle of the same laundry program works synergistically to reactively remove malodors from fabrics, and disinfect fabrics and impart Antimicrobial Durability into the fabrics that continues after the laundry program for up to 48-hours when the fabrics are treated during the wash-cycle of the laundry program and the fabrics are treated during the rinse cycle of the same laundry program in accordance with the dosage instructions presented in Table 27, below.
TABLE-US-00027 TABLE 27 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.5 of Composition B. Dosage (Ounces) Fabric Weight Water Composition Composition Type (lbs.) (gals.) A B Cotton 2.0 0.70 1.00 0.75 Fabrics 5.0 1.80 1.75 1.50 and 7.0 2.50 2.50 2.25 Cotton/ 10.0 3.50 3.50 3.25 Polyester 12.0 4.20 4.25 4.00 Blends 16.0 5.30 5.25 5.00 17.0 6.00 5.75 5.50 20.0 7.00 6.75 6.50 25.0 8.70 8.25 8.00
[0265] When an extra rinse cycle is programmed to occur between the wash-cycle of the laundry program containing an embodiment of Composition A and the final-rinse-cycle of the same laundry program containing an embodiment of Composition B, less than an appreciable amount of the unused antimicrobial efficacy of Composition A will pass-through into the final-rinse-cycle of the same laundry program containing an embodiment of Composition B. Accordingly, a method to synergistically remove malodors from fabrics, and disinfect fabrics and impart Antimicrobial Durability into the fabrics that continues after the laundry program for 24 48 hours consists of treating the fabrics during the wash-cycle of the laundry program with an embodiment of Composition A, and treating the fabrics during the final-rinse-cycle of the same laundry program with an embodiment of Composition B in accordance with the dosage instructions provided in Table 28, below.
TABLE-US-00028 TABLE 28 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.3 of Composition B. Dosage (Ounces) Fabric Weight Water Composition Composition Type (lbs.) (gals.) A B Cotton 2.0 0.70 1.00 1.00 Fabrics 5.0 1.80 1.75 1.75 and 7.0 2.50 2.50 2.50 Cotton/ 10.0 3.50 3.50 3.50 Polyester 12.0 4.20 4.25 4.25 Blends 16.0 5.30 5.25 5.25 17.0 6.00 5.75 5.75 20.0 7.00 6.75 6.75 25.0 8.70 8.25 8.25
A Synergistic Method to Remove and Prevent Biofilm Formation in the WM and Impart Antimicrobial Durability into the Fabrics During the Laundry Program
[0266] A method to reactively remove malodors and disinfect fabrics, and deactivate existing biofilms in the WM and prevent the formation of new biofilms in the WM, and impart Antimicrobial Durability into the fabrics that continues after the laundry program for 24-48 hours, consists of treating the fabrics and the abiotic surfaces in the WM during the wash-cycle of the laundry program with an embodiment of Composition A and treating the fabrics and the abiotic surfaces in the WM during the rinse-cycle of the same laundry program with an embodiment of Composition B in accordance with the dosage instructions presented in Table 29, below.
TABLE-US-00029 TABLE 29 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.3 of Composition B. Dosage (Ounces) Fabric Weight Water Composition Composition Type (lbs.) (gals.) A B Cotton 2.0 0.70 1.00 1.00 Fabrics 5.0 1.80 1.75 1.75 and 7.0 2.50 2.50 2.50 Cotton/ 10.0 3.50 3.50 3.50 Polyester 12.0 4.20 4.25 4.25 Blends 16.0 5.30 5.25 5.25 17.0 6.00 5.75 5.75 20.0 7.00 6.75 6.75 25.0 8.70 8.25 8.25
[0267] When an extra rinse-cycle is programmed to occur between the wash-cycle of the laundry program containing an embodiment of Composition A and the final-rinse-cycle of the same laundry program containing an embodiment of Composition B, less than an appreciable amount of the unused antimicrobial efficacy of Composition A will pass-through into the final-rinse-cycle of the same laundry program containing an embodiment of Composition B. Accordingly, a method to synergistically remove malodors from the fabrics, disinfect the fabrics, deactivate existing biofilms in the WM, prevent the formation of new biofilms in the WM and impart Antimicrobial Durability into the fabrics that continues after the laundry program for 24-48-hours consists of treating the fabrics during the wash-cycle of the laundry program with an embodiment of Composition A, and treating the fabrics during the final-rinse-cycle of the same laundry program with an embodiment of Composition B in accordance with the dosage instructions provided in Table 30, below.
TABLE-US-00030 TABLE 30 Formulations TA08.3 or TA08.5 of Composition A used Synergistically with Formulation TA01.3 of Composition B. Dosage (Ounces) Fabric Weight Water Composition Composition Type (lbs.) (gals.) A B Cotton 2.0 0.70 1.25 1.00 Fabrics 5.0 1.80 2.00 1.75 and 7.0 2.50 2.75 2.50 Cotton/ 10.0 3.50 3.75 3.50 Polyester 12.0 4.20 4.50 4.25 Blends 16.0 5.30 5.50 5.25 17.0 6.00 6.00 5.75 20.0 7.00 7.00 6.75 25.0 8.70 8.50 8.25
Methods to Investigate the Mechanism of Action of Antimicrobial Laundry Compositions
[0268] Scope: To examine the mechanism of action and antimicrobial effects, compositions are subjected to cellular, molecular and biophysical analysis using S. aureus ATCC 9144, E coli K-12, Salmonella enterica serovar Typhimurium LT2, Pseudomonas aeruginosa CDC 0248 and acid-fast Mycobacterium smegmatis MC2155 as model bacterial cell systems. Cell membrane activities are examined using composition-fluorophore conjugates together with cell growth, microscopy and physiological assays. Composition/nucleic acid interactions are also be examined using biophysical methods.
Determine the Activity of the Test Compositions on the Bacterial Cell Membrane
[0269] Scope: If the antibacterial activity of the Test Composition is due solely to membrane disruption, it would be expected to permeabilize bacterial cell barriers at growth inhibitory and sub growth inhibitory concentrations. To assess cell membrane activities of the Test Composition, establish the minimal inhibitory concentration and time kill properties of the Test Composition against S. aureus ATCC 9144, E coli K-12, Salmonella enterica serovar Typhimurium LT2, and Pseudomonas aeruginosa CDC 0248. Each bacterial system should be grown in Mueller Hinton Broth at 37° C. overnight. Cells are then examined using bright field light microscopy to determine if the Test Articles permeated the cell membrane.
[0270] Minimum Inhibitory Concentration Assay: Determine MIC's by serial dilution of the Test Composition in 200 ul MHB containing 10.sup.5 (CFU)/ml of the test organism using 96-well plates. Incubate the plates for 18-hours at 37° C. in a BMG Labtech Spectrophotometer with shaking for 5 seconds every 5 minutes followed by record absorbance at 550 nm. The MIC is scored as the lowest concentration of the Test Composition at which no-growth is observed.
[0271] Minimum Bactercidal Concentration: To determine the minimal bactericidal concentration (MBC>10.sup.3 CFU/ml reduction), bacterial cultures at 10.sup.5 CFU/ml were treated or not treated with the Test Composition and at the 0, 1, 2, 4 and 8-hour time-points samples were serially diluted and plated on LB agar. CFUs are counted after 18 hours of incubation at 37° C.
Fractional Inhibitory Concentrations are Determined Using the Synergy Measurement by Checkerboard Assay Using Hoechst 33258.
[0272] Bacteria Cell Membrane Permeability Assay: Transfer E-coli K-12 mid-log phase (10 μl of culture, OD.sub.600 adjusted to 0.1) to 96-well plates containing the Test Composition, polymyxin B or triclosan (0-8 μg/mL) in 100 μL phosphate buffered solution (PBS), and incubate in a spectrophotometer with shaking for 5 seconds every 5 minutes. To generate cells with maximum permeability to SYTOX Green, incubate untreated cultures for 10 minutes in a heating block maintained at 70° C. Add the dye SYTOX Green to a final concentration of 1 μM, and monitor changes in florescence emission at 575 nm upon excitation at 485 nm using a Multi label counter. SYTOX Green fluoresces strongly upon binding to DNA, and fluorescence is taken as an indication of membrane permeabilization.
[0273] Assess Cell Barrier Damage: To further assess cell barrier damage that might be invisible to microscopy, grow fresh cultures of the model bacterial systems to mid-log phase and treat with the Test Composition or with Polymyxin B, in the presence of the fluorescent membrane integrity probe SYTOX® Green, and then monitor using fluorimetry. SYTOX® Green is useful as an indicator of membrane damage because it is normally excluded from intact bacteria and its fluorescence quantum yield increases upon DNA binding. Therefore, intact bacteria are expected to display low fluorescence, and fluorescence is expected to increase following cell barrier damage. If the results from the activity of the Test Composition on the bacterial cell membrane indicate an increase in fluorescence quantum yield, then the primary target of the Test Composition is not exclusively cell barriers and the mechanism of action is likely internal which would require cell entry.
[0274] Bacteria Cell Entry Assay: To test for bacterial cell entry, synthesize the Test Composition with FITC by creating three solutions containing 2 mg Fluorescein isothiocyanate (FITC) dissolved in 800 μL dimethyl formamide, and 50 μL of N, N-diisopropylethylamine, and combine each solution with 200 μL of the Test Composition and shake overnight at room temperature. Dialyze the resulting solution using a molecular weight cut off membrane (MWCO) 3.5 KDa against 50% aqueous ethanol for 5 days with intermittent change of dialysate (10 times, 500 mL), lyophilize to obtain Test Composition-FITC. Confirm that the terminal amino groups in the Test Composition are chemically conjugated with FITC; confirm the formation of the thiourea bond by infrared spectroscopy, IR (Nujol), v (cm.sup.−1): 756 cm.sup.−1 (C═S stretching).
[0275] Assess Uptake: Assess uptake into the model bacterial systems using microscopy and flow cytometry by treating overnight cultures of the model bacterial systems with the Test Composition-FITC conjugate (2 μg/ml) for 90 minutes. Quantify the number of cells scoring positive for cell-associated fluorescence using flow cytometry. Treat cultures of each model bacteria system (10.sup.8 CFU/ml) with the Test Composition-FITC conjugate (0-6 μg/ml) at 4° C. or 37° C. and measure cell associated fluorescence by fluorimetry. Treat cultures of each Test Composition (10.sup.8 CFU/ml) with the Test Composition-FITC conjugate (4 μg/ml) at 37° C. for 90 minutes, stain with DAPI and observe by using epifluorescence microscopy. Observe cell movement at 0. 10, 20 and 30 second time points. To examine cell localization more thoroughly treat Bacillus megaterium cultures with the Test Composition-FITC conjugate, counter stain with wheat germ agglutinin (WGA-red) and examine by fluorescence microscopy.
[0276] Observe and Report: the effects of Test Composition, heat, Polymyxin B, (positive control) and triclosan (negative control) on cell permeability to SYTOX Green, plotting on a graph the RFU and MIC values for the Test Composition; the fluorescence microscopy results of each bacteria system untreated and treated with Test-Composition-FITC counterstained with DAPI; a confocal image showing localization of Test Composition-FITC (green) in B. Megaterium, and bacteria counterstained with the membrane localizing probe wheat-germ agglutinin (WGA) conjugated to Alexa Fluor-555 (red) and visualized as live and fixed cells, Bar=5 μm; a fluorescence intensity profile plot analysis (mean fluorescence intensity v. width of the cell in μm) of cellular localization of Test Composition-FITC and WGA fluorescence depicting the cross section used for analysis, the level of Test Composition-FITC and position within the cell, and the level of Test Composition-FITC and position within the membrane.