A Review of Polysaccharide-Zinc Oxide Nanocomposites as Safe Coating for Fruits Preservation

Safe coating formulated from biopolymer can be an alternative for better packaging for fruits. Among biopolymers used for safe coating, polysaccharides attracted more attention due to its biocompatibility and edibility. However, polysaccharide-based materials have weaknesses such as low water barrier and mechanical properties which result in lower capability on preserving the coated fruits. Hence, the incorporation of nanoparticles (NPs) such as zinc oxide (ZnO) is expected to increase the ability of polysaccharide-based coating for the enhancement of fruit shelf life. In this review paper, the basic information and the latest updates on the incorporation of ZnO NPs into the polysaccharide-based safe coating for fruit are presented. Various research has investigated polysaccharide-ZnO nanocomposite safe coating to prolong the shelf life of fruits. The polysaccharides used include chitosan, alginate, carrageenan, cellulose, and pectin. Overall, polysaccharide-ZnO nanocomposites can improve the shelf life of fruits by reducing weight loss, maintaining firmness, reducing the ripening process, reducing respiration, reducing the oxidation process, and inhibiting microbial growth. Finally, the challenges and potential of ZnO NPs as an active agent in the safe coating application are also discussed.


Introduction
The main function of food packaging is to protect food products from physical, biological, and chemical impact. The ability of food packaging to preserve food is by delaying food deterioration, retaining and prolonging the food's beneficial effect that comes from processing, and conserving the quality and safety aspect of the food by improving its shelf life [1]. However, the standard materials used for manufacturing food packaging (synthetic petroleum-based polymer) have their drawbacks, such as non-renewable, non-biodegradable, carcinogenic, and non-eco-friendly [2,3]. Therefore, it is crucial to develop biopolymer-based packaging that is inexpensive, biodegradable, renewable, available in nature, and harmless to the environment [4].
Safe coating is a layer-forming material used to enrobe food products. Safe coating can fortify or replace natural layers on food that can be consumed or not afterward [5]. The uses of safe coating can overcome many problems in food marketing. Most of the issues are associated with food safe coating and film may be produced from the same gelling agent, their characteristics may differ significantly [21].
There are at least three ways to apply safe coating on food, including dipping, spraying, and vacuum impregnation. The dipping technique is one of the most used methods in coating food. Food is immersed in the coating solution and withdraw from to drain excessive solution. In some cases, such as alginate coating, second immersion into the crosslinking solution can be performed to form film on the food [22]. The dipping technique usually requires a short duration, ranging between 0.5-5 min. This technique's main advantage is its ability to coat the food surface thoroughly, even if the food has a rough and complex surface [23] (Figure 1). However, it is essential to note that this simple dipping technique also comes with limitations. Excellent adhesion of the coating solution to the food surfaces may be hard to achieve if the surface of the food products is hydrophilic. These cases usually occur on cut fruit. It usually requires a layer-by-layer technique, in which food is dipped in polyelectrolyte solution with opposite charges to increase adhesion with the actual coating [24][25][26][27]. The dipping method also has weaknesses such as thick coating and diminished efficacy due to dilution and dissolving effect [28]. Residual accumulation of coating materials and microbiological contamination can also occur [29]. Some process parameters that need to be controlled in the dipping method are the type of solvent, temperature and viscosity of the coating solution, immersion and withdrawal speed, and dipping repetition and immersion time [30,31].
Coatings 2020, 10, x FOR PEER REVIEW 3 of 15 though both safe coating and film may be produced from the same gelling agent, their characteristics may differ significantly [21]. There are at least three ways to apply safe coating on food, including dipping, spraying, and vacuum impregnation. The dipping technique is one of the most used methods in coating food. Food is immersed in the coating solution and withdraw from to drain excessive solution. In some cases, such as alginate coating, second immersion into the crosslinking solution can be performed to form film on the food [22]. The dipping technique usually requires a short duration, ranging between 0.5-5 min. This technique's main advantage is its ability to coat the food surface thoroughly, even if the food has a rough and complex surface [23] (Figure 1). However, it is essential to note that this simple dipping technique also comes with limitations. Excellent adhesion of the coating solution to the food surfaces may be hard to achieve if the surface of the food products is hydrophilic. These cases usually occur on cut fruit. It usually requires a layer-by-layer technique, in which food is dipped in polyelectrolyte solution with opposite charges to increase adhesion with the actual coating [24][25][26][27]. The dipping method also has weaknesses such as thick coating and diminished efficacy due to dilution and dissolving effect [28]. Residual accumulation of coating materials and microbiological contamination can also occur [29]. Some process parameters that need to be controlled in the dipping method are the type of solvent, temperature and viscosity of the coating solution, immersion and withdrawal speed, and dipping repetition and immersion time [30,31]. The spraying technique is also often used in coating food. This technique distributes the coating solution on food surface in the form of droplets with the aid of nozzles [23]. The advantage of this technique is that only a small amount of coating solution is required to coat food due to the high pressure used during the spraying process-up to 60-80 psi [32]. There are still more advantages of coating with spraying techniques such as uniform coating, controllable thickness, possibility of multiple layer coating, minimalized coating solution contamination, controllable temperature of the coating solution, and the possibility to work with a large surface area [23]. However, certain conditions need to be controlled to apply the spraying technique properly. The viscosity of the coating solution should not be high. The characteristics of the spray flow are very dependent on the liquid properties (density, viscosity, and surface tension), operating conditions (flow rate and air pressure), and system conditions (nozzle design and spray angle) [23,33].
The vacuum impregnation technique is usually used when the coating is also used to enrich the food with vitamins and minerals. This technique produces a thicker and more compelling film than the previous coating technique mentioned. The vacuum impregnation is also able to incorporate solutes into foods that have air containing porous matrices such as vegetables and fruits [34,35]. The steps for this technique are similar to the dipping method. However, instead of immersing in the dipping tanks, the food is submerged into a coating solution within two airtight vacuum chambers connected to a vacuum pump. Afterward, the food is conditioned to atmospheric pressure while remained immersed in the coating solution. It is crucial to monitor and control the vacuum period, The spraying technique is also often used in coating food. This technique distributes the coating solution on food surface in the form of droplets with the aid of nozzles [23]. The advantage of this technique is that only a small amount of coating solution is required to coat food due to the high pressure used during the spraying process-up to 60-80 psi [32]. There are still more advantages of coating with spraying techniques such as uniform coating, controllable thickness, possibility of multiple layer coating, minimalized coating solution contamination, controllable temperature of the coating solution, and the possibility to work with a large surface area [23]. However, certain conditions need to be controlled to apply the spraying technique properly. The viscosity of the coating solution should not be high. The characteristics of the spray flow are very dependent on the liquid properties (density, viscosity, and surface tension), operating conditions (flow rate and air pressure), and system conditions (nozzle design and spray angle) [23,33].
The vacuum impregnation technique is usually used when the coating is also used to enrich the food with vitamins and minerals. This technique produces a thicker and more compelling film than the previous coating technique mentioned. The vacuum impregnation is also able to incorporate solutes into foods that have air containing porous matrices such as vegetables and fruits [34,35]. The steps for this technique are similar to the dipping method. However, instead of immersing in the dipping tanks, the food is submerged into a coating solution within two airtight vacuum chambers connected to a vacuum pump. Afterward, the food is conditioned to atmospheric pressure while remained immersed in the coating solution. It is crucial to monitor and control the vacuum period, vacuum pressure, and the atmospheric restoration time for the vacuum impregnation technique [36]. This technique provides enhanced retention, uniformity, and adherence of the coating solution compared to the conventional dipping method [35].
Safe coating can be loaded with additives to modify its mechanical, functional, organoleptic, and nutritional characteristics. Additives can include plasticizer, surfactants, antimicrobials, antioxidants, antibrowning agents, flavor, and or pigments. Among the marvelous additives for food coating is ZnO NPs. It has excellent antibacterial, antifungal, and antibiotic nanoscale agents [8,37]. Therefore, in the following section, the effect of ZnO NPs on properties of some polysaccharides for safe coating are discussed.

Effects of Zinc Oxide Nanoparticles on Properties of Polysaccharide-Based Coating
Polysaccharide-based coating selectively allows the transfer of gas, which makes it an excellent barrier to gasses. This advantage property enables the formation of the modified atmospheric condition within the coated food. Even if the gas transfer between the surrounding and the coated food is minimized, it is not completely terminated. The coated fruit is still allowed to respirate aerobically at a lower rate. If the fruit only respires anaerobically, the production of different compounds may occur. These various compounds can affect the coated fruit quality in an unpleasant way. As aerobic respiration occurs at a lower rate, the process of senescence also occurs at a lower rate. Thus, the shelf life of the fruit increases [38].
Even though polysaccharide-based coating provides an excellent barrier against gasses, its hydrophilic nature results in a poor barrier against moisture. Therefore, the polysaccharide-based coating is less suitable for food with high moisture [38]. It is necessary to improve the water barrier capability and other properties of polysaccharide-based coating to increase its functionality as a food coating. Many studies tried to search for a way that increases the functionality of the polysaccharide-based coating. Incorporating ZnO NPs became an alternative to do so. The incorporation of ZnO NPs into polysaccharides provides coating with excellent mechanical, structural, and barrier properties [20,39]. As mentioned in the previous sections, ZnO NPs also provides excellent antimicrobial activities. There are some proposed antimicrobial activity mechanisms for ZnO NPs, such as the induction of oxidative stress through the formation of reactive oxygen species (ROS) and the release of Zn 2+ ion that could penetrate the cell wall and reacts with the cytoplasmic content [20,[39][40][41] (Figure 2).
Coatings 2020, 10, x FOR PEER REVIEW 4 of 15 vacuum pressure, and the atmospheric restoration time for the vacuum impregnation technique [36]. This technique provides enhanced retention, uniformity, and adherence of the coating solution compared to the conventional dipping method [35]. Safe coating can be loaded with additives to modify its mechanical, functional, organoleptic, and nutritional characteristics. Additives can include plasticizer, surfactants, antimicrobials, antioxidants, antibrowning agents, flavor, and or pigments. Among the marvelous additives for food coating is ZnO NPs. It has excellent antibacterial, antifungal, and antibiotic nanoscale agents [8,37]. Therefore, in the following section, the effect of ZnO NPs on properties of some polysaccharides for safe coating are discussed.

Effects of Zinc Oxide Nanoparticles on Properties of Polysaccharide-Based Coating
Polysaccharide-based coating selectively allows the transfer of gas, which makes it an excellent barrier to gasses. This advantage property enables the formation of the modified atmospheric condition within the coated food. Even if the gas transfer between the surrounding and the coated food is minimized, it is not completely terminated. The coated fruit is still allowed to respirate aerobically at a lower rate. If the fruit only respires anaerobically, the production of different compounds may occur. These various compounds can affect the coated fruit quality in an unpleasant way. As aerobic respiration occurs at a lower rate, the process of senescence also occurs at a lower rate. Thus, the shelf life of the fruit increases [38].
Even though polysaccharide-based coating provides an excellent barrier against gasses, its hydrophilic nature results in a poor barrier against moisture. Therefore, the polysaccharide-based coating is less suitable for food with high moisture [38]. It is necessary to improve the water barrier capability and other properties of polysaccharide-based coating to increase its functionality as a food coating. Many studies tried to search for a way that increases the functionality of the polysaccharidebased coating. Incorporating ZnO NPs became an alternative to do so. The incorporation of ZnO NPs into polysaccharides provides coating with excellent mechanical, structural, and barrier properties [20,39]. As mentioned in the previous sections, ZnO NPs also provides excellent antimicrobial activities. There are some proposed antimicrobial activity mechanisms for ZnO NPs, such as the induction of oxidative stress through the formation of reactive oxygen species (ROS) and the release of Zn 2+ ion that could penetrate the cell wall and reacts with the cytoplasmic content [20,[39][40][41] ( Figure 2). Several studies have investigated the application of polysaccharide nanocomposite safe coating for fruits. The studies are summarized in Table 1 and further discussed in the following subsection. Several studies have investigated the application of polysaccharide nanocomposite safe coating for fruits. The studies are summarized in Table 1 and further discussed in the following subsection.

Chitosan
Chitosan is a copolymer of N-acetyl-d-glucosamine and d-glucosamine with linear and semicrystal polymer structures [46,47]. The presence of amine groups in chitosan contribute to its excellent solubility in dilute acid solution (pH < 6) [48,49]. Chitosan has a lot of interesting properties such as biodegradability, natural origin, abundance, and antimicrobial and antifungal activities, which led to its utilization in many fields, such as in the food industry [50,51]. Chitosan film can be utilized as food packaging with excellent protection for diverse kinds of food, especially when it is combined with other film-forming material [52][53][54].
The research conducted by Arroyo et al. investigated chitosan-ZnO NPs coating toward the shelf life of guava (Psidium guajava L.) [10]. Safe coating was prepared with a composition of chitosan 5% w/v dissolved in acetic acid 0.5% v/v, glycerol 2% v/v, and ZnO NPs 1% v/v gel. The coating formulations were applied to guavas by dipping inside the coating solution for 3 min and dried at 25 • C. Guavas were then stored in a controlled environment for 20 days, with a temperature of 21 ± 1 • C and 80% relative humidity (RH). The result of the experiment showed that the chitosan coating with ZnO NPs could reduce weight loss, indicating reduced water loss and ripening process. The firmness and green colour of the coated fruit were better maintained, also indicating a reduced ripening process. The ripening index ratio (soluble solid/titratable acid) was reduced compared to the uncoated fruit. External injuries were not detected on the coated guavas until the end of storage time, while the uncoated guavas already showed lesions from day 12. The authors concluded that coating with chitosan enhanced by ZnO NPs could delay the fruit ripening process and protect the fruit from weight loss and damaged appearance, resulting in the guavas still being edible until 20 days of storage.
The study by Lavinia et al. also explored the application of chitosan-ZnO NPs as fruit coating, but mainly focused on microbiological activities toward fresh-cut papaya [42]. The coating formulations were prepared by mixing 0.1 L ZnO NPs solution (0.005%, 0.006%, 0.010%, 0.020%, and 0.027% w/w of total solution weight inside 1% acetic acid; size 611.30 nm) with 0.3 L chitosan solution (3 g chitosan dissolved in acetic acid 1%). The coating was applied to fresh-cut papaya (size 3-4 cm 3 ) through dipping for 10-20 s and drained afterward. The coated papaya was then stored in a refrigerator for 12 days at 10 • C. The result showed that chitosan-ZnO NPs coating could retard microorganism growth. Based on the maximum limit of microbial count (5.00 log CFU/g) set by the Indonesia National Agency of Drug and Food Control (BPOM), the coated fresh-cut papaya was still safe to consume up until four days after storage. At the same time, the uncoated one was no longer safe to consume. The author mentioned that the antimicrobial activities of the coating were the result of synergistic interaction between chitosan and ZnO NPs, and concluded that the nanocomposite coating can maintain the quality of the fresh-cut papaya through the reduction in microbial growth.

Alginate
Alginate is one of the most abundant polysaccharides that can be found on brown algae and seaweed. Alginate is a polymer composed of (1→4)-linked α-l-guluronic acid (G) and β-d-mannuronic acid (M) monomeric unit. Alginate has the potential to be utilized as active packaging due to its biodegradability, biocompatibility, and null toxicity [55][56][57].
Emamifar and Bavaisi observed the effect of alginate-ZnO NPs coating, together with cold storage temperature, toward the shelf life of fresh strawberries (Fragaria × ananassa Duch.) [43]. Coating solutions were composed of sodium alginate 1.5% w/v, glycerol 1% v/v, and ZnO NPs (size 30-50 nm) with different concentrations (0.25, 0.75, and 1.25 g/L). Coatings were applied to fresh strawberries by dipping inside the coating solution at 25 • C for 5 min. The coated strawberries were then dried for an hour under fans. The coated strawberries were then stored at 1 • C and 95% RH for 20 days. The result showed that coated strawberries had reduced microbiological growth, including yeast, mold, and aerobic bacteria. It was mentioned by the authors that reduced microbiological growth may be attributed to the antibacterial properties of ZnO NPs or the improved barrier properties to oxygen and moisture by ZnO NPs. The coated strawberries also reduced weight loss, indicating reduced fruit surface evaporation and respiration rate. The coated strawberries also exhibited better-maintained firmness, resulted from CO 2 buildup inside the fruit that caused inhibition of enzymes responsible for fruit softening. The strawberries with coating had lower increases in total soluble solid and lower decreases in acidity, showing that the coating can control the fruit maturity. The coated strawberry exhibited lower decreases in ascorbic acid, anthocyanin and phenolic content, and antioxidant activity, showing that the coating has antioxidant and antimicrobial properties. The coated strawberries showed lower increases in peroxidase activity and lower decreases in superoxide dismutase activity, which indicated the strawberries were better maintained from various damages or stresses. The sensory evaluation also showed that the coated strawberries had better acceptance compared to the uncoated. The authors concluded that alginate coating could improve the physicochemical, sensory, and microbial qualities of fresh strawberries within cold storage conditions. The addition of ZnO NPs into alginate coating showed a synergistic effect toward the shelf life of the strawberries. Increasing concentration of ZnO NPs, up to 1.25 mg/L, showed improvement in the reduction in microbial numbers, physicochemical properties, and overall sensory acceptance. The authors also highlighted that cold storage temperature was also an important factor for strawberry preservation.
In the research conducted by Arroyo et al. (2020), the coating solution used consists of sodium alginate 5% w/v, glycerol 2% v/v, and ZnO NPs 1% v/v gel to coat guavas by dipping for 3 min, and later dried at 25 • C [10]. The coated and uncoated guavas were then stored for 20 days with a temperature of 21 ± 1 • C and 80% RH. The experiment demonstrated that weight loss of coated and uncoated guavas was not significantly different after 20 days of storage. Hence, the author highlighted that the incorporation of ZnO NPs in the alginate coating system did not provide a water vapor barrier to prevent water and weight loss of guavas. The poor barrier of alginate coating was due to the little adherence of the coating solution toward the guavas epicarp, which might be caused by the low viscosity of the coating solution or the concentration of alginate used in the coating solution.
Aside from pure alginate coating with ZnO NPs, Arroyo et al. also fabricated a coating solution that consists of mixed alginate and chitosan (90-10%) in the concentration of 5% w/v to coat guavas [10].
The coating system showed improvement in reducing weight loss. Additional parameters observed including firmness, color, and ripening index ratio were also tested. The coated guavas had better maintained firmness, but the color was not significantly different to control guavas. The ripening index ratio was not significantly different to controlled guavas. The author concluded that the coating could delay the ripening process, reduce weight loss, and maintain the appearance of the guavas more effectively. However, it was not the best formulation pointed out by the authors.

Carrageenan
Carrageenan is a polysaccharide with gel-forming and thickening abilities originated from certain species of red seaweed. Carrageenan structure consists of an alternating unit of D-galactose and 3,6-anhydrogalactose (3,6-AG), which is connected through α-1,3 and β-1,4 glycosidic links. Based on its solubility on potassium chloride solution, carrageenan is classified into various types such as kappa (κ), iota (ι), and lambda (λ) carrageenan. Carrageenan can be utilized as an safe coating due to its potential as a film-forming material [58].
Meindrawan et al. investigated the application of carrageenan incorporated with ZnO NPs toward mangoes' (Mangifera indica L.) shelf-life [15]. Coating solutions were made with composition of the ZnO NPs (0%, 0.5%, and 1% w/w of carrageenan), 0.8 g carrageenan, and 0.5 mL of glycerol inside a 100 mL solution. The coatings were then applied to the selected mangoes by dipping inside the coating solution for 30 s and drained afterwards. Mangoes were then subjected to a controlled environment with a temperature of 20 • C and 61% RH. The incorporation of ZnO NPs into the carrageenan coating could reduce the weight loss of the coated mangoes, resulted from decreased water loss due to improved moisture barrier. However, carrageenan nanocomposite could not maintain the firmness of the coated mangoes compared to the uncoated mangoes. The authors mentioned that this may happen because the formation of ZnO NPs aggregation during production of the coating may influence the barrier ability of the coating, and there are many factors that cause mango softening that is not inhibited by the coating. Carrageenan coating with ZnO NPs reduced CO 2 production and maintained total acidity. These results showed the decreased in respiration rate. The appearance of coated mangoes was also better maintained in terms of color and textural appearance. The coated mangoes maintained their color better and showed less emergence of black color due to microbiological damages. Textural appearance of the coated mangoes tended to be more rigid, compared to the wrinkled texture of the uncoated. Overall, the author declared that carrageenan coating, fortified with ZnO NPs, could maintain the shelf life of mangoes. The author highlighted that the best formulation of the coating was 1% w/v ZnO NPs.

Carboxymethyl Cellulose
Cellulose is one of the most abundant organic compounds mainly found on biomass. However, utilization of cellulose itself is quite limited due to its low reactivity and solubility [59]. Hence, chemically modified cellulose is invented to improve its utilization and application. One of the most common industrially produced chemically modified cellulose is carboxymethyl cellulose (CMC). CMC is made through the carboxymethylation process of cellulose. CMC has good solubility and reactivity that are accredited to the reactive carboxyl and hydroxyl groups that cellulose do not have [59]. CMC application mainly includes thickener, emulsifier, and suspension agent [59]. It can also be utilized as a gelling agent in some circumstances. The advantages of CMC includes its non-toxicity, biodegradability, and biocompatibility [59]. Hence, CMC has potential to be utilized as safe coating.
Corporations of ZnO NPs in carboxymethyl cellulose (CMC) coatings for pomegranate (Punica granatume L.) arils were proposed by Saba and Amini [44]. The arils were dipped in ZnO NPs solution (0.1% and 0.2% w/v; size 30-100 nm) at 20 • C for 4 min, and excess water was removed afterwards. ZnO NPs treated arils were then dipped in CMC 0.5% w/v. As a control experiment, the arils were only dipped 4 min in distilled water. Arils were then air-dried at room temperature and stored for 12 days at 4 • C and 90% RH. The results showed that yeast, mold, and aerobic mesophilic bacteria growth were detected lower on coated arils until the end of storage time. The authors mentioned that the antimicrobial activities of the coating were resulted from ZnO NPs antimicrobial properties. Soluble solid concentration (SSC) of the arils decreased regardless of the treatments and the TA value increased with no significant difference between coated or uncoated arils at the end of storage time. The coating managed to reduce weight loss of the arils, indicating less water loss. However, amount of water loss did not cause a difference in juice content between coated and uncoated arils. Vitamin C loss across storage time was lower in coated arils compared to uncoated ones ( Figure 3A). This was due to reduce endogenous oxygen by the coating, resulting in less ascorbic acid oxidation. Anthocyanin and phenolic content reduced along the storage time. However, the coated arils exhibited lower reduction than uncoated arils ( Figure 3B,C). Antioxidant activities of arils increased along storage time, but coated arils showed higher antioxidant activities than the uncoated one ( Figure 3D). Reproduced with permission from [44]. Copyright 2017, Elsevier.

Pectin
Pectin is a complex polysaccharide found in plant cell walls that acts as a hydrating agent and cement for the cellulosic network. It is located on the primary cell wall and the main middle lamella, contributing to the firmness of plant tissue. Pectin is a block copolymer, which consists of block containing heteropolymer of rhamnogalacturonan ([1à2]α-L-rhamnosyl-[l-L]α-D-galactosyluronic acid) with side chains such as galactan and arabinan on rhamnose unit, and block of homopolymer galacturonic acid with varying degrees of methyl esterified carboxyl group. Pectin became among the growing interests in the food packaging industry due to its non-toxic, biocompatible, and biodegradable properties [60].
The application of pectin-ZnO nanocomposite safe coating for star fruit (Averrhoa carambola) has been investigated by Romadhan and Pujilestari [45]. The safe coating was composed of 0.1 g ZnO NPs, 10 g pectin, and 1 mL of glycerol inside 1 L aquadest. The coating was applied to star fruits by dipping for 20 s, followed draining the excess solution from the star fruit. The star fruits were then subjected to storage at room temperature (25 °C). The result of the experiment showed that the coated star fruits did not exhibit significant difference in weight loss compared to uncoated star fruits. The authors mentioned that single polysaccharide could give significant differences in maintaining weight loss because it contains many hydrophilic groups. The addition of ZnO NPs in small amounts only gave small reduction in weight loss (~1% compared to uncoated star fruits). The star fruits exhibited reduced browning index and redness value increases, showing decreases in ripening process. Physical damage on coated star fruit are also reduced. Until the end of storage time, the coated star fruits were still edible because physical damage was minimal. On the other hand, the uncoated star fruits were not edible at the end of storage time. The authors mentioned that damages on the star fruit may be caused by brown rot diseases that are caused by mold. The authors concluded that safe coating could increase the star fruit shelf life up to 2-3 days longer than the uncoated one.

Pectin
Pectin is a complex polysaccharide found in plant cell walls that acts as a hydrating agent and cement for the cellulosic network. It is located on the primary cell wall and the main middle lamella, contributing to the firmness of plant tissue. Pectin is a block copolymer, which consists of block containing heteropolymer of rhamnogalacturonan ([1à2]α-l-rhamnosyl-[l-L]α-d-galactosyluronic acid) with side chains such as galactan and arabinan on rhamnose unit, and block of homopolymer galacturonic acid with varying degrees of methyl esterified carboxyl group. Pectin became among the growing interests in the food packaging industry due to its non-toxic, biocompatible, and biodegradable properties [60].
The application of pectin-ZnO nanocomposite safe coating for star fruit (Averrhoa carambola) has been investigated by Romadhan and Pujilestari [45]. The safe coating was composed of 0.1 g ZnO NPs, 10 g pectin, and 1 mL of glycerol inside 1 L aquadest. The coating was applied to star fruits by dipping for 20 s, followed draining the excess solution from the star fruit. The star fruits were then subjected to storage at room temperature (25 • C). The result of the experiment showed that the coated star fruits did not exhibit significant difference in weight loss compared to uncoated star fruits. The authors mentioned that single polysaccharide could give significant differences in maintaining weight loss because it contains many hydrophilic groups. The addition of ZnO NPs in small amounts only gave small reduction in weight loss (~1% compared to uncoated star fruits). The star fruits exhibited reduced browning index and redness value increases, showing decreases in ripening process. Physical damage on coated star fruit are also reduced. Until the end of storage time, the coated star fruits were still edible because physical damage was minimal. On the other hand, the uncoated star fruits were not edible at the end of storage time. The authors mentioned that damages on the star fruit may be caused by brown rot diseases that are caused by mold. The authors concluded that safe coating could increase the star fruit shelf life up to 2-3 days longer than the uncoated one.

Safety Issue of Zinc Oxide Nanoparticles as a Safe Coating Material
Food packaging is an essential part of food manufacturing. Due to the expanding mindfulness of consumers in terms of health issues, the migration level of food packaging materials is a crucial point in commercial food packaging. Consumers may hesitate and worry about consuming foods that are treated with ZnO NPs. However, little study about the migration of ZnO NPs from polysaccharide is available.
ZnO NPs migration from low-density polyethylene (LDPE)-ZnO nanocomposite film reported by Bumbudsanpharoke et al. in varying conditions [61]. Conditions used were distilled water to simulate aqueous food, acetic acid 4% w/v to simulate acidic food, ethanol 50% v/v to simulate alcoholic food, and n-heptane to simulate fatty food. The result showed that ZnO NPs performed the highest migration on acetic acid 4% w/v. The high migration rate of ZnO NPs might be accredited to its solubility on acetic acid, which was the highest compared to other simulating solutions used. On the other hand, there was no zinc detected on n-heptane simulating solution.
Another study also focused on ZnO NPs migration from commercially available polypropylene (PP)-ZnO nanocomposite food containers (Nano center, Ltd., Shanghai, China). Simulating solution placed on the container for migration test was distilled water, acetic acid 4% v/v, and n-heptane. The test was conducted with varying temperatures (30,45, and 60 • C). The result showed that as storage time increased, the amount of migrated ZnO NPs also increased [62]. The rate of migration was higher under high-temperature conditions [62]. The migration of ZnO NPs was found to be higher on acetic acid and n-heptane [62]. The authors mentioned that organic food simulating solution had a swelling effect on polypropylene which caused a high migration concentration of ZnO NPs.
Even though ZnO is GRAS substance which is approved by the Food and Drug Administration (FDA) [63], the nano size of ZnO may generate toxic actions. Therefore, an assessment of ZnO NPs toxicity is needed. Research by Barkhordari et al. specifically tested the cytotoxicity of ZnO NPs (size 30-70 nm) toward the human spermatozoa cells [64]. The authors mentioned that ZnO NPs caused sperm cell death in a dose-and time-dependent manner. The result showed that ZnO NPs concentration up to 100 µg/mL, incubated up to 90 min with the spermatozoa, resulted in cell death less than 10%. Elsewhere, Wahab's research group introduced ZnO NPs to malignant cells (T98G Gliomas, and KB) and non-malignant cell (HEK) [65]. The result showed that ZnO NPs presented a depressing effect of the T98G cell growth, moderately effective on KB cells and least toxic on normal HEK cell. Namvar et al. also showed the similar effect of ZnO NPs on mice cells [66]. Namvar et al. tested ZnO against normal fibroblast cell of mice. For comparison, ZnO NPs were also tested against various cancer cells (4T1, CRL-1453, CT-26, and WEHI-3B), and cancer drugs (paclitaxel/PTX) were tested against cancer cell WEHI-3B. The results showed that the presence of ZnO NPs inhibited the proliferation of the various cancer cells (Figure 4a). Meanwhile, the presence of PTX also showed the inhibitory effect on WEHI-3B cancer cells (Figure 4b). On the other hand, ZnO NPs (100 µg/mL) did not show any toxicity toward normal fibroblast cell lines of mice using the MTT assay (Figure 4c).

Conclusions
There is a lot of added value in safe coating incorporated with ZnO NPs. Polysaccharide-ZnO nanocomposite can inhibit the ripening process. Varying polysaccharides have been used, including chitosan, alginate, carrageenan, cellulose. The deterioration process due to water loss and oxidation can also be prevented due to the improved gas and water barrier properties of nanocomposite material. The antimicrobial activity of ZnO NPs from the nanocomposite has also proven to be effective on coated fruits. The migration of ZnO NPs into the food does not pose a dangerous risk, as various cytotoxicity tests showed low toxicity of ZnO NPs. Hence, there is great potential for the utilization of ZnO NPs as a component for food packaging, as it has proven its ability to extend the shelf life of the food products, and safety issues are minimal. As for the future development of polysaccharide nanocomposite safe coating, it is necessary to investigate the migration of ZnO NPs from biopolymer instead of synthetic petroleum-based polymer to address the safety issue concerning even more accurately ZnO NPs migration.

Conclusions
There is a lot of added value in safe coating incorporated with ZnO NPs. Polysaccharide-ZnO nanocomposite can inhibit the ripening process. Varying polysaccharides have been used, including chitosan, alginate, carrageenan, cellulose. The deterioration process due to water loss and oxidation can also be prevented due to the improved gas and water barrier properties of nanocomposite material. The antimicrobial activity of ZnO NPs from the nanocomposite has also proven to be effective on coated fruits. The migration of ZnO NPs into the food does not pose a dangerous risk, as various cytotoxicity tests showed low toxicity of ZnO NPs. Hence, there is great potential for the utilization of ZnO NPs as a component for food packaging, as it has proven its ability to extend the shelf life of the food products, and safety issues are minimal. As for the future development of polysaccharide nanocomposite safe coating, it is necessary to investigate the migration of ZnO NPs from biopolymer instead of synthetic petroleum-based polymer to address the safety issue concerning even more accurately ZnO NPs migration.
Funding: This research received no external funding.
Acknowledgments: This work was supported by the Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia.

Conflicts of Interest:
The authors declare no conflict of interest.