Description:FIELD OF THE INVENTION
The present invention relates to an organo-nanoparticle composite composition with excellent surface adhesion and antimicrobial properties against a diverse range of pathogenic bacteria and fungi. More particularly, the present invention relates to an antimicrobial coating composition and a method of preparing the same.
BACKGROUND OF THE INVENTION
Surface contamination from virulent pathogens is a major source of disease outbreak. Recent global pandemic of SARS-COV-19 has emphasized the need to maintain hygienic, pathogen free surfaces. An alternative and more efficient route to continuous disinfection of common surfaces, is provided by antimicrobial surface coater that provides long term protection against pathogens. Coating formulations can vary to a great degree based on their chemical composition ranging from transition metal nanoparticles, polycationic gels, polymer brushes etc. The need for designing new antibacterial materials has led to the emergence of various organic derivatives, nanomaterials, metal complexes, amino acids, and peptides, with potential bactericidal activity. However, utility all known materials is limited mainly due to their activity being effective only against smaller group of pathogens and thus lack in broad-spectrum activity or they are highly toxic such that they adversely affect many different types of organisms other than pathogens. Materials known in the art also lack biocompatibility with many different materials. Thus, main concept in designing of potential antibacterial agent is to ensure that it is selectively lethal to bacterial cells but non-toxic to mammalian cells. Naturally derived molecules provide a useful starting point for antibacterial solution. Molecules that show anti-bacterial activity are known to exist in natural products such as edible fruits, nuts, honey, garlic, ginger, clove, etc. Collection of such functionally active antibiotic found in the natural products is termed as a library of “antibiotic-ome” that includes different classes of polyphenols, dicarbonyls, carboxylic compounds such as allicin, polyphenol compounds like 6-gingerol, 12-gingerol, eugenol etc, which are used for antibacterial drug discovery. One such natural product is polyphenol derivative, tannic acid. Tannic acid, a specific form of tannin, is a mixture of poly galloyl glucoses or poly galloyl quinic acid esters with 2 to 12 galloyl moieties per molecule depending on the plant source used for extracting the tannic acid.
Tannic acid is an efficient antimicrobial agent, and tannic acid also acts as an anchoring group for a multitude of surfaces with exposed hydroxyl groups (e.g., glass, ceramic, paper etc) as it can form strong H-bonds with such surfaces. The polyphenolic groups also strongly bind with transition metals, making it an excellent choice for capping nanoparticles, preferably having antibacterial activity such as Ag NPs, TiO2 NPs, ZnO2 NPs etc, while simultaneously adhering to metal surfaces. This has led to widespread use of tannic acid based composite material for antibacterial applications.
Modified derivatives of tannic acid have been used for surface protection through various modes such as self-cross linking (Ren Liu et al., 2014), cross-linking through metal coordination (J.H Park et al., 2017), covalent attachment through polymeric anchor groups (Cheng et al., 2020), and being directly absorbed on the surface (Zhang et al., 2019).
However, in most of these cases, elaborate surface preparation and stringent self-polymerization conditions such as heat/UV radiation etc. are required for effective surface adhesion. This seriously limits the scope of tannic acid as an antimicrobial agent application. Although, crosslinking tannic acid through transition metal coordination can offer a more practical approach towards surface protection, this typically results in coloured films which increases opacity of surface and can often lead to aggregate formation. Moreover, protective films containing tannic acid alone does not offer adequate protection against a wide spectrum of pathogens.
To enhance antimicrobial efficiency of tannic acid films, metal nanoparticles such as silver (Cheng et al., 2020), gold (Han et al., 2020) are often introduced. These offer better resistance against infectious microorganisms. However, significant cytotoxicity arising out of these nanoparticles pose a serious challenge to its use.
Inventors of inventions described herein have developed an antimicrobial coating composition, which overcome the limitations of related compositions known in the art and does not require additional surface adhesion motif, alkaline pH. The antimicrobial coating composition described herein overcome the drawbacks known in the art by using nanoparticle-tannic acid composites, which allowed a wider applicability to said composition due to its transparent surfaces and minimum cytotoxicity, without a compromise on antimicrobial spectrum being covered.
 
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention.
The present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the tannic acid-alpha amino acid esters is selected from a group comprising: tannic acid- butyloxycarbonyl-glycine, tannic acid-glycine, tannic acid-alanine, tannic acid-phenylalanine, tannic acid-arginine, tannic acid-lysine.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the zinc salt is selected from a group comprising: zinc nitrate, zinc nitrate hexahydrate, zinc acetate.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the tannic acid-alpha amino acid esters is tannic acid-glycine and wherein the zinc salt is zinc nitrate hexahydrate.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the zinc oxide nanoparticles are of the diameter in a range of 15 to 25 nanometres.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein tannic acid-alpha amino acid esters is in a range of 0.3 to 0.6 wt.%; wherein zinc salt is in a range of 0.03 to 0.06 wt.%; and wherein the zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters is in a range of 0.05 to 0.2 wt.%.
In one of the aspects, the present invention provides an antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters have the tannic acid-alpha amino acid esters functionalised in any one of the ratios selected from: 16:10, 19:7, 21:5, 23:3 for free hydroxyl to amino acid esters.
In another aspect, the present invention provides a method for preparing an antimicrobial coating composition, the method comprising: - synthesizing a tannic acid-alpha amino acid esters; - deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and - capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; - adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters.
In one of the aspects, the present invention provides a method for preparing an antimicrobial coating composition, the method comprising: - synthesizing a tannic acid-alpha amino acid esters by preparing a tannic acid-butyloxycarbonyl-glycine; - deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and - capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; - adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters.
In one of the aspects, the present invention provides a method for preparing an antimicrobial coating composition, the method comprising: - synthesizing a tannic acid-alpha amino acid esters by preparing a tannic acid-butyloxycarbonyl-glycine; - deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and - capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; - adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, and wherein the tannic acid-butyloxycarbonyl-glycine is synthesized by: - dissolving tannic acid in dimethylformamide, followed by addition of sodium carbonate and butyloxycarbonyl-glycine with stirring under nitrogen; - allowing the mixture to cool followed by dissolving in water, and extraction into ethyl acetate; and - washing extracted layer with water followed by drying over sodium sulphate, to evaporate solvent and to obtain tannic acid-butyloxycarbonyl-glycine as a white solid.
In one of the aspects, the present invention provides a method for preparing an antimicrobial coating composition, the method comprising: - synthesizing a tannic acid-alpha amino acid esters; - deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and - capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; - adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the tannic acid-alpha amino acid esters is deprotected by - dissolving the tannic acid-alpha amino acid esters in ethanol, and adding hydrochloric acid dropwise; - stirring the mixture at room temperature and evaporating the solvent under vacuum; and - washing the solid obtained in above step with cold water to obtain deprotected tannic acid-alpha amino acid esters.
In one of the aspects, the present invention provides a method for preparing an antimicrobial coating composition, the method comprising: - synthesizing a tannic acid; - deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and - capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; - adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the zinc oxide nanoparticle is capped with tannic acid-alpha amino acid esters by - dissolving zinc salt in water by stirring at room temperature; - dissolving deprotected tannic acid-alpha amino acid esters in water, and slowly added to zinc solution from the above step at room temperature while stirring; and - obtaining the zinc oxide nanoparticles with tannic acid-alpha amino acid esters by filtering and vacuum drying.
In another aspect, the present invention provides a method of application of the antimicrobial coating composition comprising: (a) tannic acid-alpha amino acid esters; (b) zinc salt; and (c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters, wherein the composition is dissolved in water prior to application to a surface.

OBJECTIVES OF THE PRESENT INVENTION
It is the primary objective of the present invention to provide an antimicrobial composition and method of synthesis.
It is further objective of the present invention to provide a composite of tannic acid derived capping agent coated zinc oxide nanoparticles and cross-linked with a transition metal ion.
It is further objective of the present invention to provide a universal antimicrobial composition to be applied at any surface at neutral pH (7) and have no potential corrosivity.
It is further objective of the present invention to provide an antimicrobial composition with excellent surface adhesion properties and antimicrobial efficiency against a wide array of pathogenic bacteria and fungi.
ABBREVIATIONS
TA: Tannic acid
BOC: Butyloxycarbonyl
TA-glycine: Tannic acid + glycine
ZnO: Zinc Oxide
H12N2O12Zn: Zinc nitrate hexahydrate
DMF: Dimethylformamide
Na2CO3: Sodium carbonate
HCL: Hydrochloric acid
Na2SO4: Sodium sulfate
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments in the specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated composition, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are illustrative only and not intended to be limiting.
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
The terminology and structure employed herein is for describing, teaching, and illuminating some embodiments and their specific features and elements and does not limit, restrict, or reduce the spirit and scope of the invention.
According to the main embodiment, the present invention describes an organo-nanoparticle composite composition with an excellent surface adhesion and antimicrobial properties against a diverse range of pathogenic bacteria and fungi. The composition is non-toxic, environmentally friendly, and remains attached to a surface for an extended period of time, thereby providing antimicrobial protection for such extended duration.
In a specific embodiment, the present invention provides an antimicrobial surface coating composition comprising: TA-glycine; Zinc nitrate hexahydrate; TA-glycine capped ZnO nano particles; and distilled water.
In another embodiment, the present invention provides an antimicrobial surface coating composition comprising of TA-glycine; Zinc nitrate hexahydrate; TA-glycine capped ZnO nano particles; and distilled water.
In still another embodiment, the present invention provides an antimicrobial surface coating composition comprising of TA-glycine in the range of 0.3 to 0.6 g; Zinc nitrate hexahydrate in the range of 0.03 to 0.06 g; TA-glycine capped ZnO nano particles in the range of 0.05 to 0.2 g; and distilled water in the range of 99 to 99.9 g.
In another embodiment, the present invention provides a method for preparing an antimicrobial composition, wherein the method comprising following steps: (1) Synthesis of TA-BOC-glycine; (2) BOC deprotection; and (3) caping ZnO nanoparticle with TA-glycine.
In another embodiment, the present invention provides a method for preparing an antimicrobial surface coating composition, said method comprising following steps:
a. Synthesis of TA-BOC-glycine
i. dissolving tannic acid-alpha amino acid esters/ tannic acid (TA) in DMF (Dimethylformamide), followed by addition of Na2CO3 and Boc-glycine with stirring under nitrogen;
ii. allowing the mixture to cool followed by dissolving in water, and extraction into ethyl acetate;
iii. washing extracted layer with water followed by drying over Na2SO4, to evaporate solvent and to obtain TA-BOC-glycine (white solid);
b. BOC deprotection
i. dissolving TA-BOC-glycine in ethanol, and adding HCL (Hydrochloric acid) dropwise;
ii. stirring the mixture at room temperature and evaporating the solvent under vacuum;
iii. washing the solid obtained in above step with cold water to obtain deprotected TA-glycine as yellowish white solid;
c. caping ZnO nanoparticle with TA-glycine
i. dissolving zinc nitrate hexahydrate in water by stirring at room temperature;
ii. dissolving TA-glycine in water, and slowly added to zinc solution of step c(i) at room temperature with stirring, and
iii. obtaining the TA-glycine capped ZnO nanoparticles as white precipitate by filtering and vacuum drying.

In another embodiment, the present invention provides a method for preparing an antimicrobial surface coating composition, wherein the mixture of step a (i) is stirred under nitrogen for 4 hours at 60 °C; and in the step b (i) HCL is added dropwise over a period of 30 minutes; and in the step c (ii) solution is stirred for 72 hours.
In another embodiment, the present invention provides a method for preparing an antimicrobial surface coating composition, wherein the TA-glycine capped ZnO nano particles have the tannic acid functionalized by a 21:5 ratio of free hydroxyl to amino acid esters.
The antimicrobial surface coating composition described herein can be universally applied to any surface without requiring any specific pre-processing steps that cannot be applied to different types of surfaces. The coating composition of the present invention demonstrate an excellent surface adhesion potential and antimicrobial effects against a diverse range of pathogenic bacteria and fungi. Importantly, the composition does not require any specific surface pre-treatment process to be performed on surfaces before it being applied and even does not demand on harsh conditions like low pH for its application, which significantly impact the type of surfaces that can be coated and durability of the surface material. The coating composition of the present invention can be advantageously applied at neutral pH 7, which not only increases its applicability and acceptability, but also avoid any kind of adverse impact on the surface material, caused by harsh coating condition required by coating compositions known in the art. The advantageous characteristics of the surface coating composition described herein are attributed to using a selection of components and processing of such components in unique manner to develop the surface coating composition, which provide significant advantages in terms of wider applicability due to its coat being transparent and that the coating composition can be applied at neural pH conditions. The surface coating composition also demonstrated significant advantages in terms of being non-toxic without compromising on anti-bacterial or anti-fungal properties and afforded protection against wide range of pathogenic organism. In the specific method for the preparation of described coating composition, first a BOC protected glycine is allowed to react with TA to obtain TA-glycine, wherein glycine is protected and represented as “TA-BOC-glycine”. Further, glycine in the TA-BOC-glycine is deprotected by removal of BOC under acidic condition to free the amine group of the glycine and make it available for interaction with ZnO nanoparticle. More specifically the coating composition is prepared by dissolving tannic acid-alpha amino acid esters/ tannic acid (TA) in DMF (Dimethylformamide), followed by addition of Na2CO3 and Boc-glycine with continuous stirring under nitrogen atmosphere. the obtained mixture is allowed to cool undisturbed and then dissolved in water and extracted with ethyl acetate. The extracted layer is washed with water and dried over Na2SO4, to evaporate solvent and obtain TA-BOC-glycine as white solid. Obtained TA-BOC-glycine is then dissolved in ethanol, by gradual dropwise addition of HCL and agitation at room temperature. Finally, the solvent is evaporated under vacuum to obtain deprotected TA-glycine. The deprotected TA-glycine solid produced is washed with cold water to obtain yellowish white solid deprotected TA-glycine and is finally used for preparation of TA-glycine caped ZnO nanoparticle. Zinc nitrate hexahydrate is separately dissolved in water with continuous stirring at room temperature. A separate stock of TA-glycine prepared by dissolving the previously obtained TA-glycine in water and gradually adding the TA-glycine solution to Zinc nitrate hexahydrate solution with continuous stirring at room temperature to obtain white precipitates. Obtained white precipitate are then filtered, and vacuum dried to yield TA-glycine capped ZnO nanoparticles. The TA-glycine capped ZnO nano particles have the tannic acid-alpha amino acid esters/ tannic acid functionalized by a 21:5 ratio of free hydroxyl to amino acid esters. A detailed stepwise method of preparing TA-glycine capped ZnO nanoparticles is described herein in the following paragraphs.
Synthesis of TA-BOC-glycine: Dissolving tannic acid-alpha amino acid esters/ tannic acid in Dimethylformamide (DMF) followed by addition of Na2CO3 and Boc-glycine to said solution with continuous stirring for 4 hours at 60 °C under nitrogen atmosphere. The reaction mixture is allowed to cool to room temperature before being dissolved in water and extracted into ethyl acetate (3X 30 ml). The extracted layer was washed with distilled water before being dried over Na2SO4. The solvent was evaporated to afford TA-BOC-glycine as a white solid.

BOC deprotection: TA-glycine prepared by deprotecting TA-BOC-glycine. TA-BOC-glycine is dissolved in ethanol, followed by dropwise addition of 1 N HCl. The mixture is stirred at room temperature for 8 hours and finally the solvent is evaporated under vacuum. The resulting solid is washed with cold water, yielding a yellowish white solid as the deprotected TA-glycine.

ZnO nanoparticle caped with TA-glycine: Zinc nitrate hexahydrate dissolved in water and stirred at room temperature. A stock of TA-glycine is separately prepared in water and added to the Zinc nitrate hexahydrate solution at room temperature, with continuous stirring for 72 hours. The resulting white precipitate are filtered, and vacuum dried to yield TA-glycine capped ZnO nanoparticles.
Synthesis of antimicrobial surface coating composition: Finally different coating compositions containing TA-glycine caped ZnO nanoparticle were prepared and tested. Such composition comprises of TA-glycine, Zinc nitrate hexahydrate, TA-glycine capped ZnO NPs, and distilled water.
The following examples and experiments are for illustrative purposes only and not intended to limit the scope of this disclosure.

EXAMPLES
Example 1:
Synthesis of TA-BOC-glycine: TA-BOC-glycine is prepared by dissolving tannic acid-alpha amino acid esters/ Tannic acid (2 g, 1.2 mmols) in 4 mL of Dimethylformamide (DMF) Followed by adding Na2CO3 (1.27 g, 12 mmol) and Boc-glycine (1.05 g, 6 mmols) to obtained solution and stirring for 4 hours at 60 °C under nitrogen atmosphere. The reaction mixture is allowed to cool to room temperature before being dissolved in 40 ml of water and thereafter extracted into ethyl acetate (3X 30 ml). The extracted layer is washed with distilled water before being dried over Na2SO4. The solvent was evaporated to afford TA-BOC-glycine as a white solid (2.3 g).
Example 2:
BOC deprotection: TA-glycine is prepared by deprotecting TA-BOC-glycine. 2g of TA-BOC-glycine is dissolved in 20 ml of ethanol, followed by dropwise addition of 2ml of 1 N HCl over 30 minutes. The mixture is stirred at room temperature for 8 hours followed by evaporated of solvent under vacuum. The obtained solid is washed with cold water to yield deprotected TA-glycine as a yellowish white solid.
Example 3:
ZnO nanoparticle caped with TA-glycine: ZnO nanoparticle caped with TA-glycine are prepared by dissolving 0.15 g, 0.5 mmols of Zinc nitrate hexahydrate in 10 ml of water and stirred at room temperature. A separate stock of TA-glycine (1.98g, 1 mmols) in 10 ml water is prepared and added to the Zinc solution at room temperature, where it is stirred continuously for 72 hours to obtain white precipitates. The resulting white precipitate are then filtered, and vacuum dried to yield TA- glycine capped ZnO nanoparticles.
Example 4:
Formulation: Two separate antimicrobial composition are prepared, as per below table 1:

Table 1: Different antimicrobial compositions
Formulation TA-glycine (g) Zinc nitrate hexahydrate (g) TA-glycine capped ZnO NPs (g) Distilled Water (g)
Composition I 0.5 0.05 0 99.45
Composition II 0.4 0.05 0.1 99.45

Antimicrobial activity of composition I and II are assessed as per procedure of ASTM 2315-16. TAA-glycine compound composition, capped NPs (to be centrifuged, dried, and redispersed into water solution).

Table 2: Antimicrobial activity composition I:

Test Microorganism
Test Method
Contact Time
CFU/ml Percent reduction compared to control at time zero Log10 reduction compared to control at time zero
Staphylococcus aureus ASTM E2315- 16 Time Zero 2.6 X 10^6 N/A
30 Mins 1.3 X 10^3 99.95% 3.30
60 Mins 1.1 X 10^3 99.96% 3.37
Escherichia coli ASTM E2315- 16 Time Zero 2.5 x 10^6 N/A
30 Mins 1.1 x 10^4 99.56% 2.36
60 Mins 4.6 x 10^3 99.82% 2.74
Pseudomonas aeruginosa ASTM E2315- 16 Time Zero 3.5 x 10^6 N/A
30 Mins 1.6 x 10^4 99.54% 2.34
60 Mins 1.0 x 10^4 99.71% 2.54
Aspergillus niger ASTM E2315- 16 Time Zero 7.5 x 10^6 N/A
30 Mins 4.5 x 10^4 99.40% 2.22
60 Mins 3.3 x 10^4 99.56% 2.36
Candida albicans ASTM E2315- 16 Time Zero 5.9 x 10^6 N/A
30 Mins 5.6 x 10^4 99.05% 2.02
60 Mins 5.4 x 10^4 99.08% 2.04

Table 3: Antimicrobial activity composition II:

Test Microorganism
Test Method Contact Time
CFU/ml Percent Reduction Compared to Control at Time
Zero Log10 reduction compared to control at Time Zero
Staphylococcus aureus ASTM E2315- 16 Time Zero 2.6 X 10^6 N/A
30 Mins 1.2 X 10^2 99.99% 4.34
60 Mins 9 X 10^1 99.99% 4.46
Escherichia coli ASTM E2315- 16 Time Zero 2.5 x 10^6 N/A
30 Mins 1.2 x 10^3 99.95% 3.32
60 Mins 1.1 x 10^3 99.96% 3.36
Pseudomonas aeruginosa ASTM E2315- 16 Time Zero 3.5 x 10^6 N/A
30 Mins 2.0 x 10^3 99.94% 3.24
60 Mins 1.8 x 10^3 99.95% 3.29
Aspergillus niger ASTM
E2315- 16 Time Zero 7.5 x 10^6 N/A
30 Mins 3.4 x 10^4 99.55% 2.34
60 Mins 9.8 x 10^3 99.87% 2.88
Candida albicans ASTM E2315- 16 Time Zero 5.9 x 10^6 N/A
30 Mins 3.1 x 10^4 99.47% 2.28
60 Mins 1.8 x 10^4 99.69% 2.52

It is evident from Table 1 and 2, that the composition 2 containing TA-glycine capped ZnO NPs, has outperformed composition 1 in terms of antimicrobial efficiency.
Example 5:
Toxicity (OECD 423): Toxicity of composition I and II is evaluated according to OECD 423. In the oral toxicity test, Wistar Rats (Rattus rattus albanicus) with body weights ranging from 190 to 240 gm were tested. 300 mg/kg B.wt. followed by 300 mg/kg in step II, and subsequently 2000 mg/kg in steps III and IV, as per OECD 423. Doses were given to rats via oral gavage with an intubation cannula linked to a syringe. The dose volumes for individual animals were calculated using their body weights.

Table 4: Mean and Individual Body Weight and Mortality Data of Male Wistar Rats with Composition 1 (No.: number; M: Male; mg/kg: milligram per kilogram)
Animal. No. Step Dose, mg/kg B.wt Weight in gram Weekly % Bodyweight Gain Mortality
BD 8D 15D I II
1M I 300 230.4 240.8 252.9 4.5 9.8 None
2M I 300 239.6 251.2 262.0 4.8 9.3 None
3M I 300 238.9 248.2 261.6 3.9 9.5 None
Mean 236.30 246.73 258.83 4.42 9.54
± ± ± ± ± ±
S.D. 5.12 5.35 5.14 0.48 0.21
4M II 300 228.5 239.9 250.1 5.0 9.5 None
5M II 300 239.6 251.9 263.4 5.1 9.9 None
6M II 300 243.8 256.8 268.6 5.3 10.2 None
Mean 237.30 249.53 260.70 5.15 9.85
± ± ± ± ± ±
S.D. 7.91 8.70 9.54 0.17 0.37
7M III 2000 256.7 269.0 280.5 4.8 9.3 None
8M III 2000 254.8 266.4 278.1 4.6 9.1 None
9M III 2000 230.3 242.4 252.8 5.3 9.8 None
Mean 247.27 259.27 270.47 4.87 9.40
± ± ± ± ± ±
S.D. 14.72 14.66 15.35 0.36 0.33
10M IV 2000 228.1 238.9 252.3 4.7 10.6 None
11M IV 2000 245.2 258.2 270.0 5.3 10.1 None
12M IV 2000 252.1 263.7 274.5 4.6 8.9 None
Mean 223.90 234.60 245.80 4.78 9.78
± ± ± ± ± ±
S.D. 1.25 2.38 2.91 0.69 0.89

For Composition I, LD50 value is in the range of 2000-5000mg/Kg, as per table 4.

Table 5: Mean and Individual Body Weight and Mortality Data of Wistar Rats with Composition II (No.: number; M: Male; mg/kg: milligram per kilogram)
A. No. Step Dose mg/kg B.wt Weight in gram Weekly % Bodyweight Gain Mortality
BD 8D 15D I II
1F I 300 225.1 233.0 237.6 3.5 5.6 None
2F I 300 222.6 229.7 236.2 3.2 6.1 None
3F I 300 224.0 232.5 237.4 3.8 6.0 None
Mean 223.90 231.73 237.07 3.50 5.88
± ± ± ± ± ±
S.D. 1.25 1.78 0.76 0.30 0.29
4F II 300 229.2 235.6 242.4 2.8 5.8 None
5F II 300 214.6 221.7 227.5 3.3 6.0 None
6F II 300 224.9 233.1 239.6 3.6 6.5 None
Mean 222.90 230.13 236.50 3.25 6.10
± ± ± ± ± ±
S.D. 7.50 7.41 7.92 0.43 0.40
7F III 2000 240.1 Dead None
8F III 2000 223.5 232.0 236.4 3.8 5.8 None
9F III 2000 229.7 238.7 244.0 3.9 6.2 None
Mean 231.10 235.35 240.20 3.86 6.00
± ± ± ± ± ±
S.D. 8.39 4.74 5.37 0.08 0.32
10F IV 2000 231.0 239.5 244.9 3.7 6.0 None
11F IV 2000 238.9 247.5 253.2 3.6 6.0 None
12F IV 2000 230.4 237.9 243.1 3.3 5.5 None
Mean 223.90 234.60 245.80 4.78 9.78
± ± ± ± ± ±
S.D. 1.25 2.38 2.91 0.69 0.89

For composition II, LD50 value is in the range of 2000-5000mg/Kg, as per table 5.

Example 6:
Efficiency of surface adhesion: Surface adhesion properties of composition I and II are evaluated using UV- visible spectroscopic test that measures absorbance between 200- 800 nm. The residual absorbance of the quartz surfaces coated with composition I and II is measured after repeated water washing and is compared with the initial absorbance of each quartz surface. For coating quartz plates were separately immersed in composition I, and Composition II, respectively and maintained at room temperature for 15 minutes. The quartz plates were then allowed to cure for 180 minutes at room temperature (25 °C). The absorbance was measured between 200-800 nm. After that, each quartz plate was carefully washed with 10 ml distilled water, dried at room temperature for 180 minutes, and the absorbance was measured. The water washing and drying cycle was repeated 5 times. Further, the quartz plates were kept at ambient conditions of 25°C and normal atmospheric pressure for up to 60 days and absorbance recorded. The results are summarised in Table 6:
Table 6: UV-Vis for surface adhesion efficiency:
Formulation tested Absorbance @ 254 nm
Blank before coating After coating After 1st wash After 2nd wash After 3rd wash After 4th wash After 5th wash After 30 days @ ambient After 45 days @ ambient After 60 days @ ambient
Composition I 0.03 0.29 0.25 0.26 0.25 0.24 0.24 0.22 0.21 0.21
Composition II 0.03 0.33 0.31 0.31 0.29 0.28 0.28 0.25 0.23 0.23
Formulation tested Absorbance @ 430 nm
Blank before coating After coating After 1st wash After 2nd wash After 3rd wash After 4th wash After 5th wash After 30 days @ ambient After 45 days @ ambient After 60 days @ ambient
Composition I 0.02 0.14 0.16 0.14 0.15 0.14 0.14 0.10 0.09 0.08
Composition II 0.017 0.17 0.18 0.16 0.15 0.16 0.16 0.16 0.12 0.11

Both composition I and composition II efficiently coated quartz plates, as shown in table 6. After numerous washing cycles, the absorbance for both free ligand region (250-260 nm) and metal-tannic acid complex region (530-570 nm) remained stable, indicating a stable coat on the quartz plate.
UV-visible spectroscopic test was also carried out with TA-glycine capped ZnO NPs where functionalization of tannic acid-alpha amino acid esters/ tannic acid is carried out at a ratio different than 21:5 for free hydroxyl to amino acid esters and are summarized in table 7.
Table 7: Different ratios of free hydroxyl to amino acid esters
Formulation TA-glycine (g) Zinc nitrate hexahydrate (g) TA-glycine capped ZnO NPs (g) Ratio of Free hydroxyl to amino acid esters Distilled Water (g)
S1 0.4 0.05 0.1 23:3 99.45
S2 0.4 0.05 0.1 21:5 99.45
S3 0.4 0.05 0.1 19:7 99.45
S4 0.4 0.05 0.1 16:10 99.45

Effect of different ratios of free hydroxyl to amino acid esters on antibacterial efficiency on Gram positive and Gram-negative bacteria were conducted to assessed and identify the surface functionalization ratio, which results in optimal antibacterial effect in the final composition. The details are summarised in below Table 8:
Table 8: Effect of different ratios free hydroxyl to amino acid esters
Strain Zone of inhibition (diameter in mm)
S1 S2 S3 S4
E. coli (Gram negative) 9 9 9 8
Bacillus cereus (Gram positive) 4 10 8 8

TA-glycine capped ZnO NPs with functionalization of tannic acid-alpha amino acid esters/ tannic acid in 21:5 ratio with respect to free hydroxyl to amino acid esters, is most potent against microbes, as depicted in Table 8.
Example 7:
Comparison of present antimicrobial composition with antimicrobial composition known in the prior art
Table 1 defines compositions I and II, while composition III is prepared according to prior art (Cheng et al., 2020).

Table 9: Details of composition III
Formulation TA-glycine (g) Zinc nitrate hexahydrate (g) TA-glycine capped ZnO NPs (g) Tris Buffer (10 mM, pH = 8.5)
Composition III 0.5 0 0 99.5

The UV- visible spectroscopic tests carried out by coating quartz surface with composition I and II and residual absorbance was measured after repeated water wash. The initial absorbance of individual quartz measured, and the quartz plates immersed separately in composition I, II and III and kept at room temperature for 15 minutes. The quartz plate removed and dried under ambient condition at room temperature (25 ℃) for 180 minutes. The absorbance measured in the range of 200-800 nm. Each quartz plate carefully washed with 10 ml of distilled water, dried under ambient condition at room temperature for 180 minutes and absorbance measured. The washing with water, drying cycle was repeated 5 times.
Table 10: Comparison of composition I, II and III
Formulation tested Absorbance @ 254 nm
Blank pH at which compo. applied After coating After 1st wash After 2nd wash After 3rd wash After 4th wash After 5th wash After 30 days @ ambient After 45 days @ ambient After 60 days @ ambient
Composition I 0.03 7 0.29 0.25 0.26 0.25 0.24 0.24 0.22 0.21 0.21
Composition II 0.03 7 0.33 0.31 0.31 0.29 0.28 0.28 0.25 0.23 0.23
Composition III (Absorbance at 270 nm; shifted@ pH 8.5) 0.02 8.5 0.38 0.16 0.07 0.03 0.02 0.03
Formulation tested Absorbance @ 430 nm
Blank pH at which composition applied to surface After coating After 1st wash After 2nd wash After 3rd wash After 4th wash After 5th wash After 30 days @ ambient After 45 days @ ambient After 60 days @ ambient
Composition I 0.02 7 0.14 0.16 0.14 0.15 0.14 0.14 0.10 0.09 0.08
Composition II 0.017 7 0.17 0.18 0.16 0.15 0.16 0.16 0.16 0.12 0.11
Composition III 0.02 8.5 0.02 0.02 0.02 0.02 0.02

As evident from the table 10, composition III undergoes self-polymerization and provides a colourless coating that can be universally applied to any surface. However, said self-polymerization of Composition III, necessitates a higher basicity pH (8.5), which may cause unwanted corrosion of metals and ceramics. Furthermore, the resulting coating is unstable and almost completely removed i.e., 97 % by 3rd water wash..
Advantages of the present invention
a) a stable, antimicrobial coating
b) can be applied universally on any surface at neutral pH (7) and have no potential corrosivity
c) is colourless
d) has high durability
e) shows improved performance through inclusion of ZnO nanoparticles
f) is non-toxic. , Claims:1. An antimicrobial coating composition comprising:
(a) tannic acid-alpha amino acid esters;
(b) zinc salt; and
(c) zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters.
2. The antimicrobial coating composition as claimed in claim 1, wherein the tannic acid-alpha amino acid esters is selected from a group comprising: tannic acid- butyloxycarbonyl-glycine, tannic acid-glycine, tannic acid-alanine, tannic acid-phenylalanine, tannic acid-arginine, tannic acid-lysine.
3. The antimicrobial coating composition as claimed in claim 1, wherein the zinc salt is selected from a group comprising: zinc nitrate, zinc nitrate hexahydrate, zinc acetate.
4. The antimicrobial coating composition as claimed in claim 1, wherein the tannic acid-alpha amino acid esters is tannic acid-glycine and wherein the zinc salt is zinc nitrate hexahydrate.
5. The antimicrobial coating composition as claimed in claim 1, wherein the zinc oxide nanoparticles are of the diameter in a range of 15 to 25 nanometres.
6. The antimicrobial coating composition as claimed in claim 1, wherein tannic acid-alpha amino acid esters is in a range of 0.3 to 0.6 wt.%; wherein zinc salt is in a range of 0.03 to 0.06 wt.%; and wherein the zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters is in a range of 0.05 to 0.2 wt.%.
7. The antimicrobial coating composition as claimed in claim 1, wherein the zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters have the tannic acid-alpha amino acid esters functionalised in any one of the ratios selected from: 16:10, 19:7, 21:5, 23:3 for free hydroxyl to amino acid esters.
8. A method for preparing an antimicrobial coating composition, the method comprising:
- synthesizing a tannic acid-alpha amino acid esters;
- deprotecting the tannic acid-alpha amino acid esters to expose active sites thereof; and
- capping zinc oxide nanoparticles with tannic acid-alpha amino acid esters at the exposed active sites thereof using a zinc salt; and
- adding 0.3 to 0.6 % tannic acid-alpha amino acid esters, 0.03 to 0.06 % zinc salt, and 0.05 to 0.2 wt. % zinc oxide nanoparticles capped with tannic acid-alpha amino acid esters.
9. A method as claimed in claim 8, wherein the tannic acid-alpha amino acid esters is synthesised by preparing a tannic acid-butyloxycarbonyl-glycine.
10. A method as claimed in claim 9, wherein the tannic acid-butyloxycarbonyl-glycine is synthesized by:
- dissolving tannic acid in dimethylformamide, followed by addition of sodium carbonate and butyloxycarbonyl-glycine with stirring under nitrogen;
- allowing the mixture to cool followed by dissolving in water, and extraction into ethyl acetate; and
- washing extracted layer with water followed by drying over sodium sulphate, to evaporate solvent and to obtain tannic acid-butyloxycarbonyl-glycine as a white solid.
11. A method as claimed in claim 8, wherein the tannic acid-alpha amino acid esters is deprotected by
- dissolving the tannic acid-alpha amino acid esters in ethanol, and adding hydrochloric acid dropwise;
- stirring the mixture at room temperature and evaporating the solvent under vacuum; and
- washing the solid obtained in above step with cold water to obtain deprotected tannic acid-alpha amino acid esters.
12. A method as claimed in claim 8, wherein the zinc oxide nanoparticle is capped with tannic acid-alpha amino acid esters by
- dissolving zinc salt in water by stirring at room temperature;
- dissolving deprotected tannic acid-alpha amino acid esters in water, and slowly added to zinc solution obtained in the above step at room temperature while stirring; and
- obtaining the zinc oxide nanoparticles with tannic acid-alpha amino acid esters by filtering and vacuum drying.
13. A method of application of the antimicrobial coating composition as claimed in claim 1, wherein the composition is dissolved in water prior to application to a surface.