A synergistic combination of an antimicrobial peptide and actives for hygiene applications

Abstract

A synergistic combination of antimicrobial peptide having Sequence ID 1 or salts and derivatives thereof and at least one active selected from thymol or caprylic acid such that said combination achieves >5 log kill in 1 minute for both Gram positive and Gram negative organisms and composition thereof.

Description

[0001]A SYNERGISTIC COMBINATION OF AN ANTIMICROBIAL PEPTIDE AND ACTIVES FOR HYGIENE APPLICATIONS Field of the invention The present invention relates to personal care compositions. More specifically the present invention relates to compositions comprising synergistic combinations for hygiene applications. Background and the prior art Antimicrobial actives are often used in personal care products like soaps, creams, lotions, and sprays to reduce the number of microbes on the skin and treat bacterial infections. The well-known and currently used antimicrobial actives such as Triclosan mediate bacterial killing by specific mechanism that leads to selective pressure on the microorganism to develop resistance. In organisms such as Pseudomonas aeruginosa the resistance is plasmid mediated leading to faster spread of resistance. World Health Organization (WHO) classifies the appearance of antibiotic resistance as one of the biggest threats to human health. Indiscriminate use of certain antimicrobial actives would exacerbate the problem. There are few active combinations that are efficacious as well as safe. The efficacious antimicrobial actives such as Triclosan are under global regulatory agency’s scanner because of safety concern. Triclosan is associated with endocrine disruption and antimicrobial resistance. The long-term systemic exposure to actives like Triclosan is an area of concern. Hence, there is a need to develop efficacious and safer antimicrobial active combinations. Antimicrobial Peptides are part of innate defense for all living organisms. Antimicrobial properties are inherent to AMPs. However, the antimicrobial property cited in the prior art is mainly related to determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). There are few reports of determination of log reduction for AMPs. However, the first time point for measuring the log reduction is 30 minutes. For hand hygiene applications, the relevant time points are 1 minute and 5 minutes. For AMPs such data is not cited in the prior art. Further, AMPs cited in prior art are tested in conditions that do not mimic real-life like scenario with respect to hand hygiene standard requirements. For example, mimicking soil conditions by testing in the presence of protein, Bovine Serum Albumin (BSA), and testing at temperature such as 20°C and for 1 minute / 5 minute as the time for testing depending on the applications. Nagaoka et al., Synergistic actions of antibacterial neutrophil defensins and cathelicidins. Inflamm Res. 2000 Feb; 49(2):73-9 discloses activated neutrophils extracellularly release antibacterial defensins and cathelicidins from the granules. In this study, to elucidate the interactions between defensins and cathelicidins in the extracellular environment, the researchers have evaluated the individual and synergistic actions of defensins and cathelicidins in the presence of physiological concentration of NaCl (150 mM). US2013210705 discloses antimicrobial metallodrugs comprising an antimicrobial peptide ("AMP") and / or an antibiotic covalently bound to a metal binding moiety. These metallodrugs combine a metal binding domain which typically catalyzes oxido-reductase chemistry or acts as a Lewis-Acid catalyst, with a member of a diverse class of antimicrobial agents currently validated in preclinical and clinical settings for the treatment of a broad spectrum of pathogenic organisms. This prior art requires antibiotics for its function. US19990318195 discloses novel anti-microbial system suitable for formulation in a wide variety of ophthalmic solutions. In particular, the composition comprises an antimicrobial peptide that is an indolicidin and a buffer compatible with application to a mammalian eye, wherein the buffer is a Good's buffer or the buffer has a halide ion concentration less than 0.85wt%. The compositions are useful for storing, cleaning, or disinfecting a contact lens. In particular, the compositions are self- preserving upon lengthy storage, effective in cleaning and sterilizing contact lenses upon exposure of the lens to the composition, do not require the need for physical or thermal treatment of the lens and enable the immediate application of the lens to the eye without the need for neutralization, deactivation or washing. US 5,043,176 discloses antimicrobial composition composed of an antimicrobial polypeptide and a hypothiocyanate component. Synergistic activity is seen when the composition is applied at between about 30 and 40 degrees Centigrade at a pH between about 3 and about 5. The composition is useful against gram negative bacteria such as Salmonella. A preferred composition is nisin, lactoperoxidase, thiocyanate and hydrogen peroxide. Such a composition is capable of reducing the viable cell count of Salmonella by greater than 6 logs in 10 to 20 minutes. US 5,043,176 discloses synergistic combinations of hypothiocyante component and antimicrobial peptides useful against Gram negative bacteria only and further the contact time is high about 10 to 20 min. US 5,736,574 discloses combinatory antimicrobial immixtures comprising at least one antimicrobial hydrolipid and / or lipid and an antimicrobially synergistically effective amount of at least one glyceryl monoalkyl ether are well suited for formulation, as preservatives, into a wide variety of pharmaceutical / cosmetic compositions. US 7,960,339 relates to peptides with antimicrobial or endotoxin-neutralizing activity having the general formula (Xaa1)M-(Xaa2)0-Xaa3-(Xaa4)P-(Xaa5)Q- (Xaa6)M-(Xaa7)R-(Xaa8)S. WO98 / 40401 discloses Compositions and methods for treating infections, especially bacterial infections, are provided. Cationic peptides in combination with an antibiotic agent are administered to a patient to enhance the activity of the antibiotic agent, overcome tolerance, overcome acquired resistance, or overcome inherent resistance. WO98 / 40401 discloses cationic peptide and antibiotic combination for antibacterial efficacy where indolicidin and its analogs have been tested. Antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food, Rai et.al., J Food Sci Technol (September 2016) 53(9):3381–3394, DOI 10.1007 / s13197-016-2318-5 discloses Antimicrobial peptides (AMPs) as diverse group of natural proteins present in animals, plants, insects and bacteria. These peptides are responsible for defense of host from pathogenic organisms. These peptides have been found to be an alternative to the chemical preservatives. Antimicrobial peptides can be used alone or in combination with other antimicrobial, essential oils and polymeric nanoparticles to enhance the shelf-life of food. It discloses Cathelicidins LL37 having sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES as an antimicrobial peptide. Antimicrobial activity of some plant essential oils and an antimicrobial-peptide against some clinically isolated pathogens, Tanhaeian et al. Chem. Biol. Technol. Agric. (2020) 7:13, discloses that the combination is effective against both gram positive and gram negative bacteria. This prior art has studied the antibacterial activity of four plant essential oils and one lactoferrin-derived peptide (cLFchimera). IN202031033995 discloses antimicrobial compositions comprising an antimicrobial peptide and an antimicrobial active. It discloses an antimicrobial composition comprising cathelicidin peptide fragment (FK-16) or derivatives thereof at a concentration ranging from 40μM to 900 μM; and an alcohol at a concentration ranging from 8% to 50%. However, there is always a need for providing a non-alcohol composition. Further British Standard EN1276 has laid out stringent protocol for antibacterial efficacy testing of the active system / formulation for hand hygiene applications. There is a need to provide an AMP containing active system / composition achieving >5 log kill in 1 minute for both Gram positive and Gram negative organisms. Object of the invention It is an object of the present invention to overcome the drawbacks of the prior art. It is another object of the present invention to provide an alcohol free composition that achieves >5 log kill in 1 minute for both Gram positive and Gram negative organisms. Summary of the invention Accordingly, the present invention provides a synergistic combination of antimicrobial peptide having Sequence ID 1 or salts thereof and at least one active selected from thymol or caprylic acid such that said combination achieves >5 log kill in 1 minute for both Gram positive and Gram negative organisms. According to another aspect, the present invention provides an antimicrobial composition comprising (a) a synergistic combination of the present invention and (b) cosmetically acceptable excipients. According to a further aspect of the present invention there is provided a sanitizer and disinfectant formulation comprising (a) the synergistic combination of the present invention and (b) cosmetically acceptable excipients. In a further aspect of the present invention there is provided an alcohol-free wet wipe formulation comprising (a) the synergistic combination of the present invention and (b) cosmetically acceptable excipients. According to yet another aspect, the present invention provides a method of disinfecting a surface, the method comprising the steps of: providing an alcohol- free wet wipe of the present invention; and wiping the surface with the wet wipe. Accordingly, to another aspect, the present invention provided an alcohol-free deodorant formulation comprising (a) the synergistic combination of the present invention and (b) cosmetically acceptable excipients. Brief Description of Accompanying drawings Figure 1 illustrates HPLC chromatogram of C1 peptide Detailed description of the invention The following description is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness. The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention as defined by the appended claims and their equivalents. It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Features that are described and / or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and / or in combination with or instead of the features of the other embodiments. It should be emphasized that the term “comprises / comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof. The term "peptide" as used herein refers to short polypeptides consisting from 2 to 50 amino acid residues, “Polypeptides” and “proteins” comprise more than 50 amino acid residues. A peptide may have a specific function. A peptide can be a naturally occurring peptide or a synthetically designed and produced peptide. The peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). The term, "antimicrobial peptide" (AMP) as used herein refers to any peptide that has microbicidal and / or microbiostatic activity. Thus, the term "antimicrobial peptide" as used herein refers in particular to any peptide having anti-bacterial, anti- fungal, anti-mycotic, antiparasitic, anti-protozoal, anti-viral, anti-infectious, anti- infective and / or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal properties. The term “log kill / log reduction” as used herein denotes the number of cells killed in a test. If a log reduction is a whole integer, then its numerical value equals 20 the number of nines in the percent reduction figure. For e.g. 1 log reduction = 90% reduction, 2 log reduction = 99% reduction, 3 log reduction = 99.9% reduction, 4 log reduction = 99.99% reduction, 5 log reduction = 99.999% reduction, 6 log reduction = 99.9999% reduction. The term “minimum inhibitory concentration (MIC)” of an antimicrobial peptide is the lowest concentration of peptide that completely inhibits growth of the organism. In general, lower MIC values are preferred. The term “Hygiene” as used herein means a series of practices performed to preserve health. According to the World Health Organization (WHO), "Hygiene refers to conditions and practices that help to maintain health and prevent the spread of diseases." The term "skin" as used herein means the entire epidermis of the human body. The term ‘salts’ as used herein includes Trifluoro acetate (TFA) or Acetate or chloride or lactate or malate counterion of peptide. The term “derivatives” as used herein includes peptide analogues, peptide fragments and modified peptides. The derivatives may be a sequence that has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1 of present invention. Accordingly, the present invention provides a personal care composition comprising specific AntiMicrobial Peptide (AMP) or salts and derivatives thereof and selected actives that demonstrates ≥5 log kill in 1 minute against both Gram positive and Gram negative bacteria in presence of rigorous conditions that simulate hand hygiene conditions. The present inventors have found that the combination of AMP (peptide C1, having sequence ID 1) or salts and derivatives thereof and atleast one active selected from thymol or caprylic acid demonstrate synergy in antimicrobial efficacy than at tested individual concentrations. It works at lower concentrations providing 99.999% reduction against Gram positive and Gram negative bacteria under rigorous, challenging testing conditions. The typical cost of chemical synthesis of peptide is on higher side. There is a significant reduction of peptide concentration achieved in presence of synergistic active. The unique and synergistic combination of AMP or salts and derivatives thereof and selected actives increases bacterial reduction. Typically, synthetic peptides are produced as salts of Trifluroacetate (TFA), Acetate, chloride, lactate. However, the peptide sequence is the active principle that is linked to its efficacy, that is, antimicrobial activity. Hence, all salt forms of the peptide (TFA, acetate etc) will have similar activities. The C1 peptide or salts and derivatives thereof can be present in an amount 0.00156 to 0.0125% by weight. Thymol can be present in an amount 0.0125 to 0.025% by weight. The caprylic acid can be present in an amount 0.05 to 0.11% by weight. According to a preferred embodiment of the present invention there is provided a combination of 0.001563 – 0.0125% of C1 peptide or salts and derivatives thereof and 0.025 – 0.05% of thymol such that the combination demonstrates synergy in terms of bacterial reduction in a ratio 0.03125:1 – 0.5:1. According to another preferred embodiment of the present invention there is provided a combination of 0.001 – 0.012% of C1 peptide or salts and derivatives thereof and 0.04 – 0.11% of caprylic acid such that the combination demonstrates synergy in terms of bacterial reduction in a ratio 0.009:1 – 0.3:1. In 1-minute contact kill experiment [in presence of 0.3% BSA (final concentration)], the composition of peptide (C1) or salts and derivatives thereof and actives lead to higher log reduction against Gram negative bacteria Pseudomonas aeruginosa, Escherichia coli and Gram positive bacteria Staphylococcus aureus, Enterococcus hirae. According to another aspect of the present invention there is provided an antimicrobial composition comprising the synergistic combination of the present invention. The composition can further include other cosmetically acceptable excipients. According to a further aspect of the present invention there is provided a sanitizer and disinfectant formulation comprising (a) the synergistic combination of the present invention and (b) cosmetically acceptable excipients. According to yet another embodiment of the present invention there is provided an alcohol-free deodorant formulation comprising (a) a synergistic combination of the present invention and (b) cosmetically acceptable excipients. According to another embodiment of the present invention there is provided an alcohol-free wet wipe formulation comprising (a) the synergistic combination of the present invention and (b) cosmetically acceptable excipients. The cosmetically acceptable excipient can be selected from the group consisting of preservative (0.2 to 1%), pH modifier as required, humectant (0.2 to 2%), fragrance (0.05 to 0.5%), fragrance solubilizer (0.1 to 2%). Preservative in accordance with the present invention can be selected from DMDM hydantoin, Sodium benzoate, Pottasium sorbate, Ethylhexyl glycerin, Benzoic acid, and Dehydroacetic acid. pH modifier in accordance with the present invention can be selected from Citric acid and Triethanolamine. Humectant in accordance with the present invention can be selected from Glycerol, Hyaluronic acid, Propylene glycol, Sorbitol and Honey. Fragrance in accordance with the present invention can be selected from art known commercially available fragrance. Fragrance solubilizer in accordance with the present invention can be selected from PEG-40 Hydrogenated Castor Oil, Tween -20, Tween-80, Castor oil, Propylene glycol, Beeswax. The composition can be provided in wipes, sanitizer, body wash and hand wash, wound healing cream / lotion and surface cleaners. The present invention also provides a method of disinfecting a surface, the method comprising the steps of: providing an alcohol-free wet wipe as described herein; and wiping the surface with the wet wipe. The present disclosure with reference to the following accompanying examples describes the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way. Examples Materials and methods: ^ Peptide C1 (FK-16): The 16 amino acid peptide with sequence FKRIVQRIKDFLRNLV (Sequence ID 1) was custom synthesized from USV Pvt. Ltd, India. The molecular weight of the peptide is 2045 Daltons and it has >98% purity based on HPLC profile. The HPLC chromatogram is enclosed (Figure 1). ^ LL-37 peptide having sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Seq ID 2) was custom synthesized from USV Pvt. Ltd. India. ^ Thymol: Sigma T0501 (USA) ^ Caprylic acid (Octanoic acid): Sigma-Aldrich C2875 (USA) ^ Caproic acid (hexanoic acid): Sigma -Aldrich 21530 (USA) ^ Capric acid (decanoic acid): Sigma- Aldrich C1875(USA) ^ Disodium EDTA: Sigma-Aldrich E5134 ^ PEG-40 Hydrogenated Castor Oil (CAS Number. 61788-85-0): CREMOPHOR® CO 40 from BASF ^ Decyl Glucoside (58846-77-8): Plantacare® 2000 UP from BASF Microorganism: The microorganisms used in the study are: 1. Pseudomonas aeruginosa, ATCC 15442 2. Escherichia coli ATCC 10536 3. Staphylococcus aureus ATCC 6538 4. Enterococcus hirae ATCC 10541 5. Staphylococcus epidermidis MTCC 435 6. Corynebacterium striatum ATCC 6940 The glycerol stock of these bacteria was stored at -800C. The culture was maintained on Tryptone soya agar (TSA) and sub-cultured every two weeks. Antimicrobial activity Assay to determine Microbicidal Efficacy: Antimicrobial activity to calculate the level of killing achieved by the test samples was determined using quantitative suspension test (contact kill) following the guidelines specified by EN 1276 Standard at 20°C using Dilution-neutralization method. Briefly, the cells were contacted with test samples for predefined time interval (generally 1 minute ±5 seconds) at 20°C, the antimicrobial activities of samples stopped dilution-neutralization method using a neutralizer solution and estimating the number of surviving bacteria using TSA plates. Bovine serum albumin (heat shock fraction, pH 5.2, ≥96%) procured from Sigma Chemical Company (Catalog no A8022) was used as interfering substance in the assay to simulate soiling conditions at dirty levels as defined by EN 1276 Standard. Tryptone Soya Agar (TSA), Tryptone soya broth (TSB) and other chemicals were procured from HiMedia Laboratories, Mumbai, India. Detailed Method: Colonies of bacteria from the TSA plate were suspended in diluent to prepare bacterial cell suspension of about 3 X 108CFU / ml. Test samples, bacterial suspension and 3% BSA solution were equilibrated at 20°C for 10 min in a dry bath (Eppendorf Thermomixer comfort). To 100 µl of bacterial suspension, 100 µl of BSA was added to simulate soiling condition. Within 5 minutes of preparation, this 200 µl bacteria was added to 800 µl of test samples and immediately mixed using a vortex mixer. At predefined contact time (generally 1 minute), the action of antimicrobial was stopped using dilution neutralization method. For this, an aliquot of assay content was added to 9X volumes of neutralizer solution (Polysorbate 80, 30g / l; Saponin, 30 g / l; Lecithin, 3 g / l in 250mM potassium phosphate buffer; pH 7.2) and left for 5 minutes. The surviving bacterial counts in the neutralized samples were estimated using 10-fold serial dilutions on TSA agar plates and incubating at 37°C for 24 hours. The colonies developed on TSA plates were counted and antimicrobial activity was calculated. Activity was evaluated in terms of log10reduction factor (Log kill or Log reduction) that was calculated as the difference between logarithms of CFU / ml before and after exposure to test samples. A log kill / reduction of 1 translates to 90% killing of bacteria. Similarly, log kill / reduction of 2, 3, 4 and 5 translates to 99%, 99.9%, 99.99% and 99.999% bacterial killing respectively. MIC determination: MIC is the lowest concentration (in μM) of the peptide that inhibits the growth of a given strain of bacteria. Minimum inhibitory concentration (MIC) of actives was determined by CLSI method. The Muller Hilton (MH) broth was used to grow bacteria at 37°C. The MIC determination was done in 96 well plate. Results and Discussion: Examples 1 Antimicrobial efficacy of C1: Antimicrobial peptide C1 shows antimicrobial activity against Escherichia coli with >4 log reduction of bacterial counts in 1 minute (Table 1) at concentrations of 0.0125% and above. However, it failed to kill Enterococcus hirae, even at concentrations as high as 0.15%. E. hirae is one of core group of mandatory bacteria specified by EN 1276 standard for demonstrating sanitization and disinfection activity of chemicals. The activity against E. hirae improved with time, yet it was still considerably lower at 2.6 log reduction (Table 2). Table 1. Antimicrobial efficacy of Antimicrobial Peptide C1 against Escherichia coli and Enterococcus hirae estimated as log reduction of its viable cell counts achieved after 1-minute contact time. Table 2. Antimicrobial efficacy of Antimicrobial Peptide C1 against Enterococcus hirae estimated as log reduction of its viable cell counts achieved at different contact time. Example 2: Antimicrobial efficacy of C1 and active combinations Synthesis of antimicrobial peptides is a complex process and requires considerable cost. To develop synergistic combination of C1 for decreasing its usage level and enhancing antimicrobial activity, we evaluated its combinations with various actives, which are core groups of molecules used in personal care applications. Table 3. Antimicrobial efficacy of combinations of Antimicrobial Peptide C1 and various Phytochemicals and Essential Oil against Escherichia coli and Enterococcus hirae estimated as log reduction of its viable cell counts achieved after contact time of 1 minute. The combination of AMP C1 (0.0125%) with various phytochemicals and essential oils (0.25%) were tested against E. coli and E. hirae for bacterial killing efficacy. The log reductions of E. coli and E. hirae obtained are presented in Table 3. C1 alone exhibited good antimicrobial efficacy against E. coli with 4.1 log reduction but had no activity against E. hirae. Terpinen-4-ol completely disrupted the activity of C1 as demonstrated by loss of antimicrobial activity against E. coli to 1 log reduction compared to the killing by C1 alone. Certain combinations of C1 such as those containing Ginger Oil, Cinnamyl Acetate, Benzyl Benzoate, Eucalyptus Oil, Eugenol etc. exhibited better activity against E. coli but lacked killing activity against E. hirae. However, combination of C1 with Thymol surprisingly demonstrated activity against both E. coli and E. hirae with >5 log reduction of both bacteria in 1 minute (Table 3). Evaluation of antimicrobial activity of Thymol used alone in test sample revealed that Thymol has strong activity against E. coli at 0.125% and 0.25% with 5 log reduction but weak antimicrobial activity against E. hirae with 1.1 and 2.8 log reduction respectively (Table 4). Table 4. Antimicrobial efficacy of thymol against Escherichia coli and Enterococcus hirae after contact time of 1 minute Example 3: Antimicrobial efficacy of C1 and Thymol combinations The present inventors have optimized the combination C1 and thymol to decrease the concentration required for exhibiting strong antimicrobial activity (3 to >5 log reduction) of E. hirae. Various combinations of AMP C1 (0.001563% - 0.0125% and thymol (0.00625% - 0.05%) covering C1 : thymol ratios from 0.03125:1 to 2:1 were tested against E. hirae Both C1 and thymol were used at levels that are not lethal to cells. Table 5 summarizes the antimicrobial activities of all these combinations. When used alone at the concentrations tested, C1 and Thymol were not bactericidal and thus do not exhibit antimicrobial activity. However, various combinations covering C1 and Thymol provided >3 log reduction in E. hirae counts in contact kill assay demonstrating their strong synergistic antimicrobial effect. The synergy was defined as enhancement in killing of bacteria by at least 1 log (i.e. 10-fold in percentage value) by a combination compared to additive effect of individual actives that are part of the combination. Further, the combination needs to exhibit at least 3 log reduction of viable counts of bacteria to be considered as strong antimicrobial for its utility. The combinations (1) 0.0125% C1 + 0.05% Thymol, (2) 0.00625% C1 + 0.05% Thymol, (3) 0125% C1 + 0.025% Thymol, (4) 0.00625% C1 + 0.025% Thymol, (5) 0.003125% C1 + 0.05% Thymol, (6) 0.001563% C1 + 0.05% Thymol, with C1 to thymol ratios ranging from 0.03125:1 - 0.5:1 exhibits synergy in their antimicrobial efficacy that kills E. hirae effectively. C1 and thymol ratios of 0.125:1 to 0.5:1 with C1 at 0.0125% and 0.0625% exhibited stronger antimicrobial activity with greater than 4.8 log reduction. Table 5. Antimicrobial efficacy: Log reduction of E. hirae viable counts achieved with various combinations of C1 and Thymol after 1 minute contact time at 20°C Example 4: Sanitizer and Disinfectant formulation Alcohol free Formulations were developed to realize the utility of synergistic activity of AMP C1 with thymol. Strong bacterial killing activity makes them useful as Sanitizers (primarily used on living surfaces for e.g., hand sanitizers) or disinfectants (used on non-living surfaces such as door knobs, kitchen surfaces, tables etc). The specific requirement of such compositions is to rapidly kill bacteria with in 1 minute to help reduce load of microorganisms with the objective of reducing chances of infections being transfer from person to person or objects to person. The antimicrobial activity of sanitizer / disinfectant formulations 1, 2 and 3 (Table 6) were tested for rapid bactericidal activity as per EN 1276 standard protocol using 4 bacteria prescribed by the standard. Bacteria tested were Staphylococcus aureus ATCC 6538, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 10536 and Pseudomonas aeruginosa ATCC 15442. The conditions used for the assay was dirty (0.3% final BSA concentration) and contact time was 1 minute. The synergistic combinations of C1 and thymol in prepared Formulation 4 exhibited superiority compared to Formulation 1 – 3 by exhibiting ≥4.8 log reduction of viable cell counts against all four tested bacteria after 1 minute contact time. Base formulation 1 (without C1 / thymol), Formulation 2 – 3 (with individual components of the invention) failed to exhibit any significant antimicrobial activity. Table 6. Alcohol free Sanitizer and Disinfectant formulation and its antimicrobial efficacy estimated as log reduction of viable counts of E. coli, S. aureus, P. aeruginosa and E. hirae achieved after 1 minute contact time at 20°C. Example 5: Antimicrobial Alcohol-free Deodorant Deodorants are used to overcome the body malodour that develops over time primarily in underarm, foot and groin areas. The malodour is developed due to microbial metabolism on the surface of skin [Rudden, M., Herman, R., Rose, M. et al. The molecular basis of thioalcohol production in human body odour. Sci Rep 10, 12500 (2020). https: / / doi.org / 10.1038 / s41598-020-68860-z]. Examples of odorant molecules include volatile molecules such as 3-methyl-3-sulfanylhexan-1- ol (MSSH), 3-methyl-2-hexenoic acid (3M2H) and 3-hydroxy-3-methylhexanoic acid (HMHA) that are produced in underarm skin area from the sebaceous secretions of human apocrine glands by some members of Corynebacterium present on human skin, including Corynebacterium striatum. Short chain fatty acids such as isovaleric acid with characteristic cheesy odour are produced by Staphylococcus epidermidis present on human skin by metabolizing leucine secreted in sweat, particularly on feet. The body odour is controlled using either antiperspirants or deodorants. Antiperspirants reduces extent of sweating thereby decreasing the food available for bacteria present on skin. The deodorant formulations work by either killing microbes through use of alcohols (ethanol) and other antimicrobials or / and by masking the malodour using fragrances. Most deodorants contain >50% ethanol and routine application causes skin dryness. Underarm skin also develops pigmentation (melanin production) resulting in darker underarms compared to other skin areas. Hence, it is recommended to use alcohol free deodorant. AMP C1 and thymol combination with potent antimicrobial activity was evaluated for application as deodorants. Deodorant spray formulation was prepared with concentration as mentioned in Table 7. The antimicrobial activity of these formulations was tested against bacteria responsible for the body odour, Staphylococcus epidermidis MTCC 435 and Corynebacterium striatum ATCC 6940. The prepared formulations 7 and 9 containing synergistic antimicrobial combinations exhibited >4 log reduction against both S. epidermidis and C. striatum after 1 minute contact time (Table 7). The present deodorant spray formulation can be applied as fine mist on skin (underarm, back, foot etc) on the area skin area of interest to kill the bacteria responsible for odour production on these sites. Table 7. Alcohol free Deodorant formulation and its antimicrobial efficacy estimated as log reduction of viable counts of E. coli, S. aureus, P. aeruginosa and E. hirae achieved after 1 minute contact time at 20°C. Example 6: Antimicrobial wipes The antimicrobial wipe formulations were tested for rapid bactericidal activity using 4 bacteria prescribed as per EN 1276 standard protocol. Bacteria tested were Staphylococcus aureus ATCC 6538, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 10536 and Pseudomonas aeruginosa ATCC 15442. The conditions used for the assay was dirty (0.3% final BSA concentration) and contact time was 1 minute. The wet wipes base, Formulation 9, do not have any antimicrobial activity. The use of synergistic combination of C1 and thymol in prepared formulation 10 provided ≥5 log reduction of viable cell counts of all four tested bacteria after 1 minute contact time. Table 8. Alcohol free Antimicrobial wipe formulation and its antimicrobial efficacy estimated as log reduction of viable counts of E. coli, S. aureus, P. aeruginosa and E. hirae achieved after 1 minute contact time at 20°C. Example 7 Screening medium-chain fatty acids (MCFA) Various medium chain fatty acids were tested in combination with C1 peptide. Antimicrobial efficacy against test bacteria as per EN1276 (1 minute, dirt condition) was carried out for screening. Medium-chain fatty acids (MCFAs) are saturated or unsaturated fatty acids, which present 6 to 12 carbon atoms. Lauric acid is solid at room temperature and was only partially soluble in water, hence was not included in the screening study. Other fatty acids, caproic, caprylic, capric acids were evaluated alone and in combination with C1 peptide. Medium-chain fatty acids caproic acid, caprylic acid and capric acid combinations was tested against the most challenging organism E. hirae. EN1276 protocol was carried out. Log reduction achieved after 1 minute in dirt conditions is reported. Table 9. Antimicrobial Efficacy of C1 peptide and combination with caproic acid, caprylic acid and capric acid in water. Log reduction achieved against E. hirae after 1minute contact time (EN1276, dirt condition) Caprylic acid is an eight-carbon chain fatty acid. Among the three tested medium chain fatty acids, caprylic acid demonstrated synergistic bactericidal activity against E. hirae in 1 minute. The ratio of C1 peptide and caprylic acid was 0.11: 1 and 0.08:1. At this ratio, caproic acid and capric acid did not show significant bactericidal activity in combination with C1 peptide. Example 8 Alcohol-free Antimicrobial Wipes Formulation example comprising C1 peptide and caprylic acid The antimicrobial wipe formulations were tested for rapid bactericidal activity as per EN 1276 standard protocol using 4 bacteria prescribed by the standard. Bacteria tested were Staphylococcus aureus ATCC 6538, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 10536 and Pseudomonas aeruginosa ATCC 15442. The conditions used for the assay was dirty (0.3% final BSA concentration) and contact time was 1 minute. Formulation for Wet wipes was prepared with base composition: Glycerin (1%), PEG-40 Hydrogenated Castor Oil (Cremophor CO-40) (0.25%), Decyl Glucoside (0.4%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Disodium EDTA (0.2%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine, pH 5. The antimicrobial efficacy of this formulation base supplemented with C1, Caprylic acid and their combination are described in below tables. Table 10. Antimicrobial Efficacy of C1 alone in wet wipes base, in 1- and 5- minute contact time and dirt condition The wet wipes base alone showed significant activity against Gram negative bacteria E. coli and P. aeruginosa and no activity against S. aureus and E. hirae (both Gram positive bacteria). The peptide C1 was titrated in the range 0.001 to 0.018% versus four test bacteria in base. Significant bactericidal activity noted against E. coli and P. aeruginosa (Gram negative bacteria) from 0.001% and higher concentrations. The activity observed in gram negative at 0.001% could be due to base itself. Against Gram positive S. aureus significant activity was noticed with C1 at ≥0.006% and against E. hirae ≥0.012%. Table 11. Antimicrobial Efficacy of caprylic acid in wet wipes base, in 1 and 5 minutes contact time and dirt condition The caprylic acid was titrated in the range 0.04 to 0.14% versus four test bacteria in base. Significant bactericidal activity noted against E. coli and P. aeruginosa (Gram negative bacteria) starting from 0.04% onwards. Against Gram positive S. aureus significant activity was noticed with caprylic acid at ≥0.07% and against E. hirae ≥0.11%. Table 12. Antimicrobial Efficacy of (C1 + caprylic acid) in wet wipes base, in 1 and 5 minute contact time and dirt condition The wet wipes base alone showed significant activity against Gram negative bacteria E. coli and P. aeruginosa and no activity against S. aureus and E. hirae (both Gram positive bacteria). The test sample mixture (caprylic acid + C1) was titrated versus four test bacteria in base. Significant bactericidal activity noted against E. coli and P. aeruginosa followed by S. aureus in all tested combination concentrations. Against E. hirae, synergistic bactericidal activity was observed in certain combinations only suggesting that cidal activity against E. hirae is challenging. Among the concentrations ranges tested, 0.05% caprylic acid + 0.012% C1 and 0.11 caprylic acid +0.003 % C1 lead to significant cidal activity against all 4 bacteria. They represent lowest caprylic acid and C1 peptide-based combinations respectively. Synergy: The synergy was defined as enhancement in killing of bacteria by at least 1 log (i.e.10-fold in percentage value) by a combination compared to additive effect of individual actives that are part of the combination. Further, the combination needs to exhibit at least 3 log reduction of viable counts of bacteria to be considered as strong antimicrobial for its utility. Since base alone led to significant log reduction of 4.94 and 5.1 respectively against Gram negative bacteria, E. coli and P. aeruginosa, synergy was considered with respect to only Gram positive bacteria E. hirae and S. aureus. Among four test bacteria, E. hirae is considered as challenging bacteria for antimicrobials owing to its membrane architecture. Hence demonstrating synergy against E. hirae is important for developing efficacious formulation. Since the both actives were fast acting i.e. demonstrating rapid cidal activity, synergy was more evident in 1 minute time point, rather than at 5 minutes. Synergy against Gram positive bacteria E. hirae Activity against E. hirae in 1 minute: The C1 + caprylic combinations of below ratios demonstrated 3 to >5 log kill against 4 test bacteria 0.027:1, 0.05:1, 0.06:1, 0.08:1, 0.17:1, 0.24:1, 0.3:1, The below mentioned C1+ caprylic acid ratio demonstrated synergy against E. hirae in 1 minute. ^ 0.05:1, 0.06:1 and 0.08:1 5 minute: Synergistic ratio 5 minute contact time: The C1 + caprylic acid ratio demonstrated synergy against E. hirae ^ 0.12:1 and 0.15:1. ^ 0.009:1 - At low peptide concentration (0.001%) in combination with caprylic acid (0.11%), synergy against E. hirae was observed at 0.009:1 (C2 peptide: caprylic acid). The below peptide + ratios achieved 4 to > 5 log kill in 5 minute against 4 test bacteria 0.027:1, 0.05:1, 0.06:1, 0.08:1, 0.12:1, 0.15:1, 0.17:1, 0.24:1, 0.3:1, In wet wipes base formulation, C1 peptide and caprylic acid demonstrated synergy against E. hirae in 3 ratios in 1 minute (0.05:1, 0.06:1 and 0.08:1) Further, after contact time of 5 minute, synergy was observed in the C1 peptide + caprylic acid at ratio 0.009:1, 0.12:1 and 0.15:1 against E. hirae. Example 9 Comparison of the activities of peptides C1 and LL-37 by MIC determination: Minimum Inhibitory Concentration (MIC) of peptides C1 and LL-37: MIC is the lowest concentration (in μM) of the peptide that inhibits the growth of a given strain of bacteria. The MIC of peptides C1 and LL-37 (having Seq ID 1 and 2 respectively) was determined against P. aeruginosa (ATCC 15442) and S. aureus (ATCC 6538). LL-37 peptide having sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Seq ID 2) was used in the experiment. The MIC of the peptide was evaluated using broth microdilution assay in 96 well microtiter plate. The results obtained are shown below in Table 13 which indicates the difference in the MIC values of both the peptides C1 against Gram negative bacteria P. aeruginosa and Gram positive bacteria S. aureus and E. hirae. Table 13 : Comparative data of C1 and LL-37 All the actives (Essential Oil and medium-chain fatty acids) used in the present invention have been commercially procured from outside India and does not include any substance sourced from India.

Claims

Claims:

1. A synergistic combination of antimicrobial peptide having Sequence ID 1 or salts and derivatives thereof and at least one active selected from thymol and caprylic acid.

2. The synergistic combination as claimed in claim 1, wherein said combination achieves >5 log kill in 1 minute for both Gram positive and Gram negative organisms.

3. The synergistic combination as claimed in claim 1, wherein said salts is selected from trifluoro acetate (TFA) salt or acetate salt or chloride or lactate or malate.

4. The synergistic combination as claimed in claim 1, wherein said Gram positive organisms is selected from Enterococcus hirae, Staphylococcus aureus.

5. The synergistic combination as claimed in claim 1, wherein said Gram negative organisms is selected from Escherichia coli, Pseudomonas aeruginosa.

6. The synergistic combination as claimed in any of the preceding claims, wherein said C1 peptide or salts and derivatives thereof is present in an amount 0.00156 to 0.0125% by weight.

7. The synergistic combination as claimed in any of the preceding claims, wherein said thymol is present in an amount 0.0125 to 0.025% by weight.

8. The synergistic combination as claimed in any of the preceding claims, wherein said caprylic acid is present in an amount 0.05 to 0.11% by weight.

9. The synergistic combination as claimed in claim 1, wherein said combination comprises 0.00156 to 0.0125% by weight of C1 peptide or salts and derivatives thereof and 0.025 to 0.05% by weight of thymol in a ratio 0.03125:1 to 0.5:

1.

10. The synergistic combination as claimed in claim 1, wherein said combination comprises 0.001 to 0.012% by weight of C1 peptide or salts and derivatives thereof and 0.04 to 0.11% by weight of caprylic acid in a ratio 0.009:1 to 0.3:

1.

11. An antimicrobial composition comprising (a) a synergistic combination as claimed in claims 1 to 10 and (b) cosmetically acceptable excipients.

12. The antimicrobial composition as claimed in claim 11, wherein said cosmetically acceptable excipients are selected from preservative, pH modifier, humectant, fragrance, fragrance solubilizer.

13. The antimicrobial composition as claimed in claim 12, wherein said composition is used in wipes, sanitizer, body wash and hand wash, wound healing cream / lotion and surface cleaners.

14. An alcohol-free wet wipe formulation comprising (a) a synergistic combination as claimed in claims 1 to 10 and (b) cosmetically acceptable excipients.

15. The alcohol-free wet wipe formulation as claimed in claim 14, further comprising at least one excipient selected from the group consisting of preservative, pH modifier, humectant, fragrance, and fragrance solubilizer.

16. A method of disinfecting a surface, the method comprising the steps of: providing an alcohol-free wet wipe as claimed in claim 14 or 15; and wiping the surface with the wet wipe.

17. An alcohol-free deodorant formulation comprising (a) a synergistic combination as claimed in claims 1 to 10 and (b) cosmetically acceptable excipients.

18. The alcohol-free deodorant formulation as claimed in claim 17 further comprising at least one excipient selected from the group consisting of preservative, fragrance, and fragrance solubilizer.

19. A sanitizer and disinfectant formulation comprising (a) a synergistic combination as claimed in claims 1 to 10 and (b) cosmetically acceptable excipients.

20. The sanitizer and disinfectant formulation as claimed in claim 19 further comprising at least one excipient selected from the group consisting of preservative, fragrance, and fragrance solubilizer.

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