Amazonian tuber starch based films incorporated with silver nanoparticles for preservation of fruits

Films and coatings based on natural polymers are used to conserve nutritional quality of fruits, vegetables and also delay their ripening. The purpose of this study was to develop films with starch extracted from Dioscorea altissima Lam. (dunguey) incorporated with silver nanoparticles for coating and preserving fruits. The films obtained by cast were characterized visually, by Scanning Electron Microscope, by Atomic Force Microscopy, by X-Ray Diffraction and by Fourier-Transform Infrared Spectroscopy. Antimicrobial activity and technological properties were also Research, Society and Development, v. 10, n. 6, e23510615304, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15304 2 evaluated. The coating of camu-camu fruits [Myrciaria dubia (Kunth) McVaugh] was carried out by immersing them in the filmogenic solution, followed by their physicochemical and microbiological analysis. The films with silver nanoparticles showed transparency, flexibility, spherical clusters and a higher average roughness. A reduction in thickness, solubility and water vapor permeability was also observed. Antimicrobial action against Staphylococcus aureus and Escherichia coli was proved as well. The fruits coated with films exhibited delay in ripening, with maintenance of quality and longevity. Uncoated fruits showed greater wilting and wrinkling. The starch film incorporated with silver nanoparticles was effective for preserving camu-camu fruit.


Introduction
In response to the growing of demanding consumers for fresh foods, with guaranteed safety and a ready-to-eat concept, a lot of research work is being carried out to maintain quality, and improve post-harvest viability. Biodegradable coating films Ag nanoparticle-based are an eco-friendly, simple, and efficient technological alternative for reducing microbial contamination and increasing the storage time for products of plant origin (Pilon et al., 2015).
Biodegradable polymers such as starch predominate in the production of coating films, due to the availability of different sources such as tubers, leaves, rhizomes, seeds and fruits, which are easy to process and have a low production cost (Mateescu, Dimov, Grumezescu, Gestal & Chifiriuc, 2015). Biodegradable films are commonly made via casting, although in large-scale manufacturing, semi-automatic molding and spread coating are used (Versino, Lopez, Garcia & Zaritzky, 2016).
Among the plant species of Amazon rainforest, D. altissima is a tuber that has stood out with a significant production of 100 kg/plant in 18 months. In addition, D. altissima is not toxic, has excellent nutritional value, and prebiotic action, characteristics that promote its use as food (Teixeira et al., 2016).
The productivity of the D. altissima and its therapeutic importance are factors that stimulate its cultivation. Moreover, in D. altissima, the content of available starch and resistant starch corresponds to approximately 70.0% and 10.0%, respectively. These characteristics boost the biotechnological and commercial exploitation of this vegetal as raw material (Silva et al., 2019).
An alternative use of D. altissima starch consists of making films for the conservation of food products, a technology still unavailable for the fruits of the Amazon region. To increase efficiency, these films can be incorporated with silver nanoparticles (AgNPs) obtained by biogenic synthesis. This green technology is less aggressive to the environment, economically viable, and has demonstrated antimicrobial efficiency against several microorganisms Silva-Vinhote et al., 2017).
Bacteria, algae, actinomycetes, filamentous fungi and yeasts are explored in bioprocesses for the production of AgNPs. Silver nanoparticles are commonly used in multiple applications due to their properties such as chemical and thermal stability, conductivity, size, shape, surface and volume ratioand high catalytic activity. Silver nanoparticles are used as deodorants and disinfectants, incorporated in fabrics, bandages, coatings and food packaging; in addition, they are used to purify drinking water and, currently as insecticide to control pest attack in agriculture (Tyagi, Tyagi, Gola, Chauhan & Bharti, 2019;Pandey, Klerk, Kim, Kang & Fosso-Kankeu, 2020).
Another raw material of great prominence in the Amazon that makes part of native people diet of is Myrciaria dubia (Kunth) McVaugh, a wild species that grows on the banks of rivers and lakes (Santos et al., 2018). The fruits are known as camu-camu, have a globose shape, and red color. The high levels of anthocyanins and vitamin C content have promoted the application of this Amazonian vegetal species in local gastronomy, as well as in pharmaceutical and cosmetics industries (Nascimento & Carvalho, 2012).
Although camu-camu has nutritional and socioeconomic importance, there is no post-harvest conservation technique available to maintain the characteristics of the fruits for its commercialization in natura. The objective of this research was to prepare coating films formulated with the starch of D. altissima, incorporated with AgNPs from actinomycetes mediated synthesis, as a new technological option for the conservation of fresh camu-camu.

Obtaining AgNPs
AgNPs solutions preserved at 4°C were used in the preparation of starch-based films and were selected based on significant antimicrobial activity among others previously reported (Silva-Vinhote et al., 2017). The AgNPs that the synthesis was mediated by Streptomyces parvulum DPUA 1549 were ceded by the Nanoparticle Bank of the DPUA Cultures Collection/UFAM.

Preparation of starch-based films and incorporation of AgNPs
To prepare the films by casting, 4 g of D. altissima starch was diluted in 100 mL of distilled water containing 3 g of glycerol. The filmogenic solution was heated until 95 °C. After complete gelatinization and cooling of the solution, AgNPs (2 µg/mL) were added and 10 mL of this solution was spread on Petri dishes (140 mm X 150 mm). These dishes were maintained at 40 °C in an oven with forced air circulation for 24 h. Two types of films were produced: standard (without AgNP) and test.

Determination of film thickness
The thickness of the films was determined using a digital caliper in three random points and the results were expressed as averages (Pagno et al., 2015).

Determination of water vapor permeability
Water vapor permeability was determined by ASTM's E96 / E96 gravimetric method (American Society for Testing and Materials-ASTM, 2010). The rate of water vapor permeability was determined at 23 °C and 75% RH. The weight gain of anhydrous chloride inside aluminum capsules, kept closed with the films was quantified on a Mettler analytical balance; model AT 400 (Greifensee, Switzerland), with a resolution of 10 -4 g.

Determination of humidity
The humidity was determined by drying the film specimens in forced air circulation oven, at 105° C, until constant weight (Association of Official Analytical Chemists -AOAC, 2006).

Determination of solubility
The water solubility of the films was determined according to the method of Gontard, Duches, Cuq & Guilbert (1994).
The films of known humidity were weighed, submerged in 50 mL of distilled water and kept under stirring at 26 ºC. After 24 hours, the samples were dried at 105 ºC for 4 hours and weighed. The results were expressed as percentages (%).

Scanning Electron Microscopy
The Scanning Electron Microscopy (SEM) of the films was performed in a compact scanning electron microscope; model JSM-6010LA, Jeol (Akishima, Japan). The samples were fixed to the sample holder with double-sided adhesive tape.
An acceleration potential of 15 kV and 20 kV was used in the analyses (Campos et al., 2017).

Atomic Force Microscopy
The Atomic Force Microscopy (AFM) of the films was performed in an Agilent equipment, model AFM/SPM 5500 Dynamics (Santa Clara, USA), with a resonance frequency of 320 kHz and a constant force of 42 N/m. A fragment of the films was added to the commercial silica cantilever and dried by evaporation at 25 °C. Images were treated in the WSXM software v.5.0 (Horcas & Fernández, 2007). The topography image was analyzed to obtain the average height of the peaks, and roughness medium square (RMS) parameters using Gwyddion software version 2.19. The RMS was defined as the standard deviation of the elevation values z, within the determined area.

Quantification of total silver in films
The quantification of total silver in films, the samples were digested at 90 °C for four hours, in 98% (v/v) nitric acid (Rolim et al., 2019). The quantification of total silver was determined on a model 7900 ICP-MS spectrophotometer from Agilent Technologies (Hachioji, Japan).

Determination of antimicrobial activity
The antimicrobial activity of the films incorporated with AgNPs was determined by the agar diffusion method (Fayaz et al., 2009), against Staphylococcus aureus ATCC 25923, Escherichia coli CBAM 001 and Candida albicans DPUA 1706. The cultures of the test microorganisms were performed on Sabouraud agar (yeasts) and Mueller-Hinton agar (bacteria). For the inoculum, a cell suspension was previously prepared with sterile distilled water, the yeast it was standardized at 105 CFU/mL and bacteria at 108 CFU/mL using spectrophotometer Uv-Vis spectra (UV-VIS Cary 50 Probe Agilent). From each suspension, 100 µL was transferred to Mueller-Hinton (MHA) and Sabouraud (SAB) agar surfaces, respectively. After absorption of the suspension, the discs of the 8 mm films were transferred to the culture medium surface. As a control, streptomycin and itraconazole solution (50 µg/mL) was used for bacteria and yeasts, respectively. The experiments were maintained at 37 ºC for 24 h and were performed in triplicate and the inhibition halo expressed in millimeters.

Obtaining and sanitizing the fruits
The camu-camu [Myrciaria dubia (Kunth) McVaugh] was obtained from Lake Iripixi, located in the city of Oriximiná-Pará. The fruits were sanitized with 1% (v/v) hypochlorite solution for 20 minutes, followed by washing them under running water to remove the hypochlorite excess (Siddiq, Ahmed, Lobo & Ozadali, 2012). The fruits were classified into four degrees of physiological ripeness: green (S1), reddish green (S2), greenish red (S3) and purple (S4). The fruits on stage 2 of ripeness were chosen for the coating analyses.

Coating of the fruits
For coating the fruits, D. altissima starch (4% w/v) was firstly diluted in distilled water. Subsequently, glycerol (3% w/v) was added and heated to 95 °C until complete gelatinization. The cooled solution was added to the colloidal solution of silver nanoparticles (2 µg/mL) from the nanoparticle bank of the DPUA Culture Collection (Fayaz et al., 2009;Pagno et al., 2015). The fruits were completely immersed in filmogenic solutions for 5 seconds at 25° C two times consecutively. Then, the fruits coated with the standard film formulated with starch and glycerol (CF), the fruits coated with the test film incorporated with AgNPs (CFN) and the control fruits without any coating film (WF) were kept under refrigeration (5 ± 2 ºC). After 12 days the fruits were evaluated according to their visual appearance, physical injuries and the presence or absence of decay.

Determination of pH, titratable acidity, total soluble solids and weight loss
The pH of the fruits was measured using a digital pH meter, model PHS-3E. Acidity expressed as citric acid was determined by titration with 0.1M NaOH solution, using 1% (w/v) alcoholic phenolphthalein solution. The content of soluble solids, expressed in degrees Brix (°Brix), was measured by direct reading of a double scale Incoterm portable refractometer (AOAC, 2006). The fruits were weighed with an analytical balance to calculate the loss of mass (Fakhouri et al., 2007).
Analyses were performed on the 1 st , 4 th , 8 th and 12 th days.

Microbiological analysis
The microbiological analysis of the fruits was performed the 1 st , 4 th , 8 th and 12 th days to assess the presence of Coliforms at 45 °C, Escherichia coli, Salmonella sp., yeast and filamentous fungi (Loy-Hendrickx et al., 2018).

Statistical analysis
The data from the experiments carried out in triplicate and were subjected to analysis of variance. The means were compared by the Tukey test (<0,05), using the Minitab version 17.0 software.

Visual aspect, Humidity, Solubility, Thickness, Water vapor permeability of coating films
The visual analysis of the films showed that the addition of AgNPs did not interfere in the filmogenic capacity of D.
altissima starch. The evaluated films showed transparency, flexibility and absence of any cracks when removed off the plates ( Figure 1). According to Fakhouri et al. (2007) edible films used to coat fresh foods must be transparent, odorless and tasteless, characteristics that do not interfere with the product's sensory properties.
The characteristics of the coating films are shown in Table 1. The humidity values in the standard film and in the test films were 8.22% and 11.16%, respectively. The values determined by Kanmani and Rhim (2014) in gelatin films without and with AgNPs were 16.9% and 17.3%, respectively. These results related to the increase in humidity in films with AgNPs are similar to those obtained in the present study. The variation in humidity in the samples may be related to the reduction of the interaction between the amylose and amylopectin chains, a factor that allows the absorption of water by free hydroxyl groups.
Additives incorporated in the films can influence the increase or decrease in the humidity of the starch films (Dantas et al., 2015).
The incorporation of AgNPs in films with D. altissima starch also caused a reduction of 15.3%, 7.6%, 15.68% and 24.67% in thickness, solubility, water vapor and permeability, respectively. Thickness control is an important parameter in film production, as it directly influences water vapor permeability and solubility (

Scanning Electron Microscopy
Figure 2a and Figure 2b shows that the standard films have a smooth and homogeneous surface. However, in films incorporated with AgNPs, spherical clusters were observed. A similar result was reported by Oliani et al. (2016) in polyethylene films formulated with AgNPs.

Atomic Force Microscopy
The three-dimensional images obtained by Atomic Force Microscopy (AFM) confirm the results obtained by SEM ( Figure 3a and Figure 3b). The D. altissima films incorporated with AgNPs present a higher average roughness of 16.40% when compared to the standard film. Ortega et al. (2017) mentions that high concentrations of AgNPs used in coating films tend to form agglomerates and, consequently, generates a rougher surface.

Fourier-Transform Infrared Spectroscopy
The FTIR spectra of standard films and test films showed similar bands and small displacements that can be explained due to the interaction of AgNPs and starch. An intense band was observed at 3293 cm -1 that refers to the hydroxyl group present in the starch and in the plasticizer (Bergo, Sobral & Prison, 2010). The peaks at 1648 cm -1 indicate the presence of water bound to the polymer (Sueiro, Faria-Tischer, Lonni & Mali, 2016). The bands found in the 1000 cm -1 region are related to the crystalline structure of starch (Ferreira, Passos & Marques, 2015).

X-Ray Diffraction
Analysis of the XRD films showed that the standard and the test films presented peaks at diffraction angles close to each other. These data confirm the presence of starch extracted from type B tubers in the preparation of D. altissima films. Similar results were found by Chevirón, Gouanvé & Espuche (2016) in potato starch-based films.

Silver concentration in films
The analysis performed in the ICP-MS showed that the AgNPs incorporated films had silver concentration of 152.50 (µg/mL). Li, Li, Zhang, Yun & Qin (2018) reported that this concentration is lower than that recommended for food by the European Food Safety Authority. The possibility of migration of AgNPs from packaging to food is very low. In addition, studies reporting the possible toxicological effects of AgNPs levels in food as a result of contamination through packaging are still scarce (Simbine et al., 2019).

Film antimicrobial activity
The films incorporated with AgNPs showed bactericidal action against S. aureus (9 mm) and bacteriostatic action against E. coli (7 mm). However, C. albicans did not express sensitivity. Similar data were observed by Silva-Vinhote et al. (2017). Yoksan and Chirachanchai (2010) reported that AgNPs attach themselves to the surface of the bacterial cell membrane and disrupts its integrity, causing damage to the metabolism and death of the microorganism.

Visual aspect of the fruits
The visual evaluation of the fruits after 12 days of storage showed that the fruits without film (WF) showed wrinkling, with inadequate physical characteristics for consumption and commercialization. The fruits coated with the standard film without AgNPs (CF) showed deterioration and those with silver nanoparticles (CFN) showed delay in ripening and preserved their integrity, which is a favorable aspect for commercialization of fresh foods (Figure 4a, Figure 4b and Figure 4c). Similar data were observed for minimally processed melons, packed in trays made of cellulose and AgNPs (Assis & Britto, 21014).
Coating films promote the filling of possible aperture on the fruit peel, reducing the permeation of O2 inside the fruit and consequently reducing ethylene production and maturation (Sanches, Silva, Moreira, Costa & Cordeiro, 2017).

Fruit weight loss
AgNPs, in addition to inhibit the growth of microorganisms in fruits, can also help to control the weight loss during storage. Figure 5 shows that the weight loss of fruits without coating film (WF), fruits coated with the standard film without AgNPs (CF) and fruits coated with the test film incorporated with AgNPs (CFN) were not significantly different (<0.05) from each other until the 4 th day of analysis. According to Sanches et al. (2017) the fruits are climacteric and have a high respiratory rate. Probably, the coating of fruits with films has reduced the respiration and consequently led to a decrease in ethylene production and loss of mass; features that contributed to a longer conservation period. The addition of AgNPs in coating films is considered an alternative to improve the mechanical, thermal, gas and water barriers of the films (Li et al., 2018).

Total soluble solids
The content of soluble solids influences the flavor and acceptance of products of plant origin. In addition, it is an important parameter used to indicate the degree of fruit ripeness (Sun et al., 2019). Figure 6 shows that the content of total soluble solids in all fruits exhibited no significant difference (<0.05) until the fourth day of storage. At the end of the process, all samples showed a significant increase of soluble solids. When compared to time zero, an increase of 28.20%, 18.45% and 9.35% of soluble solids was observed in WF, CFN and CF, respectively. These results demonstrated that the coating films provide a novel alternative to delay ripening, and, maintain the quality and longevity of camu-camu fruits. Oliveira, Silva, Silva & Spoto (2014) also found an increase in total soluble solids in the fruits of Myrciaria dubia, which were not packed with BOPP (Bioriented Polypropylene) and PVC (Polyvinyl Chloride). Likewise, Fernandez, Picouet & Lloret (2010) observed that fruits packaged without the addition of AgNPs showed an increase in the content of soluble solids. The greater loss of turgidity by these unpackaged or packaged fruits without the addition of AgNPs influences the concentration of the pulp and consequently the content of soluble solids. The films with AgNPs probably decreased the ethylene synthesis and the respiratory activity of the fruits, delaying the physiological disorders and consequently their deterioration.

Titratable acidity and fruit pH
Throughout the storage period, the pH of the fruits remained acidic, in the range of 2.9 to 3.2. These pH values probably inhibited the growth of microorganisms in all fruits, with and without the coating films. Figure 7 shows the titratable acidity of the camu-camu fruits. In CF and CFN there was a gradual increase during the storage period. However, in WF, significant variations of this physicochemical parameter were observed. At the end of the storage period, the titratable acidity levels, in decreasing order, were 2.18%, 2.08% and 1.97%, in CF, WF and CFN, respectively. Organic acids tend to decrease with fruit ripening, as they are used as a substrate for respiration (Beber, Álvares & Kusdra, 2018). On the other hand, among other factors, the content of organic acids in fruits can also vary according to species, soil and stress conditions (Carvalho et al., 2020).

Microbiological analysis of fruits
The data from the microbiological analysis of the fruits confirmed the absence of microorganisms. The presence of Coliforms, E. coli, Salmonella sp., yeast and filamentous fungi was not verified. These results are likely to be related to the intrinsic conditions of the fruits, such as acidic pH. The most favorable pH for the growth of most of the pathogenic microorganisms is in the range of 6.5 -7.0 (Sanches et al., 2017). Moreover, according to Arellano-Acuña, Rojas-Zavaleta & Paucar (2016), camu-camu naturally shows antimicrobial activity against Staphylococcus aureus, Streptococcus mutans, S. sanguinis and C. albicans.

Conclusion
The results showed that the films prepared with D. altissima starch are transparent and flexible. However, the presence of AgNPs determines the formation of roughness, being a desirable characteristic of coating films for fresh foods.
Films with AgNPs have bactericidal action against S. aureus, bacteriostatic against E. coli and without expression of sensitivity by C. albicans. Fruits coated with the film without AgNPs present deterioration. On the other hand, films with AgNPs maintain integrity, delay ripening and increase the shelf life of fruits. As a suggestion, it is proposed to investigate the efficiency of other methods for the film's elaboration, the use of silver nanoparticles of different origins and characteristics to increase the antimicrobial activity and the shelf life of alimentary products.