Description:FORM 2
THE PATENT ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
Title of the invention: “Bioedible nanocoating using zinc oxide nanoparticles”
Applicant:
NAME NATIONALITY ADDRESS
1. Marwadi University
2. Dr. Sejal Shah
INDIA

MARWADI UNIVERSITY, Rajkot-Morbi Highway, At Gauridad, Rajkot – 360003, Gujarat, India
(M) 9879740982
shreedattalawconsultancy@gmail.com
Chothani18preeti@gmail.com

3. Ms Amisha Patel
INDIA RK University, Rajkot - Bhavnagar Highway, Kasturbadham, Rajkot 360020
Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the Invention:
The present invention relates to the field of food and nanobiotechnology. More specifically, the present invention relates to the preparation of Nano bio edible fruit coating by using Carica Papaya zink oxide nanoparticles. This is green approach to use Carica papaya leaf extract as a reducing agent. Furthermore, the present invention also relates to the increase in the shelf-life of the strawberry by using bioedible nanocoatings zinc oxide nanoparticles.
Background of the Invention:
Fruits are an integral part of the human diet, providing essential nutrients, vitamins, and antioxidants. However, the inherent perishability of fruits, marked by their limited shelf life and susceptibility to post-harvest deterioration, poses significant challenges to the agricultural industry, retailers, and consumers. Fruit ripening, a complex physiological process comprising various stages, plays a pivotal role in determining the optimal harvest time, flavor development, and overall quality of the fruit. Repining involves the intricate interplay of various factors, including hormonal regulation, enzymatic activities, gene expression, and environmental cues. Understanding the molecular, biochemical, and physiological events that occur during this stage is essential for implementing effective strategies to control and manipulate fruit ripening for extended shelf life. The ripening process of fruits can lead to various losses in fruit yield, both during pre-harvest and post-harvest stages. These losses occur due to several factors associated with fruit ripening such as pre-harvest fruit drop, physiological disorders, pathogen attack, mechanical damage, loss of firmness and weight.

The ripening process of climacteric fruits have been managed using various technologies like ozone treatment, 1-Methylcyclopropene, controlled atmosphere storage, and low-temperature storage. In this connection, edible coatings plays an important role to control the ripening process and also edible coatings are easy to develop, inexpensive and becoming popular for managing climacteric fruit ripening. Therefore, it is essentially required to develop the efficient method for the improvement of the shelf-life of the perishable fruits.

In this connection, nanocoatings play a crucial role in improving the shelf life of various products by providing a range of protective functions. These coatings are applied at the nanoscale level, typically between 1 and 100 nm, and offer unique properties that can extend the longevity of items in different industries. Nanocoatings have an advantages such as barrier protection, anti-corrosion properties, antimicrobial and antifungal properties, improved mechanical strength and enhanced stability as compared to the conventional coatings. In this invention, zinc oxide (ZnO) nanoparticles based edible nanocoatings have been developed for the shelf-life improvement of the perishable fruits.

Generally, zinc is a nutritional need which influences human physiology. Zinc element has the structural, functional, and regulatory roles via its interactions with a plethora of enzymes and proteins. Zinc deficiency causes various physiological disorders. It is naturally found in animal products like beef, poultry, and pork, but the body doesn't absorb it well from plant diets. Zinc fortified foods and supplements can boost zinc intake. Zinc oxide (ZnO) is one type of material that might be suitable for this purpose. The green plant based nanoparticles synthesizing is a nature-effective as well as cheap method to producing NPs compared to the other technique. The washing of fruit may help to reduce microbial growth, but not enough to prevent microbiological and non-microbial degradation during storage. ZnO NPs are one type of inorganic nanoparticle with the ability to inhibit growth of microorganisms.

Due to the extraordinary advantages, nanocoatings have been widely used for the shelf-life improvement of the harvested perishable fruits.

Hence, the present invention deals with the preparation of the edible nanocoatings using zinc oxide nanoparticles prepared from the Carica papaya leaf extract. The prepared nanocoatings are low cost and effective as compare to the conventional coatings. It also improves the shelf-life of fruits.
Object of the Invention:
The main objective of the present invention is to prepare eco-friendly bioedible nanocoatings from zinc oxide nanoparticles for the shelf-life improvement of perishable fruits.

Another objective of the present invention is to prepare the zinc oxide nanoparticles from the Carica papaya leaf extract used for the bioedible nanocoatings.

Yet another objective of the present invention is to characterize the Zinc Oxide nanoparticles using UV-Visible spectrophotometer, FT-IR (Fourier Transform Infrared), XRD (X-Ray diffraction), SEM (Scanning Electron Microscopy), EDAX (Energy Dispersive X-Ray Analysis) and PSA (Particle Size Analysis).

Yet another objective of the present invention evaluates the antimicrobial and antifungal activities of zinc oxide.

Yet another objective of the present invention is to analyse the effect of bioedible nanocoatings on the strawberry based on physical parameters such as effect on texture and effect on weight.

Yet another objective of the present invention is to provide cost-effective and easy to scale up techniques for the preparation of bioedible nanocoatings of the shelf-life improvement of perishable fruits.

Summary of the Invention:
The present invention relates to green synthesized zinc oxide nanoparticles-based edible nanocoatings for the shelf-life improvement of perishable fruits. Especially, the present invention is related to the preparation of zinc oxide nanoparticles from the Carica papaya leaf extract and prepared ZnO NPs were used as the edible nanocoatings which delays the ripening of fruits. The developed edible nanocoatings also possess excellent antimicrobial properties.
Brief description of Drawings:
For the better understanding of the present invention, there are shown in the drawings as below:
Figure 1 shows the synthesis of zinc oxide nanoparticles (ZnO NPs) using Carica papaya leaf extract as precursor.
Figure 2 shows the UV–Visible spectrum of ZnO NPs synthesized from Carica papaya leaf extract.
Figure 3 shows the FT-IR spectrum of ZnO NPs synthesized from Carica papaya leaf extract.

Figure 4 shows the particles size analysis of ZnO NPs synthesized from Carica papaya leaf extract.
Figure 5 shows the scanning electron microscope (SEM) analysis of ZnO NPs synthesized from Carica papaya leaf extract.
Figure 6 shows the Energy Dispersive X-Ray Analysis (EDAX) for elemental composition of ZnO NPs from Carica papaya leaf extract.
Figure 7 shows the XRD analysis of ZnO NPs from Carica papaya leaf extract.
Figure 8 shows the antimicrobial activity of ZnO NPs that describe different zone of inhibition comparing to positive and negative control.
Figure 9 shows the antioxidant activity of ZnO NPs coated fruit extract with compare to ZnO NPs and Ascorbic Acid.
Figure 10 shows the effect of ZnO NPs enabled nanocoatings for the shelf-life improvement of strawberry.

Detailed Description of the Invention:
Various aspects of the present invention will be described in detail in connection with the accompanying drawings, in order to provide a better understanding of the present invention.
Synthesis of zinc oxide (ZnO) nanoparticles
Preparation of Carica papaya leaf extract
The Carica papaya leaves were washed with distilled water and dried to remove the residual moisture for the formulation of the extract. The dried leaves were finely grinded using a mixer. The leaf extract was prepared by using 5.0 gm of dried powder in 100 ml of sterile distilled water followed by the boiling of mixture at 100°C for 20 to 30 minutes with continuous stirring. The mixture was cooled at room temperature and filtered out with Whatman No. 1 filter paper and stored at 4°C for further use.
Synthesis of zinc oxide (ZnO) nanoparticles from Carica papaya leaf extract
Briefly, 1.0 mM of zinc acetate was dissolved in distilled water followed by the addition of sodium hydroxide to maintain the pH. The solution was kept on a magnetic stirrer at 70° C temperature and 800 rpm for 4 hr. Then, 5.0 ml of plant extract was added drop wise to the 1.0 mM of zinc acetate solution at 60°C temperature with further stirring. The color change of the solution confirms the formation of ZnO nanoparticles. The solution was allowed to cool and subjected to the centrifuge at 5000 rpm for 10 min. The resulted pellets were collected and washed with the acetone followed by the preheating at 100° C temperature for 24 hr in crucible. Further, the calcination of product was carried out 400° for 4.0 hr to get the powder form of zinc oxide nanoparticles. Figure 1 depicts the process of formation of ZnO nanoparticles using Carica papaya leaf extract as a precursor.
Characterization of zinc oxide nanoparticles (ZnO NPs) prepared from Carica papaya leaf extract
UV-Visible analysis
ZnO NPs were dissolved in distilled water and sonicated using ultrasonicator probe prior to take the UV-visible spectra between 300 to 600 nm wavelengths by using UV-Visible spectrophotometer (Shimadzu UV-1900 Model no. 80595). Figure 2 shows the UV-Visible spectra of ZnO nanoparticles with the ?max value of 360 nm. The band ascribed to excitation of valence electrons of ZnO arranged in the nanoparticles (nanocrystal/nanosphere). The shape of the band is symmetrical which suggest the uniform scattering of spherical shape nanoparticles.
FT-IR analysis
FT-IR analysis is carried out to identify the functional group of ZnO NPs prepared from the Carica papaya leaf extract. The prepared ZnO NPs demonstrated the biological activity of the molecules due to stabilization of ZnO NPs in aqueous medium. Figure 3 shows the FT-IR spectrum of ZnO NPs. The sample has absorption peaks in the range of 670-3330 cm-1. The peaks at 2925.8 cm-1 and 3437.8 cm-1 are ascribed to the stretching vibration of hydroxyl compounds. Furthermore, peak at 670.01 cm-1 is the characteristic absorption of Zn-O band which confirms the hexagonal phase of ZnO NPs and broad absorption peak at 3328.06 cm-1 can be attribute to the -OH stretching vibrations. The peaks at 2912.3 cm-1 and 2883.1 cm-1 can be attributed to the C-H stretching in alkenes and O-H stretching in carboxylic acid, respectively. The peak at 1451.8 cm-1 can be attributed to the C=C stretch in aromatic ring and C=O stretch in poly phenols present in the compound. The C-N stretch of amid-1 in protein and C-O stretching in amino acid peaks are observed at 1328.8 cm-1 and 1381.0 cm-1, respectively.
Particle size analysis (PSA)
The synthesized ZnO NPs were analyzed using PSA which provided the size and zeta potential of the NPs. It is a device which count the PSA through beam of light making a size spreading from smaller to larger dimension. Figure 4 shows the particle size analysis of ZnO NPs which confirms that the dimensions of the ZnO NPs are in micron size.
Morphological analysis
The ZnO NPs derived from leaf extract were characterized by SEM and EDAX analysis to understand the morphology of the nanoparticles. The SEM analysis was carried out by applying a thin coating of gold on ZnO NPs which is largely invisible to electrons and X-rays at particular energies, though minor peaks of the coating metal can be seen in the spectrum. SEM micrographs (Figure 5) reveals that the small particles of ZnO are present surrounded by relatively large agglomerated regions. However, it is possible to predict the average size of the individual particle by selecting the specific regions. The average size of the particles calculated by analyzing the SEM micrograph is 230 nm. Furthermore, stereo chemistry and compositional analysis of ZnO NPs were carried out by the energy dispersive X-ray spectrometer (EDAX) as shown in Figure 6 which confirms the presence of Zn and O in the ZnO sample. The EDAX results revealed that the element present in the synthesized sample is ZnO nanomaterial as the atomic percentage of ZnO element is 20.09% which is greater than the other elements. The presence other peaks are due to the noise peaks.
X-ray diffraction analysis (XRD)
The purity and crystallinity of the ZnO nanoparticles was analyzed by subjecting the ZnO NPs to the X-ray diffractometer (Bruker D8 Advance Kß radiation) with a scan rate ranging from 20 to 80?. The obtained XRD spectra confirms the presence of base centred monoclinic phase of ZnO nanoparticle which corresponds to the JCPDS card number 36-1451. As shown in Figure 7, it is clearly observed that the synthesized ZnO NPs from Carica papaya leaf extract are pure and highly crystalline in nature.
Antimicrobial activity of ZnO NPs:
1.0 gm of ZnO NPs was dissolved in 100 ml of distilled water and sonicated for 5.0 min in ultrasonicator prior to check the antimicrobial activity of ZnO NPs. Antimicrobial activity was carried out using agar well diffusion method. Nutrient agar plates were used to check antimicrobial activities. Briefly, 100 µl of test culture was spread using sterile glass spreaders on the Nutrient agar plate and it was incubated for 10 min for the inoculums to absorb into an agar medium followed by diffusion well made by using sterile cup borer. Further, 1.0 µl of ZnO NPs was added into the well. Zinc Acetate solution without plant extract was used as a negative control whereas penicillin disk used as a positive control for the comparison of antimicrobial activities. The following table shows the zone of inhibition for the antimicrobial activities of tested compound against (A) Serratia, (B) Pseudomonasaeruginosa, (C) S. epidermise, (D) P. aerogenase. Figure 8 shows the antimicrobial activity of tested compounds.

Table 1: Antimicrobial activity of ZnO NPs, Penicillin antibiotic, zinc acetate, plant extract and distilled water against pathogens.
Pathogens Zone of Inhibition
ZnO NPs Penicillin antibiotic Zinc Acetate Plant Extract D/W
Serratia 10mm±5mm 10mm±3mm 10mm±3mm -- --
Pseudomonas aeruginosa 8mm±2mm 10mm±2mm 8mm±2mm -- --
Streptococcus
Epidermise 8mm ±3mm 10mm±2mm 8mm ±2mm -- --
Streptococcus
Pneumoniae 8mm±4mm 8mm±5mm 8mm±3mm -- --
From the results, it was observed that the ZnO NPs provided good antimicrobial activity as compare to the penicillin antibiotic, zinc acetate, plant extract and distilled water against both gram negative and gram-positive bacteria.
Coating of ZnO NPs on strawberry:
To provide the edible coating to the fruits, 5 % solution of ZnO NPs in distilled water is used as nanocoatings on strawberry to evaluate the efficiency of coatings.
Table 2: Physical Morphology of ZnO NPs coated strawberry and non-coated strawberry
Morphology Coated Strawberry Control
(Non- Coated Strawberry)
Size 1.4 cm 1.4 cm
Colour Red Red
Colour Change Red Dark Red
Weight 7.73 gm 7.71 gm
Weight loss (after 48 hours) 0.6 gm 0.80 gm
Texture Non Sticky Non Sticky
Texture (after 48 hours) Non Sticky Sticky

Antioxidant activity:
Antioxidant activity for the removal of free radical was performed using DPPH (1,1-diphynyle-2-picrylhydrazyl) assay. DPPH is considered as stable radical based on electron transfer that results in violet solution in methanol, which provides strong absorption band at 517 nm. The sets of various concentrations of various set, zinc oxide nanoparticle coated fruit extract and ascorbic acid solution were prepared in absolute methanol. 0.2 mM (0.007gm) solution was added to all the tubes and incubated in dark for 30 min. Absorbance was measured at 517nm using UV-Visible spectrophotometer. This test done in triplicates and % inhibition was calculated by the given equation.

% Inhibition=(OD of control-OD of sample)/(OD of control)×100
Figure 9 shows the antioxidant activity of ZnO NPs coated fruit extract with compare to ZnO NPs and Ascorbic Acid. In the graph, Blue color shows the inhibition of ZnO NPs coated fruit extract, orange color shows the inhibition of ZnO NPs and grey color shows the inhibition of ascorbic acid.
Table 3: Antioxidant activity of ZnO NPs coated fruit extract nanoparticle synthesized by Carica papaya leaf extract.
Concentration
(µg/ml) %Inhibition at 517 nm
Plant Extract NPs Coated Fruit extract NPs Coated Fruit extract Ascorbic acid
10 298±0.3 366±0.4 395±0.48 396±0.52
20 299±0.4 369±0.38 398±0.58 416±0.64
30 304±0.28 380±0.47 396±0.28 446±0.58
40 312±0.40 399±0.38 408±0.35 459±0.67
50 328±047 405±0.56 416±0.42 494±0.71

Main embodiment of the present invention is a bioedible nanocoating using zinc oxide nanoparticles comprising of:
(a) Zinc Oxide nanoparticles prepared from Carica papaya leaf extract; and
(b) Edible nanocoatings using 5 % of ZnO nanoaprticles solutions in distilled water; wherein said nanoparticles based edible coating is durable, eco-friendly, cost-effective, comfortable, and sustain antimicrobial and antioxidant properties.
Another embodiment of the present invention is a bioedible nanocoating using zinc oxide nanoparticles is prepared from Carica papaya leaf extract comprising the steps of:
(a) Preparation of Carica papaya leaf extract by washing Carica papaya leaves with distilled water and dried to remove the residual moisture for the formulation of the extract, the dried leaves were finely grinded using a mixer, the leaf extract was prepared by using 5.0 gm of dried powder in 100 ml of sterile distilled water followed by the boiling of mixture at 100°C for 20 to 30 minutes with continuous stirring, then the mixture was cooled at room temperature and filtered out with Whatman No. 1 filter paper and stored at 4°C for further use;
(b) Synthesis of zinc oxide nanoparticles from Carica papaya leaf extract by dissolving 1.0 mM of zinc acetate in distilled water followed by the addition of sodium hydroxide to maintain the pH;
(c) The said solution was kept on a magnetic stirrer at 70° C temperature and 800 rpm for 4 hr;
(d) 5.0 ml of plant extract was added drop wise to the 1.0 mM of zinc acetate solution at 60°C temperature with further stirring provides the color change which confirms the formation of nanoparticles;
(e) Centrifugation of solution at 5000 rpm for 10 min to get the pellets of the product;
(f) Pellets were collected and washed with the acetone followed by the preheating at 100° C temperature for 24 hr in crucible; and
(g) Calcination of product was carried out 400° for 4.0 hr to get the powder form of zinc oxide nanoparticles.
Another embodiment of the present invention the nanocoating prepared from ZnO nanoparticles was applied on strawberry at the 5 % concentration of ZnO in distilled water.
Another embodiment of the present invention a zinc oxide nanoparticles synthesized from Carica papaya leaf extract exhibit high anti-bacterial and antioxidant potential.
Effect on texture:
Moisture loss was the main factor for fruits damage. It may occasionally occur that a nanocoatings blocks the pores on a fruit's surface which restrict the respiration and may damage the physiological properties of the fruits. It was observed that the ZnO NPs did not obstruct the fruit's surface pores. Additionally, ZnO NPs have provided an antibacterial and antioxidant benefit without harming fruit surfaces. It was also observed that the ZnO dissolved in water provided the better results as compared to the ZnO dissolved in alcohol. Hence, ZnO NPs dissolved in water is suitable for coating as they don't damage the fruit's surface pores. Moreover, the texture of non-coated strawberry turns non-sticky to moist and sticky while coated strawberry remains unchanged for its physiological properties (Figure 10).
Effect on weight:
To identify the impact of coatings on strawberry, the weight loss study was carried out for coated and non-coated strawberries. It was observed that the coated strawberry lost 0.6 gm of weight from the original weight of 7. 73 gm whereas the non-coated strawberry lost 0.8 gm weight from the original weight of 7.71 gm. Hence, the nanocoatings have proved to be the effective against the environmental effects and early ripening process. The result of weight loss and colour changing indicates that the ZnO NPs derived from the Carica papaya leaf extract provided the optimum coating on the fruits to improve the shelf-life of the perishable fruits. , Claims:We claim,
1. A bioedible nanocoating using zinc oxide nanoparticles comprising of:
(a) Zinc Oxide nanoparticles prepared from Carica papaya leaf extract; and
(b) Edible nanocoatings using 5 % of ZnO nanoaprticles solutions in distilled water;
Wherein said nanoparticles based edible coating is durable, eco-friendly, cost-effective, comfortable, and sustain antimicrobial and antioxidant properties.
2. A bioedible nanocoating using zinc oxide nanoparticles is prepared from Carica papaya leaf extract comprising the steps of:
(a) Preparation of Carica papaya leaf extract by washing Carica papaya leaves with distilled water and dried to remove the residual moisture for the formulation of the extract, the dried leaves were finely grinded using a mixer, the leaf extract was prepared by using 5.0 gm of dried powder in 100 ml of sterile distilled water followed by the boiling of mixture at 100°C for 20 to 30 minutes with continuous stirring, then the mixture was cooled at room temperature and filtered out with Whatman No. 1 filter paper and stored at 4°C for further use;
(b) Synthesis of zinc oxide nanoparticles from Carica papaya leaf extract by dissolving 1.0 mM of zinc acetate in distilled water followed by the addition of sodium hydroxide to maintain the pH;
(c) The said solution was kept on a magnetic stirrer at 70° C temperature and 800 rpm for 4 hr;
(d) 5.0 ml of plant extract was added drop wise to the 1.0 mM of zinc acetate solution at 60°C temperature with further stirring provides the color change which confirms the formation of nanoparticles;
(e) Centrifugation of solution at 5000 rpm for 10 min to get the pellets of the product;
(f) Pellets were collected and washed with the acetone followed by the preheating at 100° C temperature for 24 hr in crucible; and
(g) Calcination of product was carried out 400° for 4.0 hr to get the powder form of zinc oxide nanoparticles.

3. The bioedible nanocoating using zinc oxide nanoparticles as claimed in claim 1, wherein the nanocoating prepared from ZnO nanoparticles was applied on strawberry at the 5 % concentration of ZnO in distilled water.

4. The bioedible nanocoating using zinc oxide nanoparticles as claimed in claim 1, wherein zinc oxide nanoparticles synthesized from Carica papaya leaf extract exhibit high anti-bacterial and antioxidant potential.

Dated 27th July, 2023

Chothani Pritibahen Bipinbhai
Reg. No.: IN/PA-3148
For and on behalf of the applicant