Influence of ZnO and CuO nanoparticle on the shelf life and physiochemical properties of guava (Psidiumguajava) fruits and juice

ABSTRACT This research was done to study the guava and guava juice treated with ZnO and CuO nanoparticles synthesized by the sol-gel method, and stored at room temperature (R.T.) for 15 days. The effectiveness of ZnO and CuO nanoparticles on all treatment was studied by using physiochemical characteristics such as pH, T.S.S. Moisture content, acidity and microbiological testing, morphological changes within the samples by S.E.M. and changes in structure by using x-ray diffraction technique at the interval of 0, 5, 10, and 15 days of storage period. The result indicates minimal changes in physicochemical parameters during the storage period, such as T.S.S. pH, acidity, and Microbial count of treated samples as compared to untreated samples. Coating ZnO and CuO nanoparticles on guava reduces the water loss and prevents microbes’ growth. Applying nanoparticles to guava and guava juice is beneficial in increasing the durability of guava and guava juice.


Introduction
Guava is a "super fruit" of significant nutrients in terms of vitamins A and C. A large amount of vitamin C in guava makes it a powerhouse for fighting against free radicals and oxidants present in the body that causes many degenerative diseases. Production of guava at a large scale, high perishable quality, and lack of proper technology are major factors responsible for the waste of many fruits in the fruit processing sector. Adding food additives and preservatives to guava fruit and guava juice improves their shelf life, which is essential for the benefit of the food industry (Queiroz, 2006). The main nutritional components of guava are vitamins, tannins, phenolic compounds, flavonoids, essential oils, sesquiterpene alcohols, and triterpenoid acids. These compounds of guava fruit have been associated with many health benefits (Haida et al., 2011).
Guava (Psidiumguajava) is a popular fruit with climacteric characteristics. It has a relatively short shelf life of (3-4 days) at tropical room temperature (28 ± 2°C) due to its physiological properties, disturbances, post-harvest infections, and aging (Lo'Ay & Doaa, 2020;Valencia-Chamorro et al., 2011). The stability of guava can be affected by many factors, such as storage temperature, relative humidity, packaging material, and the nature of the coating (Kocira et al., 2021;Lo'Ay & El-Khateeb, 2018). Guava fruits preserved in traditional packagings, like paper and/or plastic materials, have a lower shelf-life of merely seven to ten days at R.T (Kocira et al., 2021). Physiologically, guava is a climatological fruit with a high degree of breath ability and transpiration similar to various commodities such as bananas (Murmu & Mishra, 2017) and mushrooms (Mahajan et al., 2008). This necessitates the development of new technologies in order to increase its shelf life (Forato et al., 2015), provide better storage conditions, and improve its characteristics.
The edible coating is characterized as a thin layer on the outer surface of the fruits, which is invisible to the naked eye and acts as a barrier against heat transfer, respiration, and transpiration (Patel & Mishra, 2020). They may contain natural additives or chemicals and play an important role in extending the shelf life of fruits, as they increase the protective action capabilities of the fruit cuticle by preventing water loss, colour change, and reducing physico-chemical reactions and microbial spoilage, and in general, give the surface a shiny appearance (Lo'Ay & Dawood, 2017;Lo'Ay & El-Khateeb, 2018). To minimize the above losses in guava fruit and guava juice, current studies have been conducted to improve the shelf life and increase the acceptability of guava in order to improve the quality during storage and increase the shelf life of guava and make guava fruit more appealing through the use of coatings done by using wax, ZnO and CuO nanoparticles and by the use of ZnO and CuO nanoparticles as preservatives in guava juice (Hayat et al., 2005;Jarma-Arroyo et al., 2019).
As different functional inorganic nanoparticles, ZnO and CuO nanoparticles are known to inhibit microbial growth and physiochemical changes in fruit and fruit juice. Due to their antibacterial properties, ZnO and CuO nanoparticles are increasingly used in the food industry and have been listed as "safe" by the U.S. Food and Drug Administration (U.S.F.D. A.) (Lakshmi et al., 2018). Against this background, the objective of this study was to investigate the influence of Zinc oxide (ZnO) and Copper Oxide (CuO) nanoparticles synthesized by gel-sol method on quality parameters of guava and guava juice such as total dissolved solids, pH, acidity, water loss, S.E.M. for morphology, XRD to study crystallinity, and strain of samples to control texture and stability of guava at different storage times and different microbial communities (Lakshmi et al., 2018).

Safety of nanoparticles
The development of nanoparticle is progressing steadily. The development of solutions to many of the limitations of nanomaterials is of utmost importance. Reports suggest that nanoparticles enter human organisms in three different ways: inhalation, skin penetration, and ingestion. NPs can cross the cell barrier, leading to inflammatory responses and oxidative damage. In particular, in some cases, NPs incorporated into the packaging film positively affect and prevent the migration of chemical products into the food. In this regard, the nanolayers slowed the migration of 5-chloro-2-(2,-dichlorophenoxy) phenol (triclosan), trans, trans-1, -diphenyl-1,3-butadiene (DPBD)) and caprolactam from polyamide matrix up to six times. Risk assessments of some nanomaterials in the food packaging industry have been reported. Currently, it is necessary to assess the safety of NPs used in food packaging in terms of their potential impact on consumers (in terms of food quality and safety), as this is a threat. Other threats. Several research groups are actively studying the shelf life and deposition of NPs from packaging materials and their negative impact on the safety and quality of packaged foods. Chaudhry et al. (2008) and Bradley et al. (2011) demonstrated that increasing the surface area of NPs has a great influence on the biological and physicochemical properties compared with bulk particles (Bradley et al., 2011;Chaudhry et al., 2008;Silvestre et al., 2011).
The use of nanoparticles on food is increasing and must be used with precautions because they cause induce toxic effects. According to a study by the British Royal Society, humanity may face a nanotoxicity disaster in the future (Amini et al., 2014). We can only find safe and beneficial foods if we fully understand the properties of ZnO and CuO nanoparticles, including solubility, size, composition, and chemistry of surface. Some of the unique properties of particles make them attractive materials for many applications. However, this may be questionable in the case of foodstuffs, where there may be a high risk for human health (Ameta et al., 2020).
The toxicity of nanoparticles is also affected by their solubility. For example, insoluble nanoparticles are more toxic than soluble (hydrophilic) nanoparticles. Some soluble nickel compounds have been identified as carcinogenic (Grimsrud & Andersen, 2010). Therefore, a good understanding of the biological activity and toxicity of nanoparticles must be considered when using nanotechnology in food and related industries. In other words, all aspects related to the toxicity and environmental behavior of nanoparticles should be investigated (Amini et al., 2014).

Materials
Fresh ripened guavas of Allahabad Safeda Variety were purchased from the local market; fruits were medium in size, round in shape with few seeds.  Figure 1 shows the systematic presentation of methodology

Sample collection and processing
Five kilograms of fresh guava (Psidiumguajava) for coating and 1 kg for juice were collected from a local market of Lucknow. The selected guava was uniform in size, color, and external appearance and free of visible defects and decay. The guavas were transported to the food science laboratory. Guavas were washed with lukewarm water and cleaned with muslin cloth before further processing. The ZnO and CuO Nanoparticle (N.P.) coating was applied to guava fruits, and these N.P. the powder used as a preservative in guava juice.

Synthesis of ZnO and CuO nanoparticles
The synthesis of nanoparticle was done by Patel et al. (2022) and the ZnO nanoparticles were synthesized by solgel process using Zinc Nitrate with Hydrogen Peroxide as Precursor, and CuO Nanoparticles were synthesized using Copper (II) Nitrate, Sodium Hydroxide pellets, and ethanol. The precise color at the end of synthesis indicated the formation of ZnO Nanoparticle, and changes in color from dark blue to black due to chemical reaction indicated the formation of CuO Nanoparticles. The Formation of ZnO and CuO nanoparticles was confirmed and characterized by x-ray diffraction, scanning electron microscope, and FT-IR (Patel et al., 2022).

Preparation of coating
Preparation of coating of the sample was prepared by melting 200 g medible wax in distilled water and maintaining the volume of 600 ml. The solution was continuously stirred at 60°C until the wax melted. The above solution was divided into two equal parts (300 ml + 300 ml), the 0.5% (w/v) of ZnO Nanoparticles was added to one part of the solution, and 0.5% (w/v) CuO Nanoparticle was added to another part. 1% (w/v) of ZnO and 1% (w/v) of CuO nanoparticles were added as a preservative in 100 ml of guava juice.

Coating of ZnO and CuO nanoparticles on guava fruit
The fresh guava of uniform size was collected from the local market and washed with running water several times to remove the dust, pesticides, and microorganism. The selected guavas were dipped in the above solution for coating for 1 min. The treated and untreated sample was kept at room temperature to check their shelf life.

Addition of ZnO particles in guava juice
The slightly acidic and sweet guavas were locally purchased from the market and washed with lukewarm water 2-3 times. Peel the guava, cut it into pieces and remove the seeds. Put the guava pieces in a juicer, add a little water and sugar, and prepare the guava juice.
Take 100 ml of juice in three beakers each. Add 1% of ZnO N.P.s in 100 ml of juice, 1% of CuO N.P.s in 100 ml of juice, and 1 beaker kept as an untreated sample and kept a room temperature to study the shelf life.

Physiochemical analysis a) Total Soluble Solids (T.S.S.):
The T.S.S. of all treatments were determined by using a hand refractometer (MA 871, Germany) ranging from 0% to 85% Brix and conveyed in Brix after making the temperature correction at 18° (El-Gioushy et al., 2022). A drop of sample (fruit and juice) were placed on a prism cleaned with distilled water, and the percentage of brix of dry substance in it read directly at room temperature for accuracy (Lakshmi et al., 2018). b) Moisture loss: Moisture loss in the guava fruits coated with nanoparticles was calculated by the difference in Sample weight before and after the storage period expressed in percentage of weight loss (Tsegay et al., 2013). A weighed sample was taken and recorded as Initial weight (M o ) and kept in a dehydrator at 80°C for 6-10 h. The sample was removed from the dehydrator and cool it at room temperature. The cooled sample was weighted and recorded as the Final weight (M 1 ) (Lakshmi et al., 2018). Moisture loss of sample was calculated by using the given formula:  U.R.E.). For checking the pH value of fruits, fruits were crushed and homogenized to make pulp, and for juice, dip the pH meter in a beaker and take the record (Marpudi et al., 2013). d) Acidity: For juice and fruit T.A., an aliquot of fruit juice was taken and dissolved in distilled water and titrated against 0.1 N NaOH with phenolphthalein as a marker to the endpoint and stated as (%) of citric acid. The appearance of the Pink Colour denotes the endpoint (Hayat et al., 2005). Acidity was calculated by using the following formula, Titrable acidity ¼ ml NaOH � N NaOH � meg: Weight of acid � 100 ml juice titrated meq = milli equivalent meq weight of citric acid = 0.06404. e) Microbial Analysis/Total colony count: Total colony count of microorganisms was done according to the method given by Lakshmi et al. (2018) and Costa et al. (2011). Total Colony Count was done by using the serial dilution method. Colonies were counted after incubation, and the number of cfu/ml samples was calculated by applying the following formula (Costa et al., 2011).
No : of colony À forming unit=ml of sample A.S. software) for diffraction analysis and the ICDD PDF-4 Axiom 2020 database. The sample in powder form has been pressed into the sample holder, which has a smooth surface and holds the sample at a 45°. Solid samples, small sample volumes glued to the microscope slide, or thin films deposited on the substrate can also be used but with varying degrees of effectiveness (Purohit et al., 2019). h) Statistical analysis: One-way ANOVA for physiochemical test followed by the use of the t-test for comparison of means of physicochemical parameters was performed at different interval. The experiment was done by using a completely randomised factorial design using three replications for the sample treated with ZnO NPs, sample treated with CuO NPs and untreated/control fruit and juice and designated as follows:

Total soluble solids (TSS)
The changes in TSS value of Guava fruits and Guava juices treated with ZnO and CuO NPs during to storage period was shown in Tables 1 and 2; Figures 2 and 3. The TSS value of untreated samples (T 0 and T 3) during the storage period were increased very rapidly as compare to treated sample during the storage period. The amount of TSS value in samples treated with ZnO were increased slightly as compared to sample treated with CuO NPs. The TSS Value of T 0 , T 1, and T 2 at 0 days were 12, 12.04, and 12.06° Brix respectively and T 3 , T 4, and T 5 were 14.1, 14.1, and 14.2°Brix respectively. The maximum TSS was recorded as 14.9°Brix in T 3 whereas minimum was recorded as 12.4° Brix in T 1 after 15 days of storage period. The increased in TSS value during the storage period was due to the conversion of the remaining polysaccharides to soluble sugars and the formation of water-soluble pectin from protopectin (Bal et al., 2014). From Tables 1 and 2, the changes in     to control in maintaining the TSS of fruits and fruits juice (p ˂ 0.0.5). Though some of the treatments were found statistically at par but majority of them were significant. Figure 2 shows the changes in weight of all guava fruits throughout the storage time. During the storage period, fruit weight loss increased and recorded maximum value after 15 days in control fruit. Weight loss in sample treated with ZnO nanoparticle (T 1 ) was 94% significantly lower as compared to sample treated with CuO nanoparticles (T 2 ) i.e. 92%. In untreated sample (T 0 ) the weight loss was recorded very rapid. During the storage period it was noticed that the by increasing the storage period there was decrease in weight loss in treated sample. It was due to edible coating on sample act as barrier against oxygen, carbon dioxide, water loss, thereby reducing the respiration process and oxidation rates which increase the shelf life of sample (Arowora et al., 2013). From Table 1, it was found that the guava treated with CuO and ZnO particles have minimum weight loss as compared to untreated guava samples (T 0 ). The statistical analysis revealed that the nanoparticles are significant in all the treatments (p ˂ 0.05) and all the treatments were found statistically significant over control.

pH value
It is an important measure of active acidity that affects the taste or palatability of a product and affects processing requirements, so pH plays an important role in product preservation and development (Akbar et al., 2016). In all treatments stored at room temperature, it was noticed in Tables 1 and 2 that there was decrease in pH value of all treatments during the 15 days of storage period. Figures 2  and 3 indicate that the pH value of control sample (T 0 and T 3 ) decreases very fast as compare to treated sample. The amount of pH of guava coated with ZnO nanoparticle was ranges between 4.1 and 3.5 and the guava coated with CuO Nanoparticles between 4.1 and 3.3 which indicate that ZnO Nanoparticle was more effective than CuO Nanoparticle. pH value dropped sharply in untreated juice sample (T 3 ) as compared to treated juice sample (T 4 and T 5 ). The pH value of all treatments during storage period was affected due to respiration process, conversion of acid into sugar, and reactions by enzyme. The decrease in pH value during storage was due to the simultaneous increase in titrated acidity (El-Gioushy et al., 2022) Statistical result of all treatments was done and data in Tables 1 and 2 revealed that all the treatments showed that the positive effect of nanoparticles on the sample during the storage period and significantly superior to control in maintaining the pH of fruits (p ˂ 0.05) and the fruit treated with ZnO NPs were highly significant (p ≤ 0.001).

Acidity
Acidity values of all samples stored at room temperature are shown in Tables 1 and 2 that was increased during the storage period. Acidity of control sample increased very rapid as compared to treated sample. The acidity increased slightly during the 15 days of storage. This may be due to   the rapid breakdown of pectin in to pectinases in samples (Hayat et al., 2005). Kausar et al. (2016) also reported that the increase in acidity in juice during storage could be partly attributed to the contribution of the inherent acid, which is naturally present in the juice, and partly by the addition of citric acid which was added to the drink at the time of preparation during the development and standardization of juice (Kausar et al., 2016). During storage period, data indicates that there was increase in acidity value of all treatments. From Figures 2 and 3, the highest acidity value was found in T 3 i.e. untreated juice after 15 days of storage. The acidity value of guava and guava juice treated with ZnO changes from 0.419 to 0.461 (T 1 ) and 0.710 to 0.772 (T 4 ), respectively and acidity value of guava and guava juice treated with CuO changes from 0.420 to 0.482 (T 2 ) and 0.711 to 0.781 (T 5 ) after 15 days of storage period. Statistical analysis of data shows in Tables 1 and 2 shows that all treatments were significant (p ˂ 0.05) over control sample and found highly significant (p ≤ 0.001) in sample treated with CuO NPs (T 5 ) during the storage period.

Microbial analysis
Tables 1 and 2 show that the growth of Microorganism was started in all treatments during the interval of storage period, after few days of storage period the growth of microorganism were started which increased by increasing the days of storage period. In control sample (T 0 and T 3 ) the growth of microorganism was very rapid. The total count in guava and guava juice treated with ZnO and CuO after 15 days of storage period were almost similar i.e. 0.46 × 10 2 (T 1 ) & 0.46 × 10 2 (T 2 ) and 0.72 × 10 2 (T 4 ) & 0.72 × 10 2 (T 5 ) respectively which indicate that both ZnO and CuO nanoparticles were act as preservative against the microorganism and slowdown the process of multiplication. During the study it was estimated that the treated sample had less growth of microbes as compared to untreated sample (Hajirasouliha et al., 2012). From Figures 2 and 3, the minimum microbial count was recorded in T 3 (Untreated juice) i.e. 1.31 × 10 2 after 15 days. Edible coating can also improve antibacterial properties as reported by Jeon et al. (2002) and Tsai et al. (2002). In addition, evaluated minimum amount of microbial growth (both fungal and microbial) indicated that treated sample had an extremely lower growth rate than untreated sample (Kausar et al., 2016).
The statistical analysis of data revealed that the nanoparticles have significant effect on microbial growth of samples. Though some of the treatments were found statistically at par but majority of them were significant (p˂0.05)

Scanning electron microscope (SEM)
SEM images were used to study surface morphology and microbial growth on the sample surface. Figures show the morphology of untreated and guava treated with ZnO and CuO nanoparticles, respectively. At high magnificent (×5000) figure shows the growth of some microorganisms on the surface of the untreated sample, the surface becomes rough and black spot occur in outer layer. Figure 4 shows that the guava treated with ZnO nanoparticles had a smooth area and the sample treated with CuO nanoparticle had crack in outer layer and no surface microorganisms in both treated sample after 15 days of storage. Both ZnO and CuO coating on the guava fruits reduces respiration, keeps the sample surface smooth and slows down the growth of fungi or any kind of microbes (Mishra et al., 2021).

X-ray diffraction (XRD)
Different peaks in figure indicate the polymorphism, degree of crystallinity, and amorphism of all guava fruit samples which control the texture and stability of guava at different storage period. Crystallinity was determined by area under the crystalline peak and total area under diffraction peak. The structural stability of dried samples of guava stored for 15 days of storage period had been calculated using wide diffraction angle of XRD at different peak. In Figure 5, defined peak of dried sample indicate the presence of sugar in sample after the storage period. Peaks observed at 21.5, 23.8 at 2θ in untreated guava sample, dried guava treated with ZnO and CuO indicate the semi-crystalline structure and shows that there were not changes in semicrystalline structure in sample even after 15 days of storage period (Purohit et al., 2019).  4. Análisis SEM de los frutos de guayaba (a) muestra sin tratar; (b) tratada con nanopartículas de ZnO + cera; (c) tratada con nanopartículas de CuO + cera, después de 15 días de almacenamiento.

Conclusion
Guava and guava juice treated with ZnO and CuO nanoparticles synthesized by sol gel method which is used as preservatives in guava and guava juice can inhibit or slow down the rate of respiration during storage, reducing the number of microorganisms in guava juice, delaying ripening of fruit by up to 10-15 days compared to untreated guava. The physicochemical parameters of untreated samples changed significantly, affecting the stability of the preservation process. pH, TSS, acidity, moisture, morphological changes, and microbial counts in untreated samples changed significantly after 15 days of storage due to respiration rate, perspiration, and increased growth of microorganisms. However, the sample treated with ZnO and CuO nanoparticles slowed the ripening of guava, retards moisture loss, a pH and TSS values were maintained throughout the storage period. Bacterial counts in guava juice treated with ZnO and CuO nanoparticles were maintained throughout the storage period and as compare to CuO nanoparticle ZnO was more effective. Application of ZnO and CuO nanoparticles to guava and guava juice has been shown to be beneficial in increasing the shelf life and maintaining the quality of guava and guava juice.