DESC:Priority Claim:

[0001] This application claims priority from the provisional application numbered 202141004180 filed with Indian Patent Office, Chennai on 30th January 2021 entitled “A composition of hand sanitizer and the process for preparation thereof”, the entirety of which is expressly incorporated herein by reference
Preamble to the Description

[0002] The following specification describes the invention and the manner in which it is to be performed:
DESCRIPTION OF THE INVENTION
Technical field of the invention
[0003] The present invention relates to the composition of a congenial sanitizer solution and the process for preparation of the same. More particularly, the invention relates to the composition of a sanitizer solution comprising of naturally occurring enzyme lysozyme i.e., lysozyme and nano silver along with excipients for prevention of the transmission of disease-causing pathogens through hand contact. The sanitizer solution shall be used as hand sanitizer or for surface disinfection.
Background of the invention
[0004] Hand sanitizer, also called as hand antiseptic or hand rub, is an agent applied to the hands for the purpose of removing common pathogens. Hand sanitizer has become one of the most desired hygiene products in today’s time when Covid 19 has emerged as a pandemic. One of the most common modes of transmission of pathogenic organisms is through hand contact. There are various kinds of hand sanitizers available in the market based on the active ingredients in the form of liquid, gel and foam. In most places hand washing with soap and water is generally preferred. However, their use is recommended when soap and water are not available for hand washing or when repeated hand washing is not preferred by the user due to skin conditions.
[0005] Most of the hand sanitizers available in the market today are less effective at killing certain kinds of pathogens and may deposit harmful chemicals on the skin. The deposited harmful chemicals on the skin are not easily removed. Further, wiping off the hand sanitizer before it has dried and less alcohol concentrations make the sanitizer less effective. Most of the sanitizers and disinfectants available in the market are of concern in terms of fire hazard, skin toxicity due to its corrosive nature and antimicrobial resistance.
[0006] Depending on the active ingredient used, hand sanitizers can be classified as one of two types: alcohol-based or alcohol-free. Additionally, there are antiseptic hand sanitizer too. Alcohol-based sanitizers are widely used, and they typically contain between 60-95 percent alcohol, usually in the form of ethanol, isopropanol, or n-propanol. Alcohol acts on dermal constituents of the skin and denatures proteins. Effectively neutralizes certain types of symbiotic microorganisms that are required for the first line of immune response against pathogenic organisms. Alcohol-free sanitizers are general disinfectants, such as benzalkonium chloride (BAC), or on antimicrobial agents, such as triclosan. Many hand sanitizers are harsh on the skin.
[0007] The effectiveness of the hand sanitizer to prevent transmission of disease depends upon the effective concentration of active ingredient in the product, quantity used, duration of exposure, frequency of use and susceptibility of the infectious agents to the active ingredients in the sanitizer. Generally, alcohol-based hand sanitizers applied on the skin for a period of 30 seconds, followed by complete air-drying can effectively reduce pathogens on the skin.
[0008] Alcohol based sanitizers are preferred to be used by many, mainly because they are cost effective. However, long term usage of alcohol-based sanitizer is not good for human skin. The long-term use of alcohol-based sanitizer causes inflammation, irritation, fastens the ageing process and removes the protective oil layer present on the skin. It adversely affects the nervous and vascular system present in the hand and the surrounding region. In addition to the adverse effects of alcohol-based sanitizer on skin, it is not easy to use as it drips through the hands easily and most of it gets evaporated and wasted while using. Further, accidental contact of alcohol sanitizer with eyes can cause a burning sensation and prolonged irritation.
[0009] Alcohol based sanitizers, being inflammable is risky especially when transported or during storage. As a precautionary measure, it is recommended to avoid lighters, candles, matchboxes and avoid contact with fire after using sanitizer.
[0010] In most countries, the use of hand sanitizer is restricted to hospitals and laboratories. After the onset of Covid 19, personal hygiene has taken a top priority. In the present scenario, hand sanitizer has become a household product. Considering the discussed facts, many people are using hand sanitizer and usage of the same is going to be on a long-term basis. There is a need to have natural and alcohol-free sanitizer composition, which is safe to use, non-inflammable, easy to store, transport, and is not harmful to the skin and at the same time it is effective against pathogens transmitted through hand contacts.

[0011] The Patent Application No. US9757328B2 entitled “Lysozyme gel formulations” discloses formulations of gelled lysozyme achieved by the addition of water to a lysozyme suspension in a solvent, such as an alcohol, with retention of enzymatic activity. It was surprisingly discovered that lysozyme itself is a gelling substance and, therefore, it can be advantageously formulated into topical compositions without the addition of other gelling substances such as cellulose, starch or other polysaccharides. The activity of the lysozyme is enhanced as compared to other formulations comprising lysozyme. The formulations contained in the present invention are useful in methods in the fields of therapeutics, disinfectants, sanitizers, personal hygiene, and cosmetics for human and veterinary use.
[0012] The Patent Application No. US20060115536A1 entitled “Glycerin based synthesis of silver nanoparticles and nanowires” discloses compositions and methods for the production of noble metal nanoparticles and nanowires conjugated to polyols or polymers. The present invention allows the incorporation of noble metal nanoparticles to a wide range of products such as body care products to exploit the biocidal properties of silver nanoparticles against bacteria, viruses and fungi.
[0013] The Patent Application No. US20030185889A1 entitled “Colloidal nano silver solution and method for making the same” discloses a colloidal nano silver solution which contains nano silver particles having diameters between 1 nm and 100 nm. The silver content in the colloidal solution is between 0.001% to 0.4% by weight. The colloidal nano silver solution also contains a gelling agent which includes, but is not limited to, starch or its derivative, cellulose or its derivative, polymer or copolymer of acrylate or acrylate derivative, polyvinyl pyrrolidone, alginic acid, and xanthogenated gel.
[0014] The Patent Application No. US20090232860A1 entitled "Colloidal metal-containing skin sanitizer" discloses disinfectant hand sanitizing compositions. In one embodiment, a hand sanitizing composition includes from 10 to 1500 ppm Nanosilver, from 0.01 wt % to 30 wt. % alcohol, at least 70 wt. % water, and a thickening agent. The hand sanitizer provides continued sanitization for an extended period of time and can be formulated to be dispensed as self-supporting foams.
[0015] The Patent Application No. CN107107199B entitled “Compositions comprising spherical and coral-shaped nanoparticles and methods of making the same” discloses nanoparticle composition includes a plurality of spherical-shaped nanoparticles and a plurality of coral-shaped metal nanoparticles, each coral-shaped metal nanoparticle having a non-uniform cross-section and a globular structure formed by a plurality of nonlinear strands joined together without right angles. The nanoparticle composition may be a one-part or multi-part composition. The nanoparticle composition can have a mass ratio of spherical nanoparticles to coral-shaped nanoparticles of about 5:1 to 20:1, about 7.5:1 to 15:1, about 9:1 to 11:1, or about 10:1, and/or a number ratio of spherical nanoparticles to coral-shaped nanoparticles of about 50:1 to 200:1, about 75:1 to 150:1, about 90:1 to 110:1, or about 100: 1. The nanoparticle compositions can be used for a variety of purposes, including as an antimicrobial agent (e.g., antiviral, antibacterial, or antifungal compositions), a fuel additive, or to treat fabrics.
Summary of the invention
[0016] The present invention overcomes the drawbacks of the existing technologies with respect to effective sanitization and disinfection. The present invention discloses a composition of a hand sanitizer and surface disinfectant for effective antimicrobial activity against bacteria, virus and fungi.
[0017] The composition of the hand sanitizer comprises lysozyme at a concentration in a range between 0.05% to 0.5% w/w, nano silver at a concentration in a range between 10 ppm to 50 ppm, a stabilizer 1 and stabilizer 2 at a concentration in a range between 5% to 10% w/w, skin conditioning agent at a concentration of 2% w/w and a phosphate buffer solution at a concentration in a range between 85% to 95% w/w. The composition also comprises vinylpyrrolidone copolymer, polythene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and the core silver nanoparticles having a diameter in the range of 3nm to 11nm. The silver content used in the composition of the present invention ranges between 10 - 50 ppm.
[0018] Lysozyme being a natural enzyme inactivates bacteria through hydrolysis of glycosidic linkages present in the peptidoglycan component of the cell wall. Peptidoglycan is a heteropolymer consisting of polysaccharides chains crosslinked by short peptide molecules. It exhibits antibacterial activity by catalyzing the hydrolysis of ß-(1-4) bond between N-acetyl muramic acid and N-acetyl glucosamine residues. Silver nanoparticles are highly potent antimicrobial agent, and it is effective against a wide range of microbes such as bacteria, fungus and virus with more than 99% antimicrobial activity at very low concentrations. Silver nanoparticles of the present invention are stable at high temperature and or in light, which makes them effective and safe during storage and transportation. Hence the combination of lysozyme with silver nanoparticles makes the composition effective in exhibiting synergistic antimicrobial activity with higher efficacy rates.

[0019] The present invention discloses a process for the preparation of the described composition. The process for the preparation of the sanitizer according to an embodiment of the invention involves preparation of silver nanoparticles and lysozyme in a phosphate buffer solution.
[0020] The composition of the present invention is useful as hand sanitizer and for surface disinfection with efficacy against a wide spectrum of micro-organisms. The composition is studied for its antimicrobial activity against bacteria such as Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella enterica and Methicillin Resistant Staphylococcus aureus. Fungi such as Candida albicans and Aspergillus niger. The antiviral activity is studied with surrogate Bacteriophage MS2.
[0021] The composition exhibited greater than 99% of efficacy against the tested organisms, both as hand sanitizer and for surface disinfection. The composition results in rapid and instant antimicrobial action with time ranging from 1 to 5 minutes and the residual efficacy on the surfaces up to 24 hours when used as surface disinfectant.
[0022] The composition is easy to use, alcohol-free, non-volatile, free of unpleasant smell, compatible with skin, non-leaching, fast-acting, and effective at low concentration and in short time application. It is effective against a wide antimicrobial spectrum and drug-resistant microbes, including Methicillin Resistant Staphylococcus aureus (MRSA), which makes it a unique product.
[0023] The hand sanitizer is prepared in the form of a free-flowing liquid, gel, foam and spray, which makes it versatile to be used depending upon user type and preferences and as it is free from alcohol, the composition is safe and non-toxic for human skin on long term usage.
Brief description of the drawings
[0024] Figure 1 illustrates the composition of the hand sanitizer according to an embodiment of the invention.
[0025] Figure 2 interprets the particle diameter distribution of nano silver by number using Dynamic Light Scattering (DLS).
[0026] Figure 3 illustrates the flow chart of a process of preparation of the composition of the sanitizer.
[0027] Figure 4 tabulates the antimicrobial activity of the sanitizer on the test organisms by ASTM E 2315 standard.
[0028] Figure 5 tabulates the antimicrobial activity of the disinfectant on the test organisms by EN 13697 Method.
[0029] Figure 6 tabulates the microbicide activity of the sanitizer composition with the surrogate Bacteriophage MS2.
Detailed description of the invention
[0030] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0031] The term “Hand sanitizer” refers to a disinfectant in the form of liquid, gel, or foam. It is generally used to decrease infectious agents on the skin, more precisely on the hands.
[0032] The term “Infectious Disease” refers to illness or disorders caused by pathogenic organisms such as bacteria, viruses, fungi or parasites.
[0033] The term “Lysozyme” refers a naturally occurring enzyme found in bodily secretions such as tears, saliva, milk and functions as an antimicrobial agent by cleaving the peptidoglycan component of the bacterial cell wall, leading to cell lysis and inactivation.
[0034] The term “Nanoparticle” refers to a particle of matter that is between 1 and 100 nanometers (nm) in diameter.
[0035] The present invention relates to a composition of a congenial sanitizer solution comprising of lysozyme and nano silver. The sanitizer composition of the present invention is effective in controlling the transmission of the pathogens prevailing in the hospitals and other public places. The invention also discloses a process for the preparation of the hand sanitizer using a specific protocol.
[0036] The present invention discloses an alcohol-free and non-inflammable sanitizer utilizing naturally occurring enzyme lysozyme and nano silver and is useful as hand sanitizer or for surface disinfection.
[0037] Figure 1 illustrates the composition of the hand sanitizer according to an embodiment of the invention. The composition of the hand sanitizer comprises lysozyme at a concentration in a range between 0.05 to 0.5% w/w, silver nanoparticles at a concentration in a range between 10 ppm to 50 ppm, a stabilizer 1 and stabilizer 2 at a concentration in a range between 5% to 10% w/w, skin conditioning agent at a concentration of 2% w/w and a phosphate buffer solution at a concentration in a range between 85% to 95% w/w.
[0038] Lysozyme used in the composition is from Hen Egg White source, with activity in the range of 10,000 to 30,000 FIP unit per milligram and it is prepared in phosphate buffer solution. The required amount of lysozyme is weighed from the stock stored at 4°C-6°C and the lysozyme solution is prepared with 0.05M phosphate buffer, pH 6.5 +/- 0.10. The nano silver solution is prepared by dissolving the specified amount of nano-silver in 0.05M phosphate buffer with pH 6.5 +/-0.10. The stabilizer 1 used in the composition of the present invention is a polyol falling in the category of C6 to C12 sugar alcohol and is colourless, odourless, hygroscopic, crystalline powder, soluble in water. The stabilizer 2 used is polyethylene glycol derivatives with molecular weight ranging from 400 to 600. The polymeric material includes, but is not limited to vinyl pyrrolidone copolymer, polyvinyl pyrrolidone and polyvinyl alcohol. The hand sanitizer composition optionally contains glycerin 2% w/w as a skin conditioning agent. This ingredient may be omitted in the composition for surface disinfection according to an embodiment of the invention.
[0039] The phosphate buffer solution used in the composition is prepared at a concentration of 0.05M, pH 6.5+/-0.10. The buffer solution is prepared by dissolving appropriate amount of disodium hydrogen phosphate and sodium dihydrogen phosphate salts in water. To start with, 0.05M sodium dihydrogen phosphate solution is prepared by adding water in the clean stainless-steel container. Sodium dihydrogen phosphate monohydrate is added under stirring condition at room temperature. The stirring is continued till the salts are dissolved thoroughly at room temperature for approximately 10 minutes. Secondly, 0.05M disodium hydrogen phosphate solution is prepared by adding water in a clean stainless-steel container by adding disodium hydrogen phosphate heptahydrate under stirring condition and allowing the solution to mix thoroughly for 10 minutes at room temperature. Finally, 0.05 M Sodium phosphate buffer is prepared by mixing 0.05M sodium dihydrogen phosphate solution and 0.05M disodium hydrogen phosphate solution in a required proportion and ratio in a stainless-steel container. The resultant solution is mixed thoroughly till a clear transparent solution is obtained.
[0040] Nano silver used in the present invention is a yellow-colored aqueous dispersion of silver nanoparticles, wherein the dispersion is characterized by having plasmonic peaks in the range of 390-420nm with missing peaks in the range of 330–335 nm and 650-720nm in UV-Vis spectrum. The dispersion has silver nanoparticles of spherical shape with majority of particles having an equivalent diameter in the range of 3-50 nm more specifically 3-11nm as depicted in the Figure 2, with the dispersion stability of the nano silver particles of the present invention is 8-12months. The Minimum Inhibitory Concentration (MIC) is lesser than 0.10 ppm.
[0041] Lysozyme (EC: 3.2.1.17) is a glycoside hydrolase, also known as muramidase or N-acetylmuramide glycanhydrolase. It is an antimicrobial enzyme produced by animals that forms a part of the innate immune system. It is a naturally occurring enzyme found in bodily secretions such as tears, saliva, milk and present in the cytoplasmic granules of macrophages and polymorphonuclear neutrophils (PMNs). High quantity lysozyme is found in Hen egg white. However, for industrial and commercial applications, lysozyme is produced from Hen egg white and using recombinant DNA technology.
[0042] Lysozyme functions as an antimicrobial agent by acting on the peptidoglycan molecule of the bacterial cell wall. Lysozyme inactivates bacteria by catalyzing the hydrolysis of the glycosidic linkages present in the peptidogylcan layer present outside the plasma membrane, forming the cell wall. The enzyme catalyzes the cleavage of ß-1,4 linkages between N-acetyl muramic acid (NAM) and N-acetyl glucosamine residues (NAG).
[0043] Silver is known for its antimicrobial activity. Silver used as nano silver in the present composition increases its antimicrobial activity multifold. The nanosized silver or silver nanoparticles are utilized in many antimicrobial applications including food packaging, wound healing, medical devices, textile, household commodities such as deodorants, paints, kitchen utensils etc. The silver nanoparticles are highly potent antimicrobial agent, and it is effective against a wide range of microbes such as bacteria, fungus and virus with approximately 99% antimicrobial activity at very low concentrations. Silver nanoparticles of the present invention are easy to synthesize and are stable at high temperature and or in light, which makes its storage process very effective.
[0044] Firstly, the silver nanoparticles exhibit antimicrobial activity by adhesion onto microbial cell surface. The silver nanoparticles being positively charged are attracted to the surface of bacteria or microbe, which is having a slight negative charge due to the presence of phosphate and carboxyl groups in the cell membrane. This electrostatic interaction facilitates the adhesion of the silver nanoparticles onto cell membranes. Upon adhesion onto the bacterial surface, the silver nanoparticles release the silver ions, which bind to cell membrane structures thus destabilizing the membrane potential and causing proton leakage. Cell wall destabilization increases bacterial permeability which in-turn leads to leakage of cellular contents and cell lysis.
[0045] The silver nanoparticles in addition to adhesion, it may also penetrate into the microbial cells and interact with cellular structures to exhibit antimicrobial action.
[0046] The combination of lysozyme and nano silver follow different mechanisms in exhibiting the antimicrobial activity. This includes adhesion of silver nanoparticles onto the microbial cell surface, penetration of silver nanoparticles inside the cell resulting in deactivation of intracellular structure of pathogenic microorganisms.
[0047] Silver and lysozyme perform independently and compliment each other performance in exhibiting the antimicrobial activity. The combination of nano silver and lysozyme produces a combined antimicrobial effect. The independent action of the key active ingredients produces a combined effect that is greater than the sum of the antimicrobial activity of the individual ingredient.
[0048] The present invention discloses a process for the preparation of the sanitizer composition. The process of the preparation of sanitizer according to an embodiment of the invention, involves preparation of silver nanoparticles and lysozyme in phosphate buffer solution.
[0049] Figure 3 illustrates the flow chart of a process of preparation of the composition of the sanitizer. The process (300) starts with a step (301) adding 0.05M phosphate buffer solution pH 6.5+/-0.10 at a concentration in a range between 85 to 95%w/w into a clean stainless-steel container. At step (302), stabilizer-1 at a concentration in a range between 4% to 8% w/w is added to the phosphate buffer with constant stirring and at a temperature of 40°C-50°C for 10 to 15 minutes. The stabilizer-1 used in the process is polyol. At step (303), the mixed solution is allowed to cool down to room temperature for 15 to 30 minutes. At step (304), the stabilizer-2 at a concentration in a range between 2% to 4% w/w is added under stirring condition and the solution is allowed to mix thoroughly for 10 to 15 minutes at room temperature. The stabilizer 2 used in the process is polyethylene glycol. At step (305), the lysozyme solution in phosphate buffer is added into the solution with gentle and constant stirring. At step (306), nano silver solution at a concentration in a range between 10ppm and 50ppm is added to the preparation with gentle and constant stirring for 10 minutes. Finally, at step (307), prepared solution is filtered and stored at room temperature. The process results in the formation of a clear pale-yellow solution.
[0050] Optionally, the process involves the addition of skin conditioning agent at a concentration of equal to or less than 2% w/w for the preparation of hand sanitizer.
[0051] The composition of congenial sanitizer of the present invention is useful as hand sanitizer and for surface disinfection. The sanitizer of the present invention is alcohol free thus is safe for human skin and devoid of any complications of storage and transportation. The hand sanitizer is noninflammable due to the absence of alcohol and provide better skin properties upon application.
[0052] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.
[0053] Evaluation of the antimicrobial performance of the sanitizer composition against bacteria, fungi and viruses as per global standard ASTM International and EN methodologies.
Example 1: Assessment of the antimicrobial activity of the composition of the present invention using a Time-Kill Procedure by ASTM E 2315 standard

[0054] The efficacy of the sanitizer of the present invention is evaluated for its antimicrobial activity against bacteria and fungi by ASTM E 2315, standard guide for the assessment antimicrobial activity by time-kill study. The liquid suspension time kill test is a standard method to measure the microbicidal performance of the liquid antimicrobial formulation. The test method uses soya bean casein digest agar and sabouraud dextrose agar as nutrient medium. Lecithin, polysorbate 80, sodium thiosulphate, histidine, saponin in phosphate buffer 0.0025 mol/l is used as diluent and neutralizer.
[0055] The microbial cultures are prepared in nutrient broth to obtain the required culture growth approximately 105 to 108 CFU/ ml. A specified volume of test product is placed in a sterile test vessel. A volume of microbial culture consisting of test organisms such as bacteria and fungi is inoculated into the sterile vessel. After the specified exposure time of 1 minute and 5 minutes, the surviving microorganisms are recovered by drawing an aliquot, neutralizing it and performing standard Pour plate Technique. The culture count is ascertained by dilution Blank and the adequate validation of neutralizing agent is also carried. The test is carried out in duplicate and average count is taken as CFU/ml.
[0056] The test organisms include bacteria such as Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella enterica and Methicillin Resistant Staphylococcus aureus and fungi such as Candida albicans and Aspergillus niger.
[0057] Figure 4 tabulates the antimicrobial activity of the sanitizer on the test organisms by ASTM E 2315 standard. The results interpreted that the sanitizer of the present invention exhibited greater than 99% reduction in the microbial load on the test organisms in 1 minute and 5-minutes exposure time.
Example 2: Quantitative nonporous surface test for the evaluation of bactericidal and fungicidal activity of a disinfectant by EN13697 Method.
[0058] The efficacy of the composition of the present invention is quantitatively analyzed for bactericidal and fungicidal activity on nonporous surface. The test organisms considered for the test includes bacteria such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus hirae and fungi namely Candida albicans and Aspergillus niger.
[0059] The composition of the present invention is analyzed for antimicrobial activity against various test organisms at 0 hour and after 24 hours as per the standard EN 13697 method. The values of the sanitizer or the disinfectant i.e., test product is compared with the activity at 0 hour and after 24 hours. The test is conducted by taking a loopful culture of each of the test organism Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus hirae and grown on soyabean casein digest agar media, incubated at 37±20C for 24 hours. The total number of cells are adjusted to 107 CFU/ml in normal saline at 620 nm. For Candida albicans, in 10 ml of diluent, a loopful of Candida albicans is suspended and the total number of cells adjusted to 107 CFU/ml. In case of Aspergillus niger, spores of Aspergillus niger are transferred into 10 ml of normal saline with 0.05% Tween-80 solution. The total number of spores adjusted to 107 spores/ml by counting under microscope.
[0060] The test is initiated by adding the test organism to the selected interfering substance and applied onto a stainless-steel metal disc. The metal disc represents an actual instrument or carrier of the test organism. The carrier refers to the material used to simulate the contaminated test surface. The metal disc is then left to air-dry to simulate the surface of the carrier of the test organism. The disinfectant of the present invention is applied to the inoculated area and allowed 5 minutes contact time. The carrier is submerged in the neutralizer solution to prevent continued activity. Immediately at the 0th hour, the sample is serially diluted and plated on soyabean casein digest agar media and incubated at 37±20C for 24 hours. The carrier is then submerged into the neutralizer solution to prevent continued activity and incubated for 24 hours. After 24 hours, the tubes are serially diluted and plated on soyabean casein digest agar media and incubated at 37±20C for 24 hours. The number of test organisms recovered are compared with the number of organisms recovered from control where the test organism is exposed to water instead of disinfectant.
[0061] Figure 5 tabulates the antimicrobial activity of the disinfectant on the test organisms by EN 13697 Method. The results indicated that the disinfectant of the present invention is 99.96% effective in reducing Escherichia coli, 99.97% effective against Staphylococcus aureus and 99.75% effective against Pseudomonas aeruginosa, 99.83% effective against Enterococcus hirae, 99.44% effective against Candida albicans and 99.14% effective against Aspergillus niger at the defined contact time.
Example 3: Assessment of the activity of microbicides against Viruses in Suspension by ASTM E 1052: 2020 method.
[0062] The efficacy of the sanitizer of the present invention is analyzed for its microbicidal activity against viruses in suspension with surrogate Bacteriophage MS2 with Permissive Host Cell, Escherichia coli. ASTM E 1052;2020 is a Standard practice to assess the microbicidal activity of the test substance against viruses in suspension. Bacteriophage MS2 is used as a surrogate for evaluation of antiviral Activity.
[0063] This test determines the efficacy of the test product to inactivate the virus in suspension. One part of the virus suspension is added to nine parts of the test substance and held at the desired temperature for the required contact time and then assayed for viable virus in an appropriate host system. Trypticase soya agar is used as a culture broth and Dey-Engley Neutralizing (DE) broth is used as a neutralizing medium.
[0064] Test and virus control substances are dispensed in 9-part equivalent volumes into sterile vessels. Test and virus control substances are each inoculated with 1-part equivalent volumes of the test virus. The test suspensions are held for the contact time of 5 minutes and 10 minutes at room temperature. Following exposure time, aliquots are withdrawn, neutralized and by tenfold serial dilutions into appropriate solution. The virus control suspension is neutralized in the same manner as the test suspensions. Following neutralization, viral suspensions are assayed to determine surviving viruses in comparison with Control without test product. Virus suspensions are assayed by plaque assay technique and quantitatively expressed as a Plaque forming unit (PFU) visible as an area of clearance.
[0065] Figure 6 tabulates the microbicides activity of the sanitizer composition with the surrogate Bacteriophage MS2. The sanitizer composition of the present invention shows greater than 99% reduction of Bacteriophage MS2 in 5 and 10 minutes, when tested as per ASTM E 1052:2020 guidelines.
[0066] The present invention discloses the composition of the sanitizer and the disinfectant to control the spread of infectious microbial diseases by sanitizing the surfaces and the human skin. This composition is easy to use, non-volatile, free of unpleasant smell, compatible with the skin, non-leaching, fast-acting, and effective at low concentration and in short time application. It is effective against a wide antimicrobial spectrum and drug-resistant microbes, including methicillin resistant Staphylococcus aureus (MRSA), which makes it a unique product.
[0067] The composition of the present invention possesses disinfecting and sanitizing capabilities through its effective antibacterial, anti-fungal properties, and antiviral properties and is safe for repeated use.
[0068] The active ingredients namely lysozyme and silver nano particles along with other excipients used in the sanitizer composition are suspended in the aqueous phase. The hand sanitizer is prepared in the form of a free-flowing liquid, gel form, foam and spray, which makes it versatile to be used depending upon user type and preferences. The active ingredients used in the composition are known to be used in the industry for oral administration and are hence compatible with human system. The hand sanitizer is safe to be applied on skin, unlike alcohol-based sanitizers that are inflammable and harsh on skin. In contrast to the alcohol-based compositions, the composition of the present invention is easier to store and handle.
[0069] The composition of the present invention is an eco-friendly sanitizer preparation with silver technology to enhance its efficacy in combination with lysozyme. The composition is safe to skin and effective against a broad spectrum of microorganisms such as gram positive and gram-negative bacteria, fungi and surrogate model Bacteriophage MS2.
[0070] The composition results in rapid and instant antimicrobial action with time ranging from 1 to 5 minutes and exhibits residual efficacy on the surface up to 24 hours when used as surface disinfectant.
,CLAIMS:We Claim,

1. A composition of sanitizer for antimicrobial activity, the composition comprises:
a. lysozyme at a concentration in a range between 0.05 to 0.5% w/w;
b. nano silver at a concentration in a range between 10ppm to 50ppm;
c. stabilizer 1 and stabilizer 2 at a concentration in a range between
5% to 10% w/w; and
d. 0.05M phosphate buffer solution, pH 6.5+/-0.10 at a concentration in a range between 85 to 95% w/w.
wherein lysozyme and nano silver are prepared in phosphate buffer and the nano silver used is a yellow-colored aqueous dispersion of silver nanoparticles.

2. The composition as claimed in claim 1, wherein the composition additionally comprises glycerin as skin conditioning agent at a concentration of 2% w/w in the hand sanitizer.

3. The composition as claimed in claim 1, wherein the stabilizer 1 used is polyol and stabilizer 2 used is Poly Ethylene Glycol (PEG).

4. The composition as claimed in claim 1, wherein nano silver exhibits antimicrobial activity in combination with lysozyme thus enhancing the antimicrobial activity of the composition.

5. The composition as claimed in claim 1, wherein the silver nanoparticles used are of spherical shape with majority of particles having an equivalent diameter in the range of 3-50 nm more specifically 3nm-11nm.

6. The composition is effective as alcohol-free hand sanitizer with antimicrobial action in the time range, starting from 1 minute to 5 minutes and the residual efficacy on surfaces up to 24 hours as surface disinfectant.

7. The composition as claimed in claim 1, wherein the sanitizer or the disinfectant exhibited greater than 99% antimicrobial activity against bacteria such as Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella enterica, Enterococcus hirae, Meticillin Resistant Staphylococcus aureus and fungi such as Candida albicans, Aspergillus niger and surrogate model Bacteriophage MS2 for antiviral activity.

8. The composition as claimed in claim 1, wherein the sanitizer is prepared in the form of free-flowing liquid, gel form, foam and spray.

9. A process for preparation of the hand sanitizer composition or for surface disinfection, the process (300) comprises the steps of:
a. adding 0.05M phosphate buffer solution, pH 6.5+/-0.10 at a concentration in a range between 85% to 95% w/w into a clean stainless-steel container (301);
b. adding a stabilizer-1 at a concentration in a range between 4% to 8% w/w to the phosphate buffer with constant stirring and at a temperature of 40°C-50°C for 10 to 15 minutes (302);
c. allowing the mixed solution to cool down to room temperature for 15 to 30 minutes (303);
d. adding a stabilizer-2 at a concentration in a range between 2% to 4% w/w under stirring and allowing the solution to mix thoroughly for 10 to 15 minutes at room temperature (304);
e. adding the lysozyme solution in the phosphate buffer to the mixed solution with gentle and constant stirring (305);
f. adding the Nanosilver solution at a concentration in a range between 10ppm to 50ppm to the preparation with gentle and constant stirring for 10 minutes (306); and
g. filtering the prepared solution and storing at room temperature (307);

wherein said process results in formation of a clear pale-yellow solution.

10. The process as claimed in claim 9, wherein lysozyme and nano silver are used in the form of solution prepared in phosphate buffer.