Modified atmosphere using propolis in post-harvested ‘Eva’ organic apple

1School of Agriculture, São Paulo State University (UNESP), 2Regional Research Alta Paulista PoleRegional APTA, 3School of Agriculture, São Paulo State University (UNESP), 4School of Agriculture, São Paulo State University (UNESP), 5Federal University of Paraíba, 6Agrarian Services Division, Experimental Farm of Agrarian Sciences, Federal University of Grande Dourados, 7Department of Horticulture, School of Agriculture, São Paulo State University (UNESP),8Department of Animal Production, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP)


INTRODUCTION
Storage is one of the most important factors in maintaining fruit quality and making it possible to supply market for long periods. Apple culture, for example, has made good progress over the years with investments in chambers and new technologies such as storage under a controlled modified atmosphere (Petri et al., 2011).
However, more accessible techniques may be used in addition to refrigeration. Among known methods which may be used to extend fruit storage, it is possible to highlight the use of passive modified atmosphere, which may be by plastic films (packaging) use or coating with special waxes (Daiuto et al., 2012), among them propolis.
Propolis is a natural resinous compound produced by Apis mellifera bees from which they extract from various plant exudates and behave as hives protection layer against microorganisms such as fungi and bacteria proliferation (Meneses et al., 2009;Silva et al., 2006). The composition of propolis is directly related to region vegetation from it is extracted, being composed of resins (phenolic and sterile compounds), essential oils, balsams, waxes, vitamins and pollen (Zahid et al., 2013;Konishi et al., 2004).
It is used medicinally for having many biological properties such as: antitumor effect, antioxidant, antimicrobial, antiinflammatory and immune modulatory, among others. These biological activities are attributed mainly to phenolic compounds in its composition, such as flavonoids. The concentration of it, in its extract, is directly dependent of used extractor, as in its composition there are soluble substances in water and oil (Mello et al., 2010;Viuda-Martos et al., 2008).
The objective of this study was to evaluate the efficacy of different propolis extracts in organic 'Eva' apple post-harvested fruits, in order to prolong storage and maintenance of fruit quality.

MATERIALS AND METHODS
The organic 'Eva' apple fruits used in study were produced in commercial production area at Mitsuo Hino Farm, located in Botucatu, São Paulo State, Brazil (22° 95' 97" S and 48° 45' 02" W), in 2015/2016 crop season. They were harvested at physiological maturation stage and then stored in a cold room at 5ºC, in the rural property itself.
Afterwards fruits were transported in plastic boxes to São Paulo State University (UNESP), School of Agriculture, in municipality of Botucatu, and conducted at Laboratory of Fruits and Vegetables of Department of Horticulture ( Fig. 1). After receiving the fruits, they were submitted to visual selection aiming lot uniformity. Then, they were washed in running water and sanitized by immersion in vinegar solution (6%) for 10 minutes, hygienizing allowed for organic products (Brasil, 2009). After this process they were placed on a laboratory bench lined with pink semi Kraft ® paper to drain water and dry fruits.
The propolis, produced by Apis mellifera L. bees was collected at Apiculture area of School of Veterinary Medicine and Animal Science from the same university, located at Edgárdia Farm (22º 82' S and 48º 39' W).
For alcoholic extract (EAP) the propolis was crushed transferring 30 g to a Griffin's cup adding ethyl alcohol (70%) until 100mL. Immediately the solution was stored in amber glass shaking daily for one minute during seven days for ethanolic propolis extract (EEP) preparation. After this period solution was purified in analytical filter and stored in amber glass (Orsi et al., 2000). For aqueous extract (EAqP) ethyl alcohol was replaced by mineral water. to facilitate preparation by producers, and following the same methodology of alcoholic extract. From the 30% extract, dilutions were performed until extracts reached concentrations of 1.5% and 2.5%.
After treatments application, apples were placed to dry in the air. Fruits were packed in rigid plastic boxes (dimension: 30 x 36 x 55 cm) and stored in cold room at temperature of 5 ± 1°C and 85 ± 1% RH for 80 days.
Fruit quality determinations were obtained through following evaluations: mass loss (%). respiratory activity (mL of CO 2 kg of fruit -1 hour -1 ) following guidelines of Bleinroth et al. (1976), luminosity, chroma, Hue angle (Minolta, 1998). The pH, soluble solids (°Brix) and titratable acidity (g of malic acid 100g -1 ) were analysed according to methodology cited by Brasil (2008), maturation index (Tressler and Joslyn, 1961) and reducing and non-reducing sugars (Somogy, 1945adapted by Nelson, 1944. The experimental design was completely randomized in a 5 x 9 factorial scheme (five treatments x 9 storage periods) with three replications. Data were submitted to F test (p <0.05) and significant interactions were deployed via regression analysis and means compared by Tukey test when the regression was not significant.

RESULTS AND DISCUSSION
Climacteric behavior (Fig 2) was observed in the study as reported by Vieites et al. (2014) and Fante et al. (2013). The climacteric peak of the control, 1.5% propolis alcoholic extract (EAP), 2.5% propolis alcoholic extract (EAP) and 1.5% aqueous propolis extract (EAqP) occurred within 70 days of storage. However, 1.5% EAP treatment and 1.5% EAqP presented the lowest peaks (36.2 mL and 39.5 mL of CO 2 kg of fruit -1 hour -1 , respectively) when compared to control treatment (56.9 mL CO 2 kg of fruit -1 hour -1, ). Possibly the extracts 1.5% EAP and 1.5% EAqP worked as fruits coating, reducing gas exchange to external environment. Daiuto et al. (2012), researching 'Hass' avocado cited delay in climacteric peak in fruits treated with propolis alcoholic extract (2%) and wax. In contrast, treatment with 2.5% EAqP advanced fruits climacteric peak for tenth day, with respiratory rate of 69.6 mL of CO2 Kg of fruit -1 hour -1 , possibly advancing fruits senescence.
In relation to period of storage it is possible to notice that mass loss behaved in an increasing linear way (Fig 4). At eightieth day of storage it has lost 13.5% of mass. This behavior is probably directly related to water loss through transpiration and solutes (carbohydrates) via glycolysis during respiration. Climacteric fruit has an intrinsic increase in respiration rate at a certain stage of its life cycle (Chitarra and Chitarra, 2005). The same authors mention that losses from 3 to 6% are capable of causing loss of quality in stored fruits. In the storage conditions of current research (5 °C ± 1 °C/80% ± 1% RH) organic 'Eva' apple fruits have lost 6% of mass at 35 days of storage.
Soluble solids (SS) contents were not influenced by propolis extracts application and even for the interaction between them (p> 0.05) ( Table 2). Santos et al. (2015), reported as current research, there was no influence of propolis extracts on SS contents in agroecological avocado. Passos et al. (2016), researching about propolis extracts and despite finding influence on levels of SS between treatments, mentioned that treatments that better conserved the contents (aqueous, wild and green) did not statistically differentiate from control.
Contents from 13.3 °Brix to 16.7 °Brix were observed in 'Eva' organic apple fruits, with a general average of 14,9 °Brix. Similar values were found by Vieites et al. (2014) working with organic 'Eva' apple from the same production area, 13.9 °Brix to 15.8 °Brix. Chagas et al. (2012) reported levels of 15.2 °Brix to grow conventional 'Eva'. Oliveira et al. (2014) observed 9.2 °Brix to 13.6 °Brix in apple 'Eva' harvested in five seasons and Fante et al. (2013) found levels of 13.2 °Brix to 14.8 °Brix in apples of same cultivar produced in a conventional system, both works performed in Minas Gerais State. The fruits have a biological character and they present numerous changes in their composition according to each variety characteristics, climatic conditions, soil, management and harvesting point. Respiratory activity (mL of CO 2 kg of fruit -1 hour -1 ) in organic 'Eva' apple with application of different propolis extracts over 80 days of storage. EAP: alcoholic propolis extract, EAqP: aqueous propolis extract. For storage period, soluble solids contents increased (Fig 5), starting from 13.3 °Brix at experiment assembly time, achieving 15.78 °Brix at the end of experiment time. This soluble solids increase tends to occur during ripening due to polysaccharides (starch) degradation reaching maximum value at the end of ripening, conferring quality to the product (Chitarra and Chitarra, 2005). The same behavior was described by Fante et al. (2013) in 'Eva' apple with 135 days of storage. Authors justify this increase to fruit maturation and/or concentration of these solutes due to the loss of fresh mass.
In relation to pH values (Table 3) there was significant difference at treatments means (p <0.01) and storage period (p <0.01) with values from 3.91 to 4.16. Vieites et al. (2014) reported lower values between 3.73 and 3.84 in fruits from the same growing site. Among treatments, the lowest value was observed in the fruits covered with 2.5% EAqP (3.88) when compared to control (4.05). However, pH value of control fruits have not statistically differed from treatment 2.5% EAP and 1.5% EAqP (3.93 and 3.90, respectively). This lower pH value in 'Eva' for 2.5% EAqP treatment may be related to the greater conservation in storage and/ or decrease of microbial contamination due to propolis action, as according to Jacxsens et al. (2003) the microbiota (bacteria) actively participates in plants degradation, resulting the increase of pH, result of protein breakdown and compounds release. These results corroborate with Izumi et al. (1996), who associated pH increase in minimally processed zucchini with microbiological contamination.
The pH values during storage period (Fig 6) presented small increase (3.91 to 3.95). The pH tends to increase with fruit ripening due to the use of organic acids during respiration process and/or increase of their microbiota. Similar behavior was described by Passos et al. (2016) in 'Prata' banana using propolis hydro alcoholic extract, increasing pH values in storage. However, Minarelli et al. (2014) observed little pH interference during storage period of strawberries treated with aqueous propolis extract.
The titratable acidity (TA) of organic 'Eva' apples (Table 4)    For maturation index (SS/TA) (Table 5), there was no statistical difference for application of propolis extract and for interaction (p> 0.05). It was observed only influence for storage time at maturation index (p <0.01), evidencing that propolis extract did not interfere in apple maturation, as behavior of treatments did not differ from control.
Values of 22.2 were observed at the beginning of the experiment and 26.8 at the end for maturation index.
Varieties of apples with maturation index lower than 20 are more suitable for agroindustry for juices and citrons production, while fruits with higher SS/TA are classified as sweet and are indicated for table purpose (Chagas et al., 2012;Czelusniak et al., 2003). Using this classification organic 'Eva' apples from current research would be destined for in natura consumption.
At storage period (Fig 7) the values of maturation index showed increase in shelf life. This behavior indicates fruit ripening, expected behavior for climacteric fruits. Fante et al. (2013) also found the same behavior, increased SS/ TA values, from 22.8 to 28.4 at 135 days of storage for the same apple cultivar. Table 6 shows the values of reducing sugar (RS) contents. There was no statistical difference between treatments and for interaction between studied factors (p> 0.05), there was influence only at storage period (p <0.01). Reducing sugar contents were observed from 6.4% to 8.8% during all period of evaluation. Fante (2011) cited similar contents, 6.4% in conventional 'Eva' apple fruits, while higher values were found by Paganini et al. (2004) of 13.2% of glucose. Sugars have an effect on sensorial properties, on nutritional value and are considered quality indicators. In apples which present high levels of fructose they are considered as functional food, because this reducing sugar is absorbed in intestine and metabolized in epithelial cells, being thus classified as dietary and can be consumed by people with metabolic diseases (Wu et al., 2007;Czelusniak et al., 2003).   For evaluated period of time there was an increase in RS contents from 6.44% to 8.05% evidencing organic 'Eva' apple fruits maturation (Fig 8). In this aspect, there is an increase in simple sugars (fructose and glucose) content until the complete maturation. This is due to hydrolysis of long-chain carbohydrates and a consequent increase in sucrose, fructose and glucose contents (Chitarra and Chitarra, 2005;Oliveira et al., 2001).
For total sugar contents (Table 7) no significant differences were observed for treatment and their interactions (p> 0.05), with influence only for storage period (p <0.05).
Total sugar values were observed from 11.5% to 23%, with an overall mean of 13.3%. Similar values in apple 'Eva' were reported in Fante (2011) survey, 10.51%.
Fig 9 also shows total sugar contents during the storage period. Small contents increase of 11.5 to 14.7% is observed, Santos et al. (2006) also observed an increase in pitanga contents, and justified this because of the loss of fresh mass. Behavior that may be confirmed in the present work, as mass loss during evaluation was similar to total sugar contents. Another hypothesis may be polysaccharides conversion from cell wall of fruits into soluble sugars (Chitarra and Chitarra, 2005).    In sucrose contents (Table 8) there was no statistical difference between treatments, storage period and interaction between factors (p> 0.05). Organic 'Eva' apple fruit ranged from 4.5 to 14.9%, with an overall mean of 5.9% sucrose. Fante (2011) cited levels of 6.5 g 100g -1 corroborating with this research.
The coloration is directly related to perceived appearance by consumer, being important that fruit presents color intensity and uniformity, being able to be evaluated at shell and pulp (Chitarra and Chitarra, 2005). The luminosity is an attribute that may vary from zero (black) to 100 (white) (Trigo et al., 2012). For organic 'Eva' apple pulp luminosity (Table 9), there was significant difference only for treatments (p <0.01). The highest values of luminosity were observed in treatments with 1.5% EAqP (88.0); 2.5% EAqP (87.8%) and 1.5% EAP (87.9), not statistically different from fruits treated with 2.5% EAP (87.5), being more enlightened (close to color white). The control had the lowest brightness value (86.8) with less brightness.
For the chroma values (Table 10) there was no statistical difference between treatments and interaction (p> 0.05). The pulp chroma was influenced by storage period (p <0.01). There were values of 19.0 to 28.8 in pulp, showing little color saturation, and colors close to gray. Results close to zero express neutral colors (ashes) and close to 60, strong colors (Mendonça et al., 2003).
In spite of statistical difference, all fruits presented yellow flesh pulp, but with low color saturation (chroma = 23.3 -general mean) and high luminosity (L = 87.6 -mean overall), resulting in pale yellow tint. Similar values were reported by Chagas et al. (2012) in work with apple varieties

CONCLUSIONS
The application of propolis extract (aqueous and alcoholic) in 'Eva' organic apple post-harvest does not prolong refrigerated storage (5 ± 1ºC and 85 ± 1% RH) and does not influence in fruit quality conservation.

Author's contributions
Flávia Aparecida de Carvalho Mariano-Nasser: experiment conduction, experiment design, data analysis and paper writing; Mauricio Dominguez Nasser: paper writing; Juliana Arruda Ramos: experiment conduction and paper writing; Karina Aparecida Furlaneto: experiment conduction; Giovanna Alencar Lundgren: experiment conduction and literature review; Maximiliano Kawahata Pagliarini: paper writing; Rogério Lopes Vieites: experiment design, data analysis and paper writing; Ricardo de Oliveira Orsi: experiment conduction and experiment design.