COMPOSITION INCLUDING I2SCN- IONS AND/OR I(SCN)2- IONS
20200361779 · 2020-11-19
Assignee
Inventors
- Françoise BAFORT (Saint-Servais, BE)
- Mohamed Haïssam JIJAKLI (Scy, BE)
- Jean-Paul PERRAUDIN (Bruxelles, BE)
Cpc classification
C12Y111/01007
CHEMISTRY; METALLURGY
C07K14/79
CHEMISTRY; METALLURGY
A01N59/24
HUMAN NECESSITIES
International classification
A01N59/24
HUMAN NECESSITIES
Abstract
A stable composition obtained by enzymatic oxidation of a halide thiocyanate mixture, including at least one ion selected from the group of the I2SCN ions and the I(SCN).sub.2 ions, the composition being free of hypothiocyanite ions. In an embodiment, the composition further includes iodine thiocyanate ISCN. This disclosure also relates to a method for preparing the stable composition and to the uses thereof.
Claims
1. A stable composition obtained by enzymatic oxidation of a halide thiocyanate mixture, comprising at least one ion selected from the group consisting of the I.sub.2SCN ions and the I(SCN).sub.2 ions, said composition being free of hypothiocyanite ions (OSCN).
2. The stable composition according to claim 1, further comprising iodine thiocyanate ISCN.
3. The stable composition according to claim 1, further comprising at least one compound selected from the group consisting of lactoferrin, lysozyme, immunoglobulins, and growth factors.
4. A method for manufacturing a stable composition according to claim 1, comprising: a step A of preparation of a reaction medium comprising at least two substrates, at least one oxidizing agent, and a catalyst, the bringing together of said catalyst and said oxidizing agent being contingent upon the bringing together of said two substrates; a reaction step B starting with the bringing together of said oxidizing agent and said catalyst; a step C of removal of said catalyst, and of recovery of a composition according to the invention comprising at least one of the I.sub.2SCN ions and/or of the ions I(SCN).sub.2 ions; said substrates being halide (X) and thiocyanate (SCN) ions, said oxidizing agent being a hydrogen peroxide (H.sub.2O.sub.2) generating system and/or hydrogen peroxide, the catalyst being at least one peroxidase, wherein the reaction step has a duration from 30 to 1800 seconds and in that it does not give rise to the formation of hypothiocyanite ion (OSCN).
5. The method according to claim 4, further comprising a step of lyophilization of the composition at the end of which a lyophilisate is obtained, said lyophilisate enabling, during a redissolution, the reconstitution of said composition which includes at least one of the I.sub.2SCN ions and/or of the I(SCN).sub.2 ions and is free of hypothiocyanite ion (OSCN).
6. The method according to claim 4, wherein the halide ion (X) is selected from the group consisting of the iodide ion (I), the bromide ion (Br), and the chloride ion (Cl).
7. The method according to claim 4, wherein the halide ion (X) is the iodide ion (I).
8. The method according to claim 4, wherein the ratio between the molar concentration of thiocyanate ion (SCN) and the molar concentration of iodide ion (I) is greater than 1.
9. The method according to claim 4, wherein the pH of the solution is from 4 to 8.
10. The method according to claim 4, wherein the contact time is from 30 to 200 seconds.
11. The method according to claim 4, wherein the peroxidase is selected from the group consisting of the lactoperoxidase (LP), the thyroid peroxidase (TPO), the myeloperoxidase (MPO), the salivary peroxidase (SPO) and the eosinophil peroxidase (EPO).
12. The method according to claim 4, wherein the peroxidase is the lactoperoxidase (LP).
13. The method according to claim 4, wherein said peroxidase has a concentration from 1 mg/L to 500 mg/L.
14. The method according to claim 4, wherein the thiocyanate ion (SCN) is present at a molar concentration from 0.1 mM to 1 M.
15. The method according to claim 4, wherein the halide ion (X) is the iodide ion (I) and is present at a molar concentration from 0.1 mM to 1 M.
Description
DESCRIPTION OF THE FIGURES
[0220]
[0221]
[0222]
[0223]
[0224]
[0225]
[0226]
[0227]
[0228]
[0229]
[0230]
[0231]
[0232]
[0233] La
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
[0243]
[0244]
[0245]
[0246]
[0247] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture have been described in the prior art, notably in EP1349457 or WO2016026946. Mixtures prepared in water according to the operating procedures described in these patent applications did not make it possible to obtain an ion selected from the group consisting of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup. ions.
[0248]
[0249] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture have been described in the prior art, notably in EP1349457 or WO2016026946. Mixtures prepared in a citrate buffer 100 mM pH 5.5 according to the operating procedures described in these patent applications did not make it possible to obtain an ion selected from the group consisting of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup. ions. For greater clarity, the signals corresponding to the carbons of the sodium citrate have been eliminated from the spectrum (see
[0250]
[0251] The sodium citrate contains 4 carbons visible in NMR with the following chemical shifts: C.sub.1 appears at 180.45 ppm, C.sub.2 appears at 175.94 ppm, C.sub.3 appears at 73.43 ppm and C.sub.4 appears at 44.64 ppm. The thiocyanate contains 1 carbon, visible at the chemical shift of 133.48 ppm.
[0252]
[0253] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture have been described in the prior art, notably in EP1349457 or WO2016026946. Mixtures prepared in a phosphate buffer 100 mM pH 7.4 according to the operating procedures described in these patent applications did not make it possible to obtain an ion selected from the group consisting of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup. ions.
[0254]
[0255] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture in the ideal ratio of 4.5 (KI with respect to KSCN) in an aqueous matrix (of the slightly mineralized spring water, moderately mineralized spring water, highly mineralized spring water or tap water type), irrespective of the mineral composition of the aqueous matrix, did not make it possible to obtain an ion selected from the group consisting of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup. ions.
[0256]
[0257] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture have been described in the prior art, notably in EP1349457 or WO2016026946. Mixtures prepared in water according to the operating procedures described in these patent applications include hypothiocyanite ions.
[0258]
[0259] Compositions obtained by enzymatic oxidation of a halide thiocyanate mixture have been described in the prior art, notably in EP1349457 or WO2016026946. Mixtures prepared in a citrate buffer 100 mM pH 5.5 according to the operating procedures described in these patent applications include hypothiocyanite ions.
[0260]
[0261] The concomitant presence of the LP and of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup. ions causes a degradation of the I.sub.2SCN.sup. and/or I(SCN).sub.2.sup.X ions with a decrease of the signal at 49 ppm after 1 h (slight decrease), 3 h of incubation (pronounced decrease of the signal), and total disappearance of the signal after 48 h.
[0262]
[0263]
[0264]
[0265]
[0266]
[0267] Circle-shaped dots: Control
[0268] Square-shaped dots: Solution A corresponding to the commercial product (Chlorhexide gluconate 2%)
[0269] Triangular-shaped dots: Solution B corresponding to the composition according to the invention, immobilized on wipes
[0270] From the curve closest to 0 ATP/CFU at 24 hours of treatment, and going up to the control curve defined by the circles-shaped points, corresponding to 1 ATP/CFU:
[0271] Lactobacillus acidophilus in the presence of solution B
[0272] Veillonella alcalescens in the presence of solution B
[0273] Fusobacterium nucleatum in the presence of solution B
[0274] Streptococcus mutans in the presence of solution B
[0275] Actinomyces viscosus in the presence of solution B
[0276] Streptococcus mutans in the presence of solution A
[0277] Actinomyces viscosus in the presence of solution A
[0278] Fusobacterium nucleatum in the presence of solution A
[0279] Veillonella alcalescens in the presence of solution A
[0280] Lactobacillus acidophilus in the presence of solution A
[0281]
[0282]
[0283]
[0284] A composition according to the invention was prepared by bringing together of 5.4 mM of potassium iodide (KI), 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) in a sodium citrate buffer 100 mM pH 6.2 (
[0285]
EXAMPLES
Example 1
Preparation of Compositions According to the Invention
[0286] In general, a certain number of compositions according to the invention were prepared under different conditions: [0287] of buffers/concentrations of buffer/pH; [0288] of pH in the absence of a buffer (in water); [0289] of I.sup./SCN.sup. ratios, [0290] of concentration of peroxidase.
[0291] The compositions according to the invention are prepared according to the general protocol described below, said protocol being accessible to the person skilled in the art without further explanation.
[0292] A first solution comprising iodide ions (I.sup.) at an appropriate molar concentration is prepared. In parallel, a second solution comprising thiocyanate (SCN.sup.) ions at an appropriate molar concentration is prepared. In parallel, a third solution of hydrogen peroxide at an appropriate molar concentration (namely the sum of the two preceding molar concentrations) is prepared.
[0293] In parallel, a teabag comprising lactoperoxidase (LP) is prepared.
[0294] The teabag is immersed in water or a buffered aqueous solution.
[0295] The first two solutions (comprising the iodide ions and the thiocyanates ions, respectively) are added to the water or the aqueous solution comprising the tea bag.
[0296] The third solution (comprising the hydrogen peroxide) is added to the mixture.
[0297] After approximately 60 seconds of presence simultaneously of the three solutions (comprising the iodide ions, the thiocyanate ions and the H.sub.2O.sub.2, respectively), and the lactoperoxidase (LP) is removed by means of the teabag.
[0298] After removal of the lactoperoxidase (LP), several analyses can be carried out on the products of the oxidation reaction: [0299] .sup.13C NMR analysis; [0300] Measurement of the oxidizing activity of the SH groups [0301] Measurement of the oxidizing activity of the NH.sub.2 groups
[0302] In the context of the present application, .sup.13C NMR was used for identifying and quantifying the ions. In addition, it was used to confirm that no hypothiocyanite ion (OSCN.sup.) was detectable in the compositions according to the invention.
[0303] The presence of the I.sub.2SCN.sup. and I(SCN).sub.2.sup. ion mixture is demonstrated by the presence of a characteristic peak at approximately 49 to 50.5 ppm. The absence of the hypothiocyanite ions is demonstrated by the absence of peaks at approximately 127 to 128 ppm
[0304] This absence of hypothiocyanite ions was also revealed by ionic chromatography. [0305] Analysis of the oxidation of the SH et NH.sub.2 functions;
[0306] The analysis of the oxidation of the NH.sub.2 functions is carried out by oxidation of TMB (tetramethylbenzydine).
[0307] The analysis of the oxidation of the SH function is carried out by oxidation of TNB (5-thio-2-nitrobenzoic acid) into DTNB (5,5-dithiobis-(2-nitrobenzoic acid)). [0308] Testing of the compositions on microorganisms
Example 1.1
influence of the Buffers/pH/Concentrations of Buffer
[0309] The recapitulative table of the tests is presented below:
TABLE-US-00001 TABLE 1 NMR Oxidation Oxidation (S13CN) Position New NMR (relative intensity) SH-M/L NH2-M/L SCN- OSCN- Peak SCN- OSCN- New peak 2.4 mM KSCN + 2.4 mM H.sub.2O.sub.2 + LP 1 Tap water 374 0 133.48 127.71 no 320 320 NA 2 Phosphate buffer-100 mM-pH 7.4 133.48 127.71 no NA 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + LP 3 Tap water 442 504 133.48 no no 160 NA NA 4 Na acetate buffer-500 mM-pH 4.4 864 1094 no no 49.8 NA NA 1425 5 Na acetate buffer-100 mM-pH 4.4 829 1080 no no 49.63 NA NA 1650 6 Na acetate buffer-10 mM-pH 4.4 827 1000 no no 49.58 NA NA 1475 7 Na acetate buffer-1 mM-pH 4.4 365 164 133.48 no 49.62 110 NA 250 8 NH.sub.4 acetate buffer-500 mM-pH 4.5 1191 1303 no no 50.05 NA NA 1800 9 NH.sub.4 acetate buffer-100 mM-pH 4.5 964 1150 no no 49.69 NA NA 1700 10 NH.sub.4 acetate buffer-10 mM-pH 4.5 962 1187 no no 49.59 NA NA 1550 11 NH.sub.4 acetate buffer-1 mM-pH 4.5 576 539 133.48 no 49.6 150 NA 660 12 Na acetate buffer-500 mM-pH 5.6 1172 1074 no no 50.51 NA NA 1725 13 Na acetate buffer-100 mM-pH 5.6 983 1056 no no 49.71 NA NA 1800 14 Na acetate buffer-10 mM-pH 5.6 964 666 133.48 no 49.61 170 NA 200 15 Na acetate buffer-1 mM-pH 5.6 659 684 133.48 no 49.61 160 NA 290 16 Citrate buffer-500 mM-pH 6.2 1260 1375 no no 51.11 NA NA 1175 17 Citrate buffer-100 mM-pH 6.2 948 1000 no no 49.97 NA NA 1125 18 Citrate buffer-10 mM-pH 6.2 781 828 no no 49.64 NA NA 852 19 Phosphate buffer-500 mM-pH 7.4 1035 1552 no no 51.16 NA NA 1035 20 Phosphate buffer-100 mM-pH 7.4 767 900 no no 50.36 NA NA 1650 21 Phosphate buffer-10 mM-pH 7.4 475 502 133.48 no no 94 NA NA 22 Phosphate buffer-1 mM-pH 7.4 271 284 133.48 no no 205 NA NA 23 Carbonate buffer-500 mM-pH 9.2 328 87 133.48 no no 185 NA NA 24 Carbonate buffer-100 mM-pH 9.2 248 106 133.48 no no 225 NA NA 25 Carbonate buffer-10 mM-pH 9.2 214 212 133.48 no no 230 NA NA
[0310] First, it is specified that no hypothiocyanite ion (OSCN.sup.) is detected for all of the compositions in which the two ions, I.sup. and SCN.sup., were introduced (tests 3-25), while such ions form in the absence of I.sup. (tests 1-2).
[0311] As explained in the introduction of Example 1, the peak in the vicinity of 50 ppm in NMR is characteristic of the new species identified, namely I.sub.2SCN.sup. and I(SCN).sub.2.sup.. Moreover, its intensity reveals the quantity of new species formed.
[0312] This peak is observed when the pH of the solution is from 4.4 to 7.4. If the pH is less than 4.4, hydrolysis of the thiocyanate occurs.
[0313] When the assayed quantity of oxidizing molecules is high and the peak at 49-50 ppm can be seen to appear, a peak associated with the thiocyanate is no longer observed, which indeed indicates its participation in the reaction.
Example 1.2
Influence of the pH on the Water Base (With LP)
[0314] The recapitulative table of the tests is presented below:
TABLE-US-00002 TABLE 2 Oxidation NMR Oxidation NH.sub.2- (S13CN) Position New NMR (relative intensity) SH-M/L M/L SCN- OSCN- Peak SCN- OSCN- New peak 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + LP 26 Water-pH 4.4 1130 1622 no no 49.61 NA NA 1300 27 Water-pH 5.5 1010 821 no no 49.62 NA NA 740 28 Water-pH 6.5 644 1098 133.48 no no 120 NA NA 29 Water-pH 7.5 505 567 133.48 no no 210 NA NA
[0315] Here again, in the presence of iodide ion, no hypothiocyanate ion (OSCN.sup.) is detected, irrespective of the compound.
[0316] When the unbuffered aqueous solution is at a pH of less than 6.5, it is observed that the new peak is detected, which is characterized by a shift in NMR (49.6 ppm), which is correlated with an increased capacity to oxidize the SH and NH.sub.2 groups.
[0317] The new peak is observed at acidic pH values: 5.5 and 4.4, with a nearly doubled intensity at pH 4.4.
[0318] In contrast, at higher pH values, the new peak is not observed.
Example 1.3
influence of the I.SUP../SCN.SUP. ratio
[0319] The recapitulative table of the tests is presented below:
TABLE-US-00003 TABLE 3 NMR (S13CN) Position New Other NMR (relative intensity) SCN.sup.- OSCN.sup.- Peak Peak SCN.sup.- OSCN- New peak Other peaks 30 9:1 5.4 mM KI + 0.6 mM no no 49.6 no NA NA 740 NA NA KSCN + 6 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 31 1:1 5.4 mM KI + 5.4 mM 133.47 no 49.65 124.63/111.94 560 NA 190 800 130 KSCN + 10.8 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 32 0.54:1 5.4 mM KI + 10 mM 133.48 no no 124.63/111.94 1400 NA NA 1750 150 KSCN + 15.4 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 33 1:1 1.2 mM KI + 1.2 mM 133.48 no no 124.63/111.93 75 NA NA 125 105 KSCN + 2.4 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 34 0.27:1 1.2 mM KI + 5.4 mM 133.48 no no 124.63 825 NA NA 750 NA KSCN + 6.6 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 35 1:2 5.4 mM KI + 2.7 mM 133.48 no 49.62 no 100 NA 950 NA NA KSCN + 8.1 mM H.sub.2O.sub.2 Na acetate buffer pH 4.4 0.1M 36 4.5 5.4 mM KI +1.2 mM no no 49.69 no NA NA 1700 NA NA KSCN + 6.6 mM H.sub.2O.sub.2 + ammonium acetate buffer 100 mM pH 4.5
[0320] Here again, in the presence of iodide ion, no hypothiocyanate ion (OSCN.sup.) is detected, irrespective of the compound.
[0321] The new peak is observed at I.sup./SCN.sup. ratios from 9/1 to strictly greater than 1/1.
[0322] In contrast, no peak is observed at I.sup./SCN.sup. ratios from 0.27/1 to 1/1.
Example 2
Efficacy of a Composition According to the Invention
[0323] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 Mm, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0324] This compound was compared with the compounds of the prior art manufactured by bringing together of: [0325] either potassium iodide (KI), lactoperoxidase (LP) and hydrogen peroxide (H.sub.2O.sub.2); [0326] or potassium thiocyanate (KSCN), lactoperoxidase (LP) and hydrogen peroxide (H.sub.2O.sub.2).
[0327] The results are given in the table below:
TABLE-US-00004 TABLE 4 KSCN + KI KSCN KI Colletotrichum lindemuthanium ++++ Fusarium avenaceum +++ Septoria tritici + Verticillium dahliae ++++ Phytophthora infestans +++ Pythium ultimum ++++ Colletotrichum musae ++++ 0 0 Pencillium italicum ++++ 0 ++++ Penicillium digitatum ++++ + ++++ Botrytis cinerea ++++ Penicillium expansum ++++ Pectobacterium atrosepticum ++++ + + Pseudomonas syringae pv syringae ++++ + 0 Pectobacterium atrosepticum ++++ + 0 Erwynia amylovora ++++ + 0 Pseudomonas syringae pv. tomato ++++ +++ + Clavibacter michiganensis ++++ ++++ +++ subsp. michiganensis Kocuria rhizolia ++++ + + Staphylococcus aureus ++++ ++ ++ Enterobacter gergoviae ++++ + + Escherichia coli ++++ + + Klebsiella pneumoniae ++++ + + Pseudomonas aeruginosa ++++ + + Pseudomonas fluorescent ++++ + + Pseudomonas putita ++++ + + Aspergillus niger ++++ + Penicillium pinophilum ++++ + ++++ Candida albicans ++++ + ++ Xylella fastidiosa subsp. fastidiosa ++++ Xylella fastidiosa subsp. multiplex ++++ Xylella fastidiosa subsp. pauca ++++ % inhibition: ++++: 79-100% +++: 60-78% ++: 41-59% +: 0-40% 0: no inhibition -: not tested
One observes that a composition according to the invention has an often much greater activity on all the microorganisms tested.
Example 3
Oxidizing Power of a Composition According to the Invention
[0328] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0329] The time of contact of the different solutions was set at 1 minute.
[0330] The oxidizing power was then measured on the SH functions by the method of oxidation of TNB (5-thio-2-nitrobenzoic acid) into DTNB (5,5-dithiobis-(2-nitrobenzoic acid)), after the following storage times: 1, 3, 5, 10, 15, 20, 30, 60 and 120 minutes.
[0331] The time T=0 corresponds to the time when the lactoperoxidase (LP) is removed.
[0332] The results are given in the table below:
TABLE-US-00005 TABLE 5 Time (minutes) Oxidation-SH 0 516.54 3 567.64 5 586.4 10 586.7 15 587.02 20 584.68 30 581.78 60 701.58 120 684.7
[0333] It is noted that the oxidizing activity increases with the storage time.
[0334] In addition, after 60 minutes, the oxidizing power is stable.
Example 4
Stability of a Composition According to the Invention
[0335] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0336] This composition was distributed in 6 flasks. The flasks are then opened (1 per month), and the bactericidal activity on Candida albicans is tested.
[0337] The graph illustrating the variation in the survival rate of the Candida albicans as a function of time, evaluated over a period of 6 months, is presented in
[0338] It is observed that no measurable decrease in the activity is detected during the period of 6 months.
Example 5
Test of a Short Time of Contact with Candida albicans (5 Minutes)
[0339] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM potassium thiocyanate (KSCN), 6.6 mM hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 Mm, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0340] The bactericidal activity on Candida albicans was tested, for the following times of contact with Candida albicans: 5 minutes or 30 minutes.
[0341] After a contact time of 5 minutes or 30 minutes with the composition (or the control), an inoculation of a culture medium takes place.
[0342] The results of these tests are illustrated in
Example 6
Efficacy with a Decreased Concentration of Oxidizing Agents
[0343] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0344] This composition was distributed in 5 flasks according to different dilutions in decreasing order: 755 m, 252 m, 75 m, 25 m. A control solution was also prepared.
[0345] After a contact time of 5 minutes with the 6 different compositions (5 dilutions and the control), an inoculation of a culture medium takes place.
[0346] The results are presented in
Example 7
Selectivity of a Composition According to the Invention Streptococcus mutans/Streptococcus salivarius
[0347] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0348] The composition is tested with regard to its bactericidal effect on Streptococcus mutans (cariogenic bacterium) and on Streptococcus salivarius (commensal bacterium).
[0349] The results are presented in
Example 8
Activity with Respect to the Biofilms
[0350] A composition according to the invention according to the protocol described in Example 1.
[0351] This composition was diluted 3-fold and has a concentration of I.sub.2SCN.sup. ions of 250 M. This composition is called solution B.
[0352] This composition was put in contact with different biofilms organized bacteria versus control and versus another other commercial composition which is Chlorexhidine gluconate 2.0% (solution A).
[0353] The compositions were put in contact with the biofilm of the different bacteria for variable durations: 5 minutes, 15 minutes, 30 minutes, 11 hours and 24 hours. This contact occurs by immersion of the strips containing the bacterial biofilm in a solution of the composition or in a solution of chlorexhidine gluconate 2% or as control in an aqueous solution (see hereafter the details of the technique applied for Candida albicans)
[0354] The activity with respect to the following bacteria was tested: Lactobacillus acidophilus, Veillonella alcalescens, Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum.
[0355] The results are presented in
[0356] Candida albicans Biofilms
Protocol of Immobilization of Biofilms of Candida albicans on Resin and Titanium. [0357] A) Resin: Material used in the fabrication of dental prostheses, obtained by polymerization of organic compounds
[0358] The pieces of resin are stored in sodium azide (0.5 g/500 mL) in order to be disinfected. This operation occurs under sterile conditions.
[0359] Transfer 3 pieces of resin into a pot.
[0360] Wash 3 in 60 mL of H.sub.2O for 5 min with stirring.
[0361] Rinse a last time with 60 mL of Sabouraud liquid for 5 min with stirring.
[0362] Transfer each piece of resin into a 10 mL round-bottom tube.
[0363] Prepare a suspension of Candida albicans at 10.sup.6 bl/mL in 10 mL of Sabouraud medium.
[0364] Prepare the 3 reaction tubes as indicated in the table below.
TABLE-US-00006 Preparation of the biofilms on resin Suspension (composition of the tubes on D.sub.1). of C.a. Sabouraud liquid (mL) (mL) Control 4 Control + 3.6 0.4 Test 3.6 0.4
[0365] Incubate for 24 to 48 h in a Rotator (3 rotations per minute). [0366] After incubation, from each of the 3 tubes, transfer 1 mL of supernatant into a cuvette. Measure the absorbance at 600 nm. [0367] Suction the culture media in the 3 tubes. [0368] Wash 3 in phosphate buffer. [0369] Prepare a phosphate buffer solution containing glucose (2 g/100 mL). [0370] Using this buffer, prepare a solution of I.sub.2SCN.sup.. [0371] Prepare the 3 reaction tubes as indicated in the table below.
TABLE-US-00007 Preparation of the biofilms on resin (composition of the tubes on D.sub.2). I.sub.2SCN.sup.- Phosphate buffer + glucose (mL) (mL) Control 4 Control + 4 Test 4 [0372] Incubate for 30 min. [0373] Inoculate each face of the resin strips successively in 4 Petri dishes. [0374] Incubate for 24 to 48 h at 37 C. [0375] B) Titanium
[0376] This operation occurs under sterile conditions.
[0377] Weigh 500 mg of titanium in 3 different tubes.
[0378] Prepare a suspension of Candida albicans at 10.sup.6 bl/mL in 10 mL of Sabouraud medium.
[0379] Prepare the 3 reaction tubes as indicated in the table below.
TABLE-US-00008 Preparation of the biofilms on titanium Suspension (composition of the tubes on D.sub.1). of C.a. Liquid Sabouraud (mL) (mL) Control 4 Control + 3.6 0.4 Test 3.6 0.4
[0380] Incubate for 24 h to 48 h in a Rotator (3 rotations per minute). [0381] After incubation, allow settling for exactly 10 min. [0382] From each of the 3 tubes, transfer 1 mL of supernatant into a cuvette. Measure the absorbance at 600 nm. [0383] Suction the culture media in the 3 tubes. [0384] Wash 3 times in phosphate buffer. [0385] Prepare a solution of I.sub.2SCN.sup.. [0386] Prepare a phosphate buffer solution containing glucose (2 g/100 mL). [0387] Prepare the 3 reaction tubes as indicated in the table below.
TABLE-US-00009 Preparation of the biofilms on titanium (composition of the tubes on D.sub.2). I.sub.2SCN.sup.- Phosphate buffer + glucose (mL) (mL) Control 4 Control + 4 Test 4
[0388] Incubate for 30 min. [0389] Suction the supernatant
Assay with MTT (*)
[0390] The living blastoconidia transform the MTT-tetrazolium into MTT-formazan which absorbs at 570 nm (Levitz & Diamond, 1985).
[0391] The different steps of the procedure are detailed below: [0392] Prepare a suspension of Candida albicans of 10.sup.6 bl/mL in 10 mL of phosphate buffer containing glucose (2 g/100 mL) [0393] (*) MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [0394] Prepare the 4 reaction tubes as indicated in the table below.
TABLE-US-00010 Preparation of the tubes for the assay with MTT. Blank Control Control + Test Suspension of C.a. (mL) MTT (mL) 1.5 1.5 1.5 1.5 Phosphate buffer + glucose (mL) 3 3 3 3
[0395] Shown in
[0396] Control (on the left): sterile resin strip.
[0397] Control + (in the middle): contaminated strip.
[0398] Test (on the right): strip contaminated and disinfected with the solution A
[0399] One observes the sterilization of a resin strip contaminated by Candida albicans ATCC 10231 after immersion in a solution containing 250 M of I.sub.2SCN.sup. ions. The biofilm formed on the resin was totally destroyed after 30 min of contact at ambient temperature.
[0400] The action of a solution of I.sub.2SCN.sup. on biofilms on (titanium) is measured by assay of the Candida biofilms with MU.
[0401] The solution containing 250 M of ions I.sub.2SCN.sup. makes it possible to destroy 70% of the biofilms formed for 24 to 48 h.
[0402] The results obtained are collected below:
TABLE-US-00011 Control Control + Test 0.003 0.949 0.319
Example 9
Stability and Activity of a Composition According to the Invention for the Application to the Problems of Contamination of Paints and Resins
[0403] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0404] The samples of paints and resins were contaminated by several series of microorganisms such as bacteria, yeasts and molds and mixtures thereof.
[0405] The tests were carried out by adding the composition according to the invention from the start. After a waiting time of 24 hours, the resin and paint samples were inoculated with a suspension of a mixture of microorganisms so as to reach a contamination level of 1,000,000 CFU/mL. The mixtures of the microorganisms consisted of bacteria, yeasts and molds such as: [0406] bacteria: Pseudomonas fluorescent ATCC 9721, Pseudomonas aeruginosa ATCC 10145, Bacillus subtilis ATCC 6984, Proteus vulgaris ATCC 9920; [0407] yeasts: Candida tropicalis ATCC 750, Kluyveromyces fragilis ATCC 8554, Candida pseudotropicalis ATCC 4135; [0408] molds: Aspergillus niger ATCC 9642, Aspergillus flavus ATCC 9643, Penicillium pinophilum ATCC 9644.
[0409] After contact times of 1, 2 and 7 days, the microbiological analyses were carried out. They are summarized in table 6 below:
TABLE-US-00012 TABLE 6 Mixture A: 1,000,000 CFU/mL 1 day 2 days 7 days Pseudomonas fluorescent ++++ ++++ ++++ Pseudomonas aeruginosa ++++ ++++ ++++ Candida tropicalis ++++ ++++ ++++ Aspergillus niger ++++ ++++ ++++ Mixture B: 1,000,000 CFU/mL Pseudomonas aeruginasa ++++ ++++ ++++ Kluyveromyces fragilis ++++ ++++ ++++ Penicillium pinophilum ++++ ++++ ++++ % inhibition ++++ 79-100% +++ 60-78% ++ 41-59% + 0-40% 0 No inhibition
[0410] One notes that there is no contamination, irrespective of the sample. This confirms that the composition according to the invention has an activity that stops the growth of the microorganisms in paints and resins.
Example 10
Activity of a Composition According to the Invention for the Problems of Contamination of Soils, Production Materials and Dental, Surgical Instruments, Etc.
[0411] In industrial processes, a cleaning referred to as CIP (Cleaning In Place) is applied, which consists in applying disinfectants at high temperature after use of the equipment.
[0412] The same applies to the instruments used in dentistry practices, in hospitals, instruments which are cleaned after their use by sterilization in an oven at high temperature.
[0413] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0414] It was demonstrated that this composition was capable of eliminating the microorganisms responsible for contaminations of this industrial equipment and other equipment and had the advantage that it could be used at ambient temperature.
Example 11
Activity of a Composition According to the Invention for the Problems of Contamination During the Cicatrization of Injuries in Humans and Animals
[0415] The resistance of microorganisms to the antibiotics is an increasing problem and seriously complicates the cicatrization of wounds caused by an injury or a burn of the skin. These microorganisms are capable of increasing the inflammatory process.
[0416] A composition according to the invention was prepared according to the protocol described in Example 1 and was diluted until a composition comprising 250 M of I.sub.2SCN.sup. was obtained.
[0417] This composition was tested on microorganisms which are resistant to the current antibiotics.
[0418] More precisely, the use of impregnated fabrics showed the potential, when said fabrics were applied to the wound, of having an antibacterial activity against bacterial species that are resistant to different antibiotics.
[0419] The results described in the table below show that the composition according to the invention which was prepared according to the protocol described in Example 1 is very effective against these microorganisms even if its concentration of I.sub.2SCN.sup. ions is 250 M.
TABLE-US-00013 TABLE 7 Bacteria 1 day 2 days 7 days Burkholderia multivorans multiresistant ++++ ++++ ++++ Pseudomonas aeruginosa multiresistant ++++ ++++ ++++ Stenotrophomonas maltophilia multiresistant ++++ ++++ ++++ Pandoraea apista multiresistant ++++ ++++ ++++ Achromobacter denitrificans multiresistant ++++ ++++ ++++ Staphylococcus aureus multiresistant ++++ ++++ ++++ Enterococcus faecium multiresistant ++++ ++++ ++++ Enterococcus faecalis multiresistant ++++ ++++ ++++ Yeasts Malassezia pachydermatis multiresistant ++++ ++++ ++++
Example 12
Activity of a Composition According to the Invention Against the Microorganisms Responsible for Respiratory Diseases in Horses and in Humans
[0420] A composition according to the invention was prepared according to the protocol described in Example 1 and was tested on microorganisms responsible for respiratory diseases in horses and in humans.
[0421] The growth curves between the exponential phase and the stationary growth phase allowed us to select the ideal conditions which corresponded to a concentration of 1,000,000 spores/mL. A composition according to the invention was prepared according to the protocol described in example 1 and showed an efficacy against the microorganisms responsible for respiratory diseases in horses.
[0422] The microorganisms were the following: Rhodococcus equi ATCC 25729-Streptococcus equi subsp equi ATCC 53185 and Streptococcus equi subsp zooepidemicus ATCC 43079. They are responsible for respiratory diseases in horses.
[0423] Taking into account the growth time of these microorganisms, the tests were carried out after 48 hours and 120 hours of growth of the microorganism.
[0424] In all the mixtures, all the hydrogen peroxide is consumed. The thiocyanate and the iodine are consumed in identical proportions.
[0425] The composition prepared according to the protocol described in Example 1 was shown to be effective in the inhibition of the microorganisms after a contact time of 5 minutes. 4 concentrations of I.sub.2SCN.sup. in the ion composition were used at different dilutions. Each number represents the results of 3 independently performed experiments.
[0426] The percentages of in vitro inhibition of the microorganisms were measured after a contact time of 5 minutes.
[0427] Controls were run with solutions without enzyme with only the substrates 5.4 mM KI+2.2 mM KSCN, on the one hand, and with 6.6 mM of H.sub.2O.sub.2. These solutions showed an absence of efficacy on the microorganisms (see table 8 below).
TABLE-US-00014 TABLE 8 Before enzymatic Streptococcus Streptococcus reaction Rhodococcus Rhodococcus Streptococcus Streptococcus equi equi KI/KSCN/ equi equi equi equi zooepidemicus zooepidemicus LPO H202 % inhibition % inhibition % inhibition % inhibition % inhibition % inhibition U/mL mM/L Dilution at 48 h at 120 h at 48 h at 120 h at 48 h at 120 h 50 5.4/1.2/6.6 - 86 88 82 87 92 97 - 82 84 80 86 92 94 1/10- 81 83 60 62 90 94 3.6/0.8/4.4 - 86 88 65 69 95 98 - 86 88 60 61 74 77 1/10- 63 70 65 69 44 48 2.7/0.6/3.3 - 82 84 60 63 79 81 - 81 81 68 72 10 13 1/10- 21 24 64 67 10 13 0.78/0.34/1.1 - 85 90 30 35 14 19 Effects of - 0 0 4 6 4 7 the - 0 0 0 0 0 0 substrates without presence of enzyme
[0428] A second test series was carried out under the same conditions with other microorganisms responsible for respiratory diseases in humans and which were detected in the cases of muscoviscidosis: tobramycin-resistant Burkholderia cepacia (ATCC BAA-245), mucoid Pseudomonas aeruginosa, Staphylococcus aureus resistant to methicillin and to oxacillin (ATCC 43300).
TABLE-US-00015 TABLE 9 Before enzymatic reaction HUMANS LPO Burkholderia Pseudomonas Staphylococcus aureus (MRSA) U/mL KI/KSCN/H202 Dilution cepacia aeruginosa (mucoid) methylcillin and oxacillin resistant Contact time Contact time Contact time 5 minutes 5 minutes 5 minutes % inhibition % inhibition % inhibition 50 5.4/1.2/6.6 1/1 100 100 100 1/3 100 100 100 1/5 78 57 56 1/10 32 38 36 Effects of the substrates 1/1 0 0 0 without presence of enzyme 1/3 0 0 0
In summary, a strong antimicroorganism activity was detected.
Example 13
Activity of a Composition According to the Invention Against the Microorganisms Responsible for the Deterioration of Plants, Fruits and Vegetables and Other Harvested Plants, in Particular in Bananas
[0429] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in an ammonium acetate buffer 100 mM, pH 4.5 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0430] This composition was tested on contaminated bananas, in particular on bananas contaminated by fungi causing anthracnose lesions and crown rot.
[0431] By dipping the bananas in the composition prepared according to the protocol described in Example 1, it was demonstrated that said composition had a great efficacy against the infections caused by Colletotricum musae, Fusarium monoliforme and Fusarium oxysporum
[0432] In addition, it was demonstrated that the composition prepared according to Example 1 was more active against fungi compared to conventional fungicides which are toxic and pollute the environment.
Example 14
Activity on Xylella fastidiosa
[0433] A composition according to the invention was prepared by bringing together of potassium iodide (KI) 5.4 mM, 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in a citrate-phosphate buffer 100 Mm, pH 6.9 in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) according to the protocol described in Example 1.
[0434] The composition is tested for its bactericidal effect on Xylella fastidiosa subsp. fastidiosa, Xylella fastidiosa subsp. multiplex and Xylella fastidiosa subsp. pauca according to the protocol described below.
[0435] The results are presented in
[0436] The inoculum is prepared by 2 successive subcultures (each subculture was carried out at 26 C. for 10 days) on a medium comprising a mixture of 3 solutions A, B and C described below; the mixture of A and B being sterilized in the autoclave before addition of the solution C sterilized by filtration. [0437] Solution A: 500 mL of distilled water (Aq. Dest) (50 C.)+10 g ACES Sigma A9758-25G [0438] Solution B: 440 mL of distilled water (Aq. Dest)+40 mL 1.0 N KOH (1N KOH: 2.24 g solution 40 mL of distilled water.)
[0439] To solution B, 2 g of charcoal (Active Charcoal Sigma C-4386), 10 g of Yeast extract oxoid and 17 g of Agar are then added.
[0440] The mixture of the two solutions is prepared, followed by sterilization in the autoclave. [0441] Solution C: cysteine HCI 0.4 g+ferric pyrophosphate 0.25 g solution 20 mL of distilled water (Aq. Dest) Cold sterilization (0.2 m filter)
[0442] 5 mL of sterile PBS are then added to a Petri dish comprising a Xylella strain; the Petri dish is plated with a sterile spatula, and the 5 mL are pipetted into a sterile flask.
[0443] The DO650 is adjusted to 1 (DO650 of 1=104 CFU/mL ref. Shi et al., 2007, Appl. Environ. Microbiol., 73 (21)) with sterile PBS. [0444] in a 15-mL Falcon tube, add:
[0445] Control: 1 mL inoculum, 1 mL isotonic H.sub.2O (NaCl 8.5 g/L) adjusted to pH 6.9 sterile, 1 mL of PD2 Broth
[0446] The PD2 broth is obtained by mixing the solutions A and B described below:
[0447] Solution A:
[0448] Distilled water 1 L: [0449] Soy peptone: 2.0 g [0450] Bacto tryptone: 4.0 g [0451] Disodium succinate: 1.0 g [0452] Trisodium citrate: 1.0 g [0453] K.sub.2HPO.sub.4: 1.5 g [0454] KH.sub.2PO.sub.4: 1.0 g [0455] Hemin chloride stock solution (0.1% in (0.05 N NaOH: 0.112 g/40 mL)): 10.0 mL [0456] MgSO.sub.4.7H.sub.2O: 1.0 g [0457] pH: 6.9
[0458] Autoclave at 121 C. for 15 min.
[0459] Solution B [0460] Bovine serum albumin fraction V (20% w/v): 10 mL. Cold sterilize (0.2 m filtration).
[0461] Mix solution A with solution B: when the autoclaved medium (A) has cooled to 50 C., add the sterilized albumin (B).
[0462] The biocontrol agent is obtained by mixing 1 mL of inoculum, 1 mL of inhibiting agent and 1 mL of PD2 broth, incubated under stirring (100 rpm) at 26 C. for 30 minutes.
[0463] The controls are obtained by mixing 4 drops (10 L)/Petri dish; left side of the dish)5 Petri dishes and incubation for 14 days 26 C.
[0464] The biocontrol agent by mixing 4 drops (10 L)/Petri dish; to right side of the Petri dish)5 Petri dishes and incubation for 14 days 26 C.
[0465] The Petri dishes are observed under the binocular microscope 30 and photographed.
[0466] The photographs are presented in
Example 15
Importance of the Removal of the Enzyme
[0467] During the production of the wanted ions, if one keeps the enzyme in the mixture, one observes a gradual loss of the wanted ions, and, after 48 h, a total loss of the wanted ions due to enzymatic oxidation of the wanted ions, as illustrated in
Example 16
Addition of Supplementary Enzymes
[0468] The presence of supplementary enzymes confers an unexpected effect, as illustrated in
[0469] One observes notably that the addition of lactoferrin (>5 mg/L) confers an improved antimicrobial activity compared to the solutions without addition of lactoferrin, when the mixture is diluted 10-fold.
Example 17
Comparison with the Prior Art
[0470] Non-obtention of the species I.sub.2SCN.sup. or I(SCN).sub.2.sup.. Compositions comprising an enzymatic mixture with a KI/KSCN ratio of 1.74 were prepared according to the protocol described in EP1349457.
[0471] Compositions comprising an enzymatic mixture with a KI/KSCN ratio of 1.55 were prepared according to the protocols described in WO00/01237.
[0472] The NMR spectra were prepared under the following conditions: a Bruker AMX-500 MHz apparatus with an 8-mm broadband probe was used. The spectra were obtained from the reaction mixture (lactoperoxidase/[.sup.13C] SCN.sup./I.sup./ H.sub.2O.sub.2 according to the described method. The sample consists of 540 L of reaction mixture, 60 L D.sub.2O (deuterium oxide), 2 L DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid). The samples were placed in an NMR tube having a length of 8 mm and an 8-inch wall. The spectra were collected using the following parameters: scanning width=15 009, number of points=32000, acquisition time=1.066 s, recycling delay of 2 s, number of scans=2000. The chemical shifts (ppm) were referenced with respect to the NMR spectroscopy calibration standard, DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid).
[0473]
[0479] The peak corresponding to KS.sup.13CN is observed regardless of which matrix is used. There are no peaks at 49-50 ppm. In the mixtures described in WO00/01237 or EP1349457, there is no production of I.sub.2SCN.sup. or I(SCN).sub.2.sup. ion.
[0480] Demonstration of the Presence of Hypothiocyanite Ions.
[0481]
[0482]
[0483] In the enzymatic mixtures prepared according to WO00/01237 or EP1349457, the hypothiocyanite ions are detected in small quantity as are the cyanate ions (OCN.sup.), perfectly identifiable thanks to its triplet signal (Gerritsen et al. 1993), which correspond to the degradation of the OSCN.sup. ions.
Example 18
Comparison of the Kinetics of the Enzymatic and Chemical Oxidations and the Antimicrobial Activities
[0484] A) Rapidity of the production of the wanted ions (enzymatic kinetics) which implies immediate antimicrobial activity for the enzymatic mixture.
[0485] Kinetics of production of the wanted ions (measurement by oxidation of the SH or NH.sub.2 groups)
[0486] Solutions comprising 5.4 mM KI+1.2 mM KSCN+6.6 mM H.sub.2O.sub.2 in a sodium acetate buffer 100 mM pH 4.4+/LP according to the invention described are prepared
TABLE-US-00016 TABLE 10 With Lactoperoxidase: Without incubation for 1 minute then Lactoperoxidase removal of the lactoperoxidase Time Oxidation-SH Time Oxidation-SH (min) (M/L) (min) (M/L) 0 4 0 517 3 49 3 568 5 74 5 586 10 133 10 587 15 168 15 587 20 183 20 585 30 197 30 582 60 203 60 702 120 235 120 705
TABLE-US-00017 TABLE 11 With Lactoperoxidase: Without incubation for 1 minute then Lactoperoxidase removal of the lactoperoxidase Time Oxidation-NH.sub.2 Time Oxidation-NH.sub.2 (min) (M/L) (min) (M/L) 1 53 1 2521 3 54 3 1924 5 107 5 1426 10 158 10 1194 15 218 15 991 20 220 20 970 30 253 30 829 60 308 60 947
[0487] One notes that the kinetics of formation of the wanted ions is entirely different: the production of the wanted ions with the enzyme is instantaneous, while the production obtained without the enzyme is rather slow, after 1 hour of incubation only approximately 1/3 of the wanted ions is obtained. This has an implication for the immediate activity of the active mixture:
[0488] Activity with respect to Penicillium expansum
TABLE-US-00018 TABLE 12 in vitro inhibition with respect to 1 10.sup.6 spores/mL of Penicillium expansum of the mixture (5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2) without Lactoperoxidase % inhibition % inhibition (chemical mixture) after 48 h after 120 h 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/3 34 4 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/5 30 33 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/10 25 32 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/15 25 7 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/20 18 3 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 tap water 1/30 18 29 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/3 17 0 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/5 9 0 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/10 14 2 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/15 16 27 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/20 14 22 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + tap water pH 4.4 1/30 15 23 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/3 16 21 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/5 31 17 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/10 21 21 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/15 26 37 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/20 29 42 5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2 + acetate buffer 0.1 M pH 4.5 1/30 35 35
TABLE-US-00019 TABLE 13 In vitro inhibition with respect to 1 10.sup.6 spores/mL of Penicillium expansum of the mixture (5.4 mM KI + 1.2 mM KSCN + 6.6 mM H.sub.2O.sub.2) with Lactoperoxidase for 1 minute % inhibition % inhibition (enzymatic mixture) after 48 h after 120 h B1 Acetate Buffer 0.5 M pH 4.5 1/3 96 97 B1 Acetate Buffer 0.5 M pH 4.5 1/5 96 97 B1 Acetate Buffer 0.5 M pH 4.5 1/10 96 98 B1 Acetate Buffer 0.5 M pH 4.5 1/15 96 98 B1 Acetate Buffer 0.5 M pH 4.5 1/20 97 98 B1 Acetate Buffer 0.5 M pH 4.5 1/30 95 60 B1 Acetate Buffer 0.5 M pH 4.5 1/50 81 37 B1 Acetate Buffer 0.1 M pH 4.5 1/3 94 96 B1 Acetate Buffer 0.1 M pH 4.5 1/5 96 97 B1 Acetate Buffer 0.1 M pH 4.5 1/10 95 97 B1 Acetate Buffer 0.1 M pH 4.5 1/15 97 98 B1 Acetate Buffer 0.1 M pH 4.5 1/20 70 36 B1 Acetate Buffer 0.1 M pH 4.5 1/30 53 22 B1 Acetate Buffer 0.1 M pH 4.5 1/50 40 4 B1 Acetate Buffer 0.01 M pH 4.5 1/3 93 96 B1 Acetate Buffer 0.01 M pH 4.5 1/5 94 96 B1 Acetate Buffer 0.01 M pH 4.5 1/10 94 97 B1 Acetate Buffer 0.01 M pH 4.5 1/15 81 99 B1 Acetate Buffer 0.01 M pH 4.5 1/20 53 0 B1 Acetate Buffer 0.01 M pH 4.5 1/30 19 0 B1 Acetate Buffer 0.01 M pH 4.5 1/50 6 0 B1 Acetate Buffer 0.001 M pH 4.5 1/3 100 100 B1 Acetate Buffer 0.001 M pH 4.5 1/5 97 99 B1 Acetate Buffer 0.001 M pH 4.5 1/10 96 99 B1 Acetate Buffer 0.001 M pH 4.5 1/15 95 99 B1 Acetate Buffer 0.001 M pH 4.5 1/20 29 0 B1 Acetate Buffer 0.001 M pH 4.5 1/30 8 0 B1 Acetate Buffer 0.001 M pH 4.5 1/50 13 0
[0489] One notes that the chemical mixture (incubation time of the reagents: 1 minute) is not effective for in vitro growth inhibition of Penicillium expansum.
[0490] On the contrary, the enzymatic mixture of (5.4 mM KI+1.2 mM KSCN+6.6 mM H.sub.2O.sub.2)+lactoperoxidase for 1 minute, then removal of the enzyme, is effective up to the 1/30 dilution for the buffer 500 mM, up to 1/15 dilution with the buffer 100 mM, 10 mM and 1 mM:
[0491] The method for preparing the active mixture containing the wanted ions, that is to say chemical or enzymatic, has an implication for the immediate antimicrobial activity of the mixture. The enzymatic mixture has an immediate antimicrobial efficacy (present as early as after 1 minute of incubation), while this antimicrobial activity is absent after 1 minute of incubation of the substrates in the chemical mixture.
Example 19
Antimicrobial Activity with Respect to E. coli of a Composition Immobilized on a Fabric and Lyophilized
[0492] A composition according to the invention was prepared by bringing together of 5.4 mM of potassium iodide (KI), 1.2 mM of potassium thiocyanate (KSCN), 6.6 mM of hydrogen peroxide (H.sub.2O.sub.2) in the presence of 50 mg/L of lactoperoxidase (LP) (1000 ABTS units per mg) in a sodium citrate buffer 100 mM pH 6.2 (
[0493] It can be seen that the lyophilized composition maintains a bactericidal action equivalent to the other non-lyophilized compositions (
[0494] It is apparent that after 24 h of incubation at 37 C. of 100 L of E. coli at 10.sup.9 CFU/mL on a culture medium in a Petri dish, the composition immobilized on a fabric stops the growth of the bacterium (halo visible).