Changes in Quality Traits and Phytochemical Components of Blueberry (Vaccinium Corymbosum Cv. Bluecrop) Fruit in Response to Postharvest Aloe Vera Treatment

ABSTRACT The effect of Aloe vera application (33% and 66% as dipping, AV) on ‘Bluecrop’ blueberry’s (Vaccinium corymbosum) quality properties such as weight loss, respiration rate, fruit color, soluble solids content (SSC), acidity, phytochemical components (vitamin C, total phenolics, and total flavonoids) and antioxidant activity was investigated during the cold storage (at 0 ± 0.5°C and 90 ± 5% RH) for 28 days. During cold storage, significantly lower weight loss was recorded in fruit treated with AV compared to control. At the end of the cold storage, the weight loss was 20% lower in AV treated fruits and AV concentration was not effective in weight loss. On the contrary, the respiration rate (except for the 7th day) and fruit firmness were higher in these fruit. It was observed that color changes were less in AV-treated fruit during storage. In general, AV-treated fruit had significantly lower SSC, while acidity and vitamin C were higher. Moreover, 66% AV application was more effective in delaying the loss of vitamin C at the end of storage. During cold storage, 33% AV-treated fruit had significantly higher total phenolics (except day 28) and total flavonoids content compared to control. In the last two measurements of the cold storage, it was determined that fruit dipped in AV had higher antioxidant activity (both DPPH and FRAP assays) compared to control fruit. As a result, it was revealed that postharvest AV gel applications can be used as an effective postharvest tool to delay the loss of quality, the loss of phytochemical components and antioxidant activity of the blueberry fruit.


Introduction
Blueberry (Vaccinium spp), which is rich in terms of bioactive compounds and minerals, is fruit species increasing production day by day (Wang et al., 2017). It has a significant effects such as anticancer, antidiabetic, cardioprotective, antioxidant, blood vessel-softening, antimicrobial, antiproliferative, immunity-enhancing, anti-inflammatory, apoptotic and lifespan-prolonging (Ge et al., 2019). However, blueberry fruit is extremely sensitive, it can be spoiled quickly due to loss of water, mechanical damage, microbial infection and loss of nutritional value (Paniagua et al., 2014). It can be stored between 1 and 8 weeks depending on the storage conditions, the method of the harvest, disease presence, and ripening stage (Duan et al., 2011). This negatively affects the shelf life, storage, transportation and marketing processes (Deng et al., 2014). To contribute to the solution of this problem and to maintain fruit quality after harvest, the various post-harvest technologies have been RH for 28 d. Measurements and analyses were performed on 7, 14, 21 and 28 th days (d) of cold storage. In each measurement period, 3 PET-clamshells were taken for each treatment. Each PET-clamshells represented a replicate. The quality characteristics mentioned below were determined in the fruit.

Weight Loss
At the beginning of cold storage, initial weights (Wi) of the fruit were determined by a digital scale with a precision of 0.01 g (Radwag, Poland). Then, on d 7, 14, 21 and 28 of the storage, final weights (Wf) were determined. The weight loss (WL) that occurs in fruit was based on the weight at the beginning of each measurement period and determined as a percentage through the equation given below (Eq.1). (1)

Respiration Rate
The 2 L airtight chambers were used to measure respiration rate. The chambers were fitted with a rubber septum and 50 g fruit were sealed in each chamber at 20 ± 1°C temperature and 90% RH for an h. The chambers were then connected to a gas analyzer (Vernier, Oregon, USA) and the amount of CO 2 produced by the fruit was considered as the respiration rate. Results were expressed as nmol CO 2 kg −1 s −1

Fruit Firmness
Twenty fruit from each replication were used for firmness measurements. The measurements were made on two opposite sides of the equatorial part of the fruit through a portable digital durometer (nondestructive device, Agrosta® 100 Field, France) with a flat cylindrical tip and with a diameter of 4.1 mm. The tip of the durometer was slightly and longitudinally pressed into the outer skin of the fruit, and the results were expressed as Durofel Units (%). In Durofel Units, 0 indicates that the fruit is very soft and 100 is very firm (Ozturk et al., 2019b).

Color Characteristics
Color measurements were performed with a color meter (Konica Minolta, CR-400, Japan). Color data of blueberry fruit were presented according to CIE system (Commission Internationale de l'Eclairage). Color characteristics were measured at two different points in the equatorial part of 20 fruit randomly selected from each replicate. 3-D color space was defined with the aid of L*, a* and b* values. Equations in parenthesis were used to calculate chroma [C� ¼ a �2 þb �2 À � 1=2 ] and hue

Soluble Solids Content, Titratable Acidity and Vitamin C
Fifty fruits in each replication were washed with distilled water. The fruit were homogenized by a blender (Model No. Promix HR2653 Philips, Turkey). Then, the homogenate was filtered through a cheesecloth, and the juice was obtained. Soluble solids content (SSC) was measured with a portable digital refractometer (Atago PAL-1, USA) and expressed as a percentage (%). For titratable acidity measurement, 10 mL juice was taken and 10 mL distilled water was added on. Then, 0.1 N NaOH (sodium hydroxide) was added until the pH of the solution reached to 8.2. Based on the amount of NaOH consumed in titration, titratable acidity was determined and expressed as g malic acid kg −1 . For vitamin C measurement, 0.5 mL juice was taken, and 5 mL of 0.5% oxalic acid was added on it. The ascorbic acid test strip (Catalog no: 116981, Merck, Germany) was then taken from a collapsible sealed gas-tight tube. Reflectometer (Merck RQflex plus 10) was started. The test strip was plunged into the solution for 2 seconds, then removed from the solution. It was then held for 8 seconds, and reading was done at the end of the 15 th second. Results were presented as g kg −1 (Ozturk et al., 2019b).

Total Phenolics, Total Flavonoids, Antioxidant Activity
During each measurement period, Fifty fruit taken from each replication were first washed with distilled water. The fruit were homogenized by a blender (Model No. Promix HR2653 Philips, Turkey). About 30 mL of homogenate was taken and placed into a 50 ml falcon tube. The prepared tubes were kept at −20 C until the time of analyses. Before the analyses, the frozen samples were dissolved under room temperature (21°C). Pulp and juice were separated from each other by a centrifuge at 12.000 × g at 4°C for 35 min. The resultant filtrate was used to determine the content of total phenolics, total flavonoids, antioxidant activity.
Spectrophotometric measurements for total phenolics, total flavonoids and antioxidant activity were performed at the UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). Total phenolics were determined in accordance with the method described by Singleton and Rossi (1965) and was expressed as g GAE (gallic acid equivalent) kg −1 fresh weight (fw). Total flavonoids were measured according to the method described by Chang et al. (2002) and was expressed as g QE (quercetin equivalent) kg −1 fw. The antioxidant activity of blueberry fruit was determined according to two different procedures of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) (Aglar et al., 2017) and Ferric Ions (Fe +3 ) Reducing Antioxidant Power (FRAP) (Benzie and Strain, 1996), and the results were expressed in mmol Trolox equivalent (TE) kg −1 fw.

Statistical Analysis
Whether the data was normally distributed was checked by Kolmogorov-Smirnov Test. Homogeneity control of the group/subgroup variances was confirmed by Levene's test. After the variance analysis of the data, Tukey's multiple-comparison test was used to check whether there were significant differences between treatments. The statistical analyses were performed by using SAS software (SAS 9.1 version, USA).

Weight Loss
It was determined that the AV application reduced the weight losses in the cold storage, and in the measurements made at 1-week intervals during the storage, there were statistically significant differences in the weight losses in the fruit of the AV and control applications. However, increasing the AV concentration did not occur changes in the effect (Figure 1).

Respiration Rate and Fruit Firmness
The respiration rate increased in the measurements made 1 week after harvest while it was determined that a decrease in the respiration rate occurred with increasing storage period. The changes in respiration rate during cold storage were more in the control application, but AV applications had the same respiration rate. The effect of AV application on respiratoion rate during cold storage was significant. In all measurement periods, significant differences occurred between AV and control applications. The respiration rate was higher in the fruit of control application on the 7 th day of the cold storage, whereas higher respiration rate in AV-treated fruit at other measurement periods was recorded. However, the changes in respiration rate did not occur depending on AV concentration ( Figure 2).
It was determined that AV application was effective in maintaining of the fruit firmness during the cold storage. While the lowest fruit firmness values were recorded in the fruit of control application on the 7 th day of cold storage, the difference between AV concentrations was not significant. However, there were statistically significant differences between all applications on 14 th days of cold storage. The highest fruit firmness value was recorded with AV, 66% application, but the lowest values were obtained with the control application ( Figure 2).

Fruit Color
In the study, a decrease in all color values (L, hue angle, chroma) occurred in proportion to the storage time. However, it was determined that this decrease was higher in the fruit of control application, but the color changes were less in AV-treated fruit. The higher L and hue angle values were recorded with AV application during all measurement periods, there were no statistically significant differences between AV concentrations. However, there was an inconsistency in the effect in chroma values. The fact that, it was determined that the effect of AV application was not significant on 7 th day of the cold storage, and the significant differences occurred between applications on other measurement days. During cold storage, the highest Chroma values were measured in 66% AV-treated fruit. The effect of AV concentration on chroma values was significant on 14 th and 21 st days of the cold storage. It was determined that the change in chroma values was less with increasing concentration. AV concentration was not effective at the end of the cold storage ( Figure 3).

Soluble Solids Content, Titratable Acidity, and Vitamin C
AV application was effective on the increase in SSC during cold storage, but there was no statistically significant difference between 33% and 66% AV applications. The highest SSC values on all measurement days were determined in control application (Figure 4). It was determined that AV application was effective in delaying the decrease in titratable acidity during cold storage, but there were no significant differences between AV concentrations (Figure 4). Vitamin C ratio decreased in proportion to the storage period. The statistically significant differences occurred between applications in terms of vitamin C content in all measurement periods. In total, 66% AV-treated fruit had the highest vitamin C on all measurement days. In the measurements on 7 th and 21 st days of the cold storage, the vitamin C ratios of AV 33% and control applications were similar whereas lower vitamin C values were recorded with control application on 14 th day of and at the end of cold storage (Figure 4).

Total Phenolics, Total Flavonoids, and Antioxidant Activities
The total amount of phenolics increased significantly in proportion to fruit maturity during storage. It was determined that the effect of AV application on total phenolic content at the cold storage is significant and varies depending on AV concentration. At the end of the cold storage, the highest total phenolic values were recorded with AV 66% application. In the measurements on 7 th , 14 th and 21 st days of the cold storage, 33% AV-treated fruit have a higher total phenolic content while the total phenolic content of the fruit of the other two applications (AV 66 and control) was similar Figure 2. Effect of Aloe vera treatments on respiration rate and firmness of blueberry fruit (Vaccinium corymbosum cv. Bluecrop) during storage at 0°C and 90% RH. nd: not determined.* The scale ranges from 0 to 100 for very soft to very firm surfaces.n = 9 for the respiration rate (three replications x three different measurements for each replication).n = 120 for the firmness (three replications x twenty fruit x two different measurements for each fruit).Means in columns with the same letter do not differ according to Tukey's test at P < .05.Vertical bars indicate the standart errors.
( Figure 5). At the end of the cold storage, it was determined that the total flavonoid content increased significantly compared to the amount measured at harvest, and the effect of AV treatment on this increase was significant and changes occurred in this effect depending on the application concentration. At the end of the cold storage, there were statistically significant differences among  Effect of Aloe vera treatments on soluble solids content (SSC), titratable acidity and vitamin C of blueberry fruit (Vaccinium corymbosum cv. Bluecrop) during storage at 0°C and 90% RH. n = 9 for the SSC, acidity and vitamin C (three replications x three different measurements for each replication). Means in columns with the same letter do not differ according to Tukey's test at P < .05.Vertical bars indicate the standart errors all application and the highest value was recorded with AV 66% application, while the fruit of the control application had the lowest total flavonoid content. In the measurements on 7 th , 14 th and 21 st days of the cold storage, there was no difference between the values of AV 66 and control applications in terms of total flavonoid amount, but the highest total flavonoid content was measured in in 33% AV-treated fruit ( Figure 5). It was determined that an increase in antioxidant activity, which was detected as DPPH and FRAP during cold storage, occurred and AV-treated fruit had higher antioxidant activity. On the DPPH and FRAP values on 7 th , 21 st and 28 th days of the cold storage, there was no effect of AV concentration, but in the 14 th days of the cold storage, antioxidant activity changed depending on the AV concentration. In this measurement period, while the highest DPPH and FRAP values were obtained in 66% AV-treated fruit, there were no statistically significant differences among other treatments ( Figure 5).

Discussion
Weight loss causes significant economic problems by leading structural and visual quality deterioration in the fruit (Miller et al., 1993). As reported in previous studies (Chen et al., 2017;Duan et al., 2011;Liu et al., 2019), in this study, weight loss increased with increasing storage time. AV applications delayed the increasing in weight loss. Many other researchers have also determined that edible coating materials are effective in reducing weight loss in fruit at the cold storage (Ali et al., 2019;Carvalho et al., 2016;Chiabrando and Giacalone, 2015;Ozturk et al., 2019a). The effect of AV Figure 5. Effect of Aloe vera treatments on total phenolics, total flavonoids and antioxidant activities (DPPH and FRAP assay) of blueberry fruit (Vaccinium corymbosum cv. Bluecrop) during storage at 0°C and 90% RH. FRAP: Ferric Reducing Antioxidant PowerDPPH: 2,2'-Diphenyl-1-picrylhydrazyln = 9 for the total phenolics, total flavonoids and antioxidant activity (three replications x three different measurements for each replication).Means in columns with the same letter do not differ according to Tukey's test at P < .05.Vertical bars indicate the standart errors. application on weight loss can be explained by the fact that the AV gel (Khaliq et al., 2019), which forms a semi-permeable barrier on the fruit surface, reduces moisture loss and dehydration in the fruit (Sogvar et al., 2016).
It is generally known that edible coating applications such as AV and chitosan reduce the respiration rate by limiting oxygen and carbon dioxide gas transfer (Mahajan et al., 2018;Tezotto-Uliana et al., 2014). In this study, the reducing effect of AV on respiration was seen only on day of storage. In the subsequent date, respiration rate of fruits coated with AV was higher than those of control fruit. This situation can be explained by findings of Falagan et al. (2020) who stated that the low O 2 concentration may induce an abiotic stress and consequently an increaase in CO 2 production in blueberry fruits.
Fruit firmness, which is the most significant quality trait that determines the storability of the product in processes such as postharvest processing, storage and marketing (Chiabrando and Giacalone, 2017), affects consumer preferences (Chen et al., 2017). Similar to the results obtained from previous studies on blueberry (Mannozzi et al., 2018) and other fruit species (Khaliq et al., 2019;Ozturk et al., 2019b;Vieira et al., 2016), AV treatments in this study also reduced firmness the loss during storage. Fruit softening occurs due to the degradation of the cell wall components such as pectin substances, hemicellulose and cellulose (Wang et al., 2015) and the decrease in turgor pressure in the cell (Mannozzi et al., 2018). It was reported that AV application delayed the fruit softening by reducing the cell wall enzyme activity (Khaliq et al., 2019) and maintain the cell turgor pressure by limiting transpiration (Mannozzi et al., 2018).
During the storage period, L*, chroma and hue values decreased regularly in both control and AV treatments. This decrease was significantly slowed down by AV treatments. Hoagland and Parris (1996) reported that the edible coating materials affected the coloring of the fruit because of causing changes in fruit surface properties and limiting the ripening process. In previous studies, it was reported that the edible coating applications such as alginate (Chiabrando and Giacalone, 2015), parka (Aglar et al., 2017) in sweet cherry and AV in mango (Carrilo-Lopez et al., 2000) delayed coloration during cold storage. Mannozzi et al. (2018) reported that edible coating application based on polysaccharide such as alginate and pectin decreased color changes in fruit in blueberry after harvest. Phenolic compounds, especially anthocyanins, play a significant role in fruit color formation (Cheynier, 2012). As the anthocyanin content, which increases with ripening, decomposes rapidly after harvest, it decreases in proportion to the storage period (Ali et al., 2019). The breakdown of the vacuoles in cell causes the degradation of the anthocyanins and loss of the cellular compartmentation (Jiang et al., 2018). Ali et al. (2019) reported that the AV-applied fruit had higher anthocyanin pigments as cellular compartmentation on these fruit was conserved, and as a result, the fruit color is maintained.
SSC is a significant quality trait that is taken as a criterion in determining the ripening stage of fruit. As fruit ripeness progresses, SSC increases as a result of hydrolysis of undissolved polysaccharides in simple sugars. Hassanpour (2015) reported that there was an increase in the rate of SSC with the increase of the metabolic activities in raspberry fruit during the maturity stage, then the SSC decreased after 8 weeks of storage, and AV-treated fruit had higher SSC. Contrary to these results, in our study, it was observed that an increase in SSC occurred in proportion to the storage time and AV application limited the increase in SSC at the cold storage. In previous studies, it has been determined that AV application is effective in maintaining the rate of SSC in storage in grapes (Chauhan et al., 2014), strawberry (Zafari et al., 2015) and plum (Martínez-Romero et al., 2017). Edible coating applications slow the respiration and maturation by controlling the exchange of important gases such as oxygen, carbon dioxide and ethylene (Embuscado and Huber, 2009). The lower SSC in fruit treated with AV was explained by its effect on the decrease of respiration rate (Khaliq et al., 2019).
At harvest, fruit have the highest titratable acidity, but after harvest titratable acidity decreases gradually (Reque et al., 2014). In our study, the titratable acidity of the fruit decreased depending on the storage period. However, titratable acidity of AV-treated fruit was higher at the end of the cold storage. It can be said that AV application is effective in maintaining the titratable acidity in storage. Bahmani et al. (2015), support our study results, reported that the titratable acidity in the fruit decreased due to high respiration after harvest, and Valverde et al. (2005) found that AV application decreases the respiration rate by regulating the inner atmosphere in the fruit and consequently slows down the decrease of titratable acidity in storage. However, Serrano et al. (2006) and Reque et al. (2014) reported that AV application had no effect on the change in titratable acidity in storage. But, Harb et al. (2010) found that oxygen uptake in fruit with AV application is limited, thus, the respiration slows down and the titratable acidity of these fruits is higher.
Vitamin C is a significant antioxidant that prevents the harmful effects of reactive oxygen species during ripening. Vitamin C content in the fruit varies depending on the maturity stage and storage period. Vitamin C content decreases in storage and AV application delays this decrease (Khaliq et al., 2019). In accordance with this explanation in our study, a decrease in vitamin C content occurred in storage and vitamin C content in AV-treated fruit was higher. In studies conducted, it has been reported that AV application is effective in maintaining vitamin C content at cold storage in strawberry (Zafari et al., 2015), orange (Radi et al., 2017), raspberry (Hassanpour, 2015) and sweet cherry (Valverde et al., 2005). Vitamin C loss in storage can be explained by increasing the activity of ascorbic acid oxidase and phenoloxidase enzymes, those activities are affected by oxygen content (Zhou et al., 2008). Since AV-treated fruit have lower gas permeability, the respiration rate is limited and all metabolism slower (Hassanpour, 2015).
Depending on the fruit species, different results have been reported regarding the change pattern of total phenol content during the storage time. Khaliq et al. (2019) determined that the total phenolic content of sapodilla fruits decreased in the cold storage was maintained by AV treatment. On the other hand, Aglar et al. (2017) reported that an increase in the bioactive compound content such as flavonoid and phenolics occured as propotional the cold storage time in sweet cherry. In this study, the total phenolics and flavonoids and the antioxidant capacity associated with them showed a fluctuating pattern according to the measurement dates. Such an irregular change in the total phenol content and antioxidant capacity of blueberry during the storage period was also reported by Li et al. (2019). Different findings in literature on the change in total phenol content during the storage process and fluctuations in the measurement date reveal that more detailed studies should be conducted on the posthervest metabolism of such substances.
Although different results were obtained according to the measurement date, at the end of 28 days of storage, the antioxidant capacities of the fruits treated with AV were higher than those of the control fruits. In previous studies, it has been reported the fruit treated with coating material in fruit species such as guava (Nair et al., 2018), apple (Synowiec et al., 2014), raspberry (Hassanpour, 2015), grapes (Serrano et al., 2006) and sapodilla (Khaliq et al., 2019) have higher antioxidant activity at the end of cold storage. Ali et al. (2019), have reported that AV-coated litchi fruits have higher bioactive compounds content during cold storage, and AV coating possibly conserved cellular compartmentation of litchi peel and maintained higher bioactive compounds pigments. AV gel contains many bioactive compounds (Ni et al., 2004), but aloeemodin is one of the main building blocks that contribute to antioxidant activity (Ni et al., 2004). The increase in antioxidant activity with AV application can be explained by the effect of the aloe-emodin compound contained in the AV gel. Also, the present of organic acids, proteins, phenolic compounds, vitamins, minerals and amino acids in Aloe vera gel (Boudreau and Beland, 2006) can explain the increasing on antioxidant activity at cold storage.
As a result, AV application was effective in maintaining fruit quality traits during cold storage in blueberry. During storage, it was observed that the weight and vitamin C loss was lower in AV applied fruits, the fruit firmness was preserved and the content of bioactive compounds in these fruits was higher. In the study, at the cold storage, with AV application, it was revealed that AV application can be used to prolong the postharvest life in blueberry.