Physical , physicochemical , microbiological , and bioactive compounds stability of low-calorie orange jellies during storage : packaging effect

Fruit jellies are widely produced as a way to utilize fresh fruits, which are highly perishable. Orange a fruit widely consumed in Brazil, it has a significant amount of bioactive compounds. Despite the great progress in the development of jellies, several factors can change its useful life, among them is the packaging. Therefore, the objective of this study was to evaluate the effect of packaging on the physicochemical, physical, microbiological and bioactive compounds stability of low-calorie orange jellies during storage. Analyses every 30 days during the 180 days of storage. The results showed that increased storage time led to a decrease in pH, reduction of the flow rate (polypropylene packaging), reduction in yellow intensity, and growth of fungi and yeasts (higher in polypropylene packaging). In contrast, luminosity, red intensity, moisture, total sugars, and the consistency index tended to remain stable during storage. The DPPH results showed an increase in the antioxidant activity and reduction of vitamin C throughout the period of storage, especially in polypropylene packaging. The total phenolic content was stable with a tendency to decrease during storage. Notably, vitamin C showed a positive correlation with antioxidant activity in jellies. Lowcalorie orange jellies packaged in glass showed the least changes during storage.


Introduction
Consumption of fruits and fruit products is of paramount importance due to their various health benefits. Fruits have anti-inflammatory and antioxidant properties, which contribute to individual health. In addition, they can promote low-energy intake, because they contain fiber, which increases satiety (Souza et al., 2014). The orange (Citrus sinensis L. Osbeck) of the family Rutaceae has a high content of bioactive compounds represented mainly by vitamin C. Vitamin C favors the elimination of free radicals, helps protect cells against oxidative damage and flavonoids that have anti-inflammatory and anticancer activities, improves blood pressure and lipid profile, and reduces risk of cardiovascular diseases (Coelho et al., 2013).
Fruit jellies are innovative, practical, and functional products that can be used to preserve fruits, which are highly perishable (Souza et al., 2014). However, because of the increase in chronic lifestyle-related diseases, such as diabetes and obesity, there is a growing need for low-calorie products, such as low-calorie jellies (Abolila et al., 2015). To obtain lowcalorie jellies, it is necessary to reduce the amount of sugar. However, it is also necessary to add sweeteners, to impart a sweet flavor; body agents, to give form to the product; and gelling agents, to aid in their formation (Nachtigall et al., 2004). It is important to verify the stability of these ingredients during storage, as their stability will directly affect the shelf life of final product .
Despite much progress, food storage stability is still a complex issue due to the numerous factors that can reduce shelf life. In fruit jellies, changes in acidity, sugar hydrolysis, increased or decreased moisture, darkening, increased or decreased consistency, syneresis, and the growth of microorganisms, mainly mold and yeasts, can cause product deterioration (Hayat et al., 2005;Javanmard & Endan, 2010). The bioactive compounds are very unstable and can be degraded during processing and storage (Igual et al., 2015). The packaging in which the food is confined may exert an influence on the stability of bioactive compounds, especially the permeability of the container to oxygen, which favors the degradation of these compounds, or the presence of a barrier to the entrance of light (Pérez- Research, Society and Development, v. 9, n. 9, e759997900, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7900 5 Vicente et al., 2004. It is important that the food industry is aware about the degradation of these bioactive compounds since this factor influences the shelf life of foods, especially those that have a functional food claim (Shimoni 2004;Zulueta et al., 2010). Therefore, the objective of this study was to evaluate the effect of packaging on the physicochemical, physical, microbiological and bioactive compounds stability of low-calorie orange jellies during storage.

Materials
The following materials were used: oranges (Pêra Rio cultivar), crystal sugar

Processing of oranges and jellies
After washing the oranges, the fruit was soaked in a 2.5% sodium hypochlorite solution for 15 minutes to sanitize them and avoid contamination of the fruit pulp. Then, orange juice was obtained by processing the fruits in an electric juicer without adding sugar or water. The juice was stored at -18 °C in polyethylene pots covered with aluminum foil.
The jellies were prepared in an open pan heated by a gas flame as follows according to the method of Lima et al. (2019). First, orange juice (60%), crystal sugar (20%), and polydextrose (18.925%) were mixed. Then, the mixture was heated at 80 °C to 30° Brix, and then the gelling agents, low methoxylated pectin (0.7%) and gum carrageenan (0.3%) dissolved in 5 mL of distilled water, were added. Next, the mixture was baked to 60° Brix.
Then, the sweeteners acesulfame-k and sucralose were added, as described by Souza et al. (2013), as well as the preservative potassium sorbate (0.05%) dissolved in 2 mL of distilled water. The jellies were cooked until a final soluble solid with a value of 65° Brix was achieved. Then, the jellies were hot packed in previously sterilized transparent glass and polypropylene (PP) jars (glass jars were sterilized in boiling water for 10 min. and polypropylene jars were sterilized in water with 2.5% sodium hypochlorite for 20 min.) and Research, Society and Development, v. 9, n. 9, e759997900, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7900 6 stored in an incubator chamber at 25 °C for 6 months.

Physicochemical evaluation of low-calorie orange jellies
The moisture, acidity, and pH were analyzed according to IAL (2008) and AOAC (2011). The method of Dische (1962) was used to determine total sugars. The analyses were performed in triplicate.

Physical evaluation of low-calorie orange jellies
The color of the jellies was determined using a Konica Minolta colorimeter, model CR 400, working in D65 (daylight) and using CIELab standards, according to the method of Lau et al. (2000) in quadruplicate.
The rheological analysis were performed in a cone/plate-type rheometer (Brookfield model RV-III) using Rheocalc software (version 3.0), spindle CP52, and 0.5 g of sample. To obtain the upward curve, the speed of rotation was 1-250 rpm, which was increased in 5 intervals at 50 rpm. To obtain the downward curve, the procedure was repeated in the reverse direction, with progressively decreasing velocities (250-1 rpm). The measurements were performed in triplicate.
Syneresis was evaluated according to the method of Licodiedoff et al. (2010).

Microbiological evaluation
The microbiological quality of the jellies was evaluated by determining the numbers of molds and yeasts that were determined by plating the homogenate on PDA (Potato Dextrose Agar), acidified with tartaric acid 10%. The results were expressed in colony forming units per gram of jelly (CFU/g) and compared with the standards established by DRC n° 12 (Brazil, 2001).

Bioactive compounds evaluation
The stability of bioactive compounds was assessed by means of determination of ascorbic acid, total phenolic content and antioxidant activity (ABTS, DPPH and β-carotene methods).

Determination of ascorbic acid (Vitamin C)
For the determination of the ascorbic acid content, standard AOAC (1984) methodology modified by Benassi & Antunes (1988) was used. Dilution of the samples was done in 100 mL of 2% oxalic acid solution, and a 25 mL aliquot was then titrated with 0.025% DCFI (2,6-dichlorophenolindophenol) solution until pink coloration was obtained.
The solution was previously standardized with L-ascorbic acid solution. The results are expressed as mg ascorbic acid/100 g of fw.

Obtaining extracts of samples for analysis of phenolic content and antioxidant activity
The extracts were obtained according to the method described in a previous study (Larrauri et al., 1997). Ten grams of the sample was weighed, followed by the addition of 40 mL of methanol/water solution (50:50 v/v). This mixture was kept under stirring (200 rpm) at room temperature for 60 min and then allowed to rest in a cooled (8 °C) environment for 30 min. The supernatant was then recovered, filtered, and transferred to a 100 mL flask. Next, Forty millilitres of acetone/water (70:30, v/v) was added to the residue, maintaining stirring (200 rpm) at room temperature for 60 min, and then allowed to stand in a cooled (8 °C) environment for 30 min. The methanol and acetone extracts were combined and brought to a final volume of 50 mL with distilled water for the determination of antioxidant activity, total phenolic content.

Antioxidant activity
The antioxidant activity was determined using the ABTS, DPPH and β-carotene methods. For the ABTS assay, the procedure followed the previous method described by Re et al. (1999) with few modifications. The 2,2-azinobis (ABTS) radical cation (ABTS•+) was generated by reaction of 5 mL of aqueous ABTS solution (7 mM) with 88 µL of 140 mM (2.45 mM final concentration) potassium persulphate. The mixture was kept in the dark for 16 h before use and then diluted with ethanol to obtain an absorbance of 0.7 ± 0.05 units at 734 nm using a spectrophotometer VIS 325-1000 nm. The jelly extracts (30 µL) or a reference substance (Trolox) was put to react with 3 mL of the resulting blue-green ABTS radical solution in the dark. The decrease of absorbance at 734 nm was measured after 6 min.
Ethanolic solutions of known Trolox concentrations were used for calibration (100-2.000 µM). The results are expressed as micromoles of Trolox equivalents (TEs) per gram of jelly (µmol of TEs g-1 of jelly).
DPPH-free radical scavenging capacity was estimated using the method reported by Brand-Williams et al. (1995). Briefly, the DPPH solution (600 µM) was diluted with ethanol to obtain an absorbance of 0.7 ± 0.02 units at 517 nm. Jelly extracts (0.1 mL) were put to react with 3.9 mL of DPPH radical solution for 120 min in the dark, and the decrease in absorbance of the resulting solution was monitored. The absorbance of the reaction mixture was measured at 517 nm. The results were expressed as EC50 (g of jelly/g of DPPH).
The antioxidant activity was also determined by the β-carotene method, following the procedure described by Marco (1968) with minor modifications. Briefly, an aliquot (50 µL) of the β-carotene chloroform solution (20 mg/mL) was added to a flask containing 40 µL of linoleic acid, 1.0 mL of chloroform, and 530 µL of Tween 40 and then mixed. The chloroform was evaporated using an oxygenator. After the evaporation, oxygenated distilled water (approximately 100 mL) was added to obtain an absorbance of 0.65 ± 0.5 units at 470 nm. An aliquot (0.4 mL) of Trolox solution (200 mg/L) or diluted jelly extract (200 mg/L) was added to 5 mL of the β-carotene solution and incubated in a water bath at 40 °C. The measurements were performed after 2 min and 120 min at an absorbance of 470 nm using a spectrophotometer. The antioxidant activity was calculated as the percent inhibition relative to the control.

Experimental design and statistical analysis
The experimental design was a complete 2×7 factorial, and the factors under study were packaging (glass and polypropylene-PP) and storage time (0, 30, 60, 90, 120, 150, and 180 days).
The data were evaluated by analysis of variance (ANOVA), Tukey's test, and regression at 5% significance using Sisvar software (Ferreira, 2014).  Table 1 presents the average physicochemical parameters of the low-calorie orange jellies over 180 days of storage in glass and PP packaging. Research, Society and Development, v. 9, n. 9, e759997900, 2020 (CC BY 4. According to the data shown in Table 1, the pH tended to decrease during storage.

Physicochemical evaluation of low-calorie orange jellies
Regarding packaging, there was a significant difference (p ≤ 0.05) in pH at 60 and 150 days, and the pH of the jelly in the PP packaging was lower than that in the glass packaging.
Several studies that analyzed the stability of jellies during storage also observed a reduction in pH over time (Hossen et al., 2009;Safdar et al., 2012;Arévalo-Pinedo et al., 2013). In this study, the acidity of the jellies tended to increase throughout the storage period (p ≤ 0.05), and the packaging significantly affected acidity (p ≤ 0.05) at 0 and 120 days. The increase in acidity is related to the reduction of pH, as discussed above. Acidity is an important parameter for the formation and stability of the gel, and it must be 0.3-0.8%, because above this value, syneresis may occur (Assis et al., 2007;Dias et al., 2011). Despite the increased acidity, the values remained within the recommended range for avoiding syneresis. It was observed by resulting from the formation of acidic compounds due to the degradation of organic compounds.
The moisture of the jellies was significantly increased (p ≤ 0.05) at 30 days of storage, but then stabilized and remained at this level until the end of the storage period (Table 1).
Regarding packaging, a significant difference (p ≤ 0.05) was only observed at the end of storage period, and the highest average moisture content was observed in the glass packaging.
As glass packaging is chemically inert, and its closure does not compromise this feature, the higher moisture content at the end of the storage period cannot be attributed to packaging failures. Therefore, the increase in moisture observed at the end of storage period may have been caused by sugar hydrolysis, which releases water molecules . These data corroborate the results of Martins et al. (2015) in a study on the stability of cajá jellies during storage. In their study, jellies stored in glass containers showed increased moisture with storage (150 days). The authors stated that was due to interruption of the gel structure formed by pectin, sugar, and water, favoring the release of water and a consequent increase in moisture.
The total sugar content of the jellies tended to remain stable during storage; however, there was a significant reduction at 120 days and a subsequent increase at 150 and 180 days in both packaging (p ≤ 0.05) ( Table 1). These data are similar to those collected in studies by Arévalo-Pinedo et al. (2013) in which the stability of araticum jellies. In these studies, there was no significant change in the total sugar content during storage.  The luminosity (L*) (Figure 1a) of the jellies was stable, but tended to decrease in both packaging. Jellies stored in PP packaging had lower L* values than those stored in glass packaging, indicating that the jelly stored in PP was darker. The PP packaging has a lower oxygen barrier, which favors the oxidation of vitamin C, producing hydroxymethylfurfural and furfural, which generate melanoidins, compounds that confer a dark coloration (Gava et al., 2008;Gliemmo et al., 2009). Was reported by Damiani et al. (2012a)  Red intensity (a*) was generally stable during storage, but showed a tendency to increase (p ≤ 0.05) at the end of the storage period (Figure 1b). Red intensity values tended to increase during storage in both packaging, probably due to non-enzymatic darkening reactions that can generate red pigments (Gliemmo et al., 2009).

Physical evaluation of low-calorie orange jellies
The yellow intensity (b*) values were close to +b (Figure 1c), which is characteristic of the fruit pulp from which the jelly was formulated (Ramalhosa et al., 2017). During storage, the b* of the jellies in both packaging tended to decrease. This reduction may be due to degradation of the compounds in the fruit base from which the jelly was formulated. Damiani et al. (2012b) and Oliveira et al. (2014) also observed a reduction in b* values in araçá and umbu-cajá jellies, respectively, during storage. Figure 2 show the effects of the glass and PP packaging on the consistency (k) and flow rate (n) of the low-calorie orange jellies. The consistency index was not affected by the packaging (Figure 2a), as the consistency index of the jellies in both packaging was stable, but showed a tendency to increase over time. An increase in consistency during storage was also reported by Vahedi et al. (2008), who studied the quality of a yogurt formulation during storage, and Garrido et al. (2015), who studied the rheological parameters, color, and acceptance of apple jelly. reduced electrostatic repulsion between pectin molecules due to a decrease in the dissociation of carboxylic groups, favoring an increase in the possibility of contact between these molecules and consequent increase of gel stiffness (Gava et al., 2008). Figure 3 show the occurrence of syneresis in low-calorie orange jellies in relation to the packaging used (PP and glass) over the storage period (180 days). Syneresis occurred after 90 days of storage ( Figure 3). Several factors may be involved in the occurrence of syneresis, including the low pH, high acidity, and excess invert sugar (Teles et al., 2017). Despite the occurrence of syneresis after 90 days, the values were not significant (p > 0.05). This limited syneresis may be due to the use of hydrocolloids, which favor the formation of a more rigid and more stable gel. Khouryieh et al. (2005) evaluated the influence of different hydrocolloids, alone and in combination, on gel stability in low-sugar jellies and observed that a combination of hydrocolloids favored a reduction in syneresis during storage.

Microbiological evaluation
The Figure 4 show the molds and yeasts counts in the low-calorie orange jellies as a function of storage time and packaging. There was molds and yeasts growth in the jellies in both packaging during storage; however, there was significantly (p ≤ 0.05) lower growth in the glass packaging over time.
Despite the molds and yeasts growth, the values were within the acceptable range under Brazilian law (maximum, 10 4 CFU/g) (Brazil, 2001). Several factors may favor the growth of molds and yeasts during storage, and the main ones are acidic pH, high moisture, high storage temperatures, the presence of oxygen, the chemical composition of the food, and the added sugars, which are used as energy source for microbial growth (Franco & Landgraf, 2001;Azeredo, 2012). The PP packaging has a lower oxygen barrier (Jorge, 2013) compared to the glass packaging, which favors the growth of molds and yeasts, since most of these microorganisms are aerobic (Franco & Landgraf, 2001). Effect of oxygen-barrier and barrierfree packaging on the characteristics of orange juice that was unpasteurized, pasteurized at 66 °C for 10 s, and pasteurized at 90 °C for 60 s was evaluated by Sadler et al. (1992). The authors observed that, for pasteurized juices, the oxygen-barrier packaging improved the microbiological stability of orange juice. Table 2 presents the average results for the bioactive compounds and the antioxidant activity of low-calorie orange jellies stored in different packaging throughout the storage.

Bioactive compounds and antioxidant activity
Research, Society and Development, v. 9, n. 9, e759997900, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7900 The antioxidant activity of low-calorie orange jellies by DPPH displayed a tendency to increase during storage since lower values of EC50 are equivalent to higher antioxidant activity (Table 2). There was a significant difference (p≤0.05) in relation to the packaging only at 90 days of storage, and the highest averages were found in the glass packaging. The factor that may have assisted the increased antioxidant activity in jellies was the degradation of fruit compounds, and the consequent formation of degradation products that may have antioxidant activity (Shinwari & Rao, 2018). The study by Damiani et al. (2012b), which evaluated the antioxidant potential of araça jelly during storage, also showed an increase in antioxidant activity up to 8 months of storage, followed by a decrease up to 12 months of storage.
Regarding the other methodologies used to evaluate the antioxidant activity, it can be observed that the methodology proposed by the reduction of the ABTS•+ radical did not present a significant difference in relation to storage time and packaging used (p> 0.05).
Moreover, the methodology proposed by the inhibition of lipid peroxidation in the βcarotene/linoleic acid system demonstrated stability at the beginning and end of storage, with small variations over time. These methodologies are limiting due to the selectivity to certain compounds (Hassimoto et al., 2005). The ABTS•+ methodology presents differences in the incubation time and low selectivity in the reaction with hydrogen donor atoms, while the methodology proposed by the percentage of lipid peroxidation is more sensitive to lipophilic antioxidant compounds. Thus, this may be the cause of the difference in the results when compared to the methodology of DPPH (Hassimoto et al., 2005).
The phenolic content present in the jellies were observed to be stable with a tendency to decrease during storage. The packaging influenced changes significantly (p≤ 0.05) in the phenolic content at 60, 90, and 120 days of storage, with the lowest averages observed in jellies stored in PP packaging. The use of low methoxylated pectin (LMP) in jellies may be related to the retention of phenolic content, as reported in studies with low-calorie and conventional jellies, the mechanism for which remains to be elucidated (Shinwari & Rao, 2018 The classification of fruits and fruit products based on vitamin C content is as follows: low (<30 mg/100 g), medium (30-50 mg/100 g) and high (> 50 mg/100 g) (Souza et al., 2012). Thus, despite the losses of vitamin C during storage, low-calorie orange jellies can be classified as high in vitamin C, as well as favoring adequate intake of vitamin C with the consumption of 100 g of jelly, according to vitamin C recommendation for adults (90 mg/day for men and 75 mg/day for women) (Cuppari, 2005).
There was a positive correlation (r = 0.84) observed between the antioxidant activity and amount of vitamin C, with regard to PP packaging and poor correlation was observed for glass packaging (r = 0.68), as shown in Figure 5. In relation to the antioxidant activity and phenolic content, there was no correlation, since the results were negative (r = -0.28 for PP packaging and r = -0.23 for glass packaging), as shown in Figure 6. Therefore, vitamin C was determined to be the compound that most contributed to the antioxidant activity of low-calorie orange jellies stored in PP packaging, corroborating the study by Souza et al. (2012) that, when determining the antioxidant activity, chemical composition and bioactive compounds of fruits of the cerrado, verified a positive correlation between vitamin C and the antioxidant activity of fruits.

Final Considerations
The pH, acidity, and mold and yeast growth in fruit jellies were influenced by storage time. Although mold and yeasts were observed, the counts were in compliance with Brazilian law. Syneresis occurred after 90 days of storage; however, it was not significantly related to storage time or packaging.
The packaging and time of storage influenced the content of bioactive compounds at the end of storage. There was a tendency to increase the antioxidant activity, determined by the DPPH method, which could not be verified by the ABTS•+ and β-carotene/linoleic acid methodologies.
There was a reduction of vitamin C observed throughout the storage, accompanied with exhibition of a positive correlation with the antioxidant activity in the polypropylene packaging, indicating that this compound influenced the antioxidant activity in this packaging.
It was demonstrated that the stability of total phenolic content reduced substantially in PP packaging. There was no correlation of these compounds with the antioxidant activity.
It can be concluded, therefore, that glass packaging was more favorable to maintain the physicochemical, physical, and microbiological character and bioactive compounds stability at the end of the storage since the most significant changes in these compounds occurred in the PP packaging. Thus, campaigns should be carried out with the fruit jelly processing industries to highlight the importance of storing these products in glass packaging, in order to promote a product with greater nutritional value, safe and with greater stability.
In the future, it is suggested that sensory tests be carried out over the storage period in order to verify product acceptability by consumers.