Analysis of bioactive compounds in umbu ( Spondias tuberosa ) by application of edible coating based on Chlorella sp during storage

The use of edible coating has been an alternative to maintain the quality of vegetables and prolong their post-harvest life. This study aims to evaluate bioactive compounds in Spondias tuberosa submitted to different coating formulations based on Chlorella sp. associated with the pomegranate seed oil, to establish the one that best maintains post-harvest quality. The experiment was set in a completely randomized design, in a 5 × 6 factorial scheme, represented by five coating techniques (T1 = control; T2 = 0.5% Chlorella sp.; T3 = 1% Chlorella sp.; T4 = 1.5% Chlorella sp.; T5 = 2% Chlorella sp.), using 0.3% of pomegranate seed oil in coatings, during five evaluation periods (0, 1, 2, 3, 4 and 5 days), stored in BDD incubator at 24 ± 2 °C and 85 ± 5% RH, with three replicates of ten fruits per plot. Chlorella sp. associated with pomegranate seed oil produced a modified atmosphere around the fruit, retarding the ripening process. Coatings preserved the content of ascorbic acid and phenolic compounds. The treatment containing 2.0% of Chlorella sp. associated with pomegranate seed oil provided the best maintenance for fruit quality.

Pomegranate seed oil (PSD) has bioactive lipids, mainly linolenic acid, in the range of 74-85% of total fatty acid content (Verardo et al., 2014). PSD has already been used as an ingredient in edible coatings and it has shown satisfactory results when associated with biopolymers (Teodosio et al., 2018a;Morais et al., 2020). On this context, the present work aimed to evaluate the post-harvest life of S. tuberosa submitted to different coating formulations based on Chlorella sp. associated with pomegranate seed oil, to establish the one that best maintains post-harvest quality.

Edible coatings
Coatings were prepared according to Dliveira et al. (2018), with modifications. Chlorella sp. was produced according to Lima (2016), in organic production tanks, at Fazenda Tamanduá (Patos, Brazil). The pomegranate seed oil was extracted according to Silva (2009) at the Federal University of Campina Grande (Pombal, Brazil). Chlorella sp. was diluted in 1L of distilled water, under constant stirring, until the solution was completely homogenized and then added with 0.3% of pomegranate seed oil according to Teodosio et al. (2018b) for the purpose of emulsifying and maximizing the plasticization properties of the coating.

Plant material
Spondias tuberosa fruits were harvested from orchards in Juazeirinho region, in the state of Paraíba, Brazil. The harvest was done at the commercial stage (mature-green), when fruits had peel color between green and shiny, and then they were transported to the Post-Harvest Technology Laboratory of UFCG, Pombal Campus. Fruits were immersed in cold and chlorinated water (200 mg sodium dichloro-s-triazinetrione dihydrate -Sumaveg  ) for 20 min. After drying, they were individually immersed in coating treatments for 1 min and left to dry at 18 °C. Fruits were then packed in trays and stored in BDD at temperature of 24 ± 2 °C with 85 ± 5% RH.

Experimental design
The experiment was set in a completely randomized design, in a 5 × 6 factorial scheme, with four coatings based on Chlorella sp. associated with pomegranate seed oil and the control (T2 = 0.5; T3 = 1.0; T4 = 1.5 e T5 = 2.0%; and T1 = control) and six times (0; 1; 2; 3; 4 e 5 days). The experimental unit comprised ten fruits, and each treatment had three replicates (trays), totaling 900 fruits. Fruits were pulped every evaluation period in order to remove the core using stainless steel knife and later they were processed in domestic centrifuge. Photos were also taken on the last day of storage to demonstrate overall appearance ( Figure 1).

Analysis of bioactive compounds
• Vitamin C (mg/100 g ascorbic acid): determined by titration in 1 mL of diluted juice for 49 mL of oxalic acid, titration under constant stirring with DFO (2,6-dichlorophenol indophenol) solution. The results were expressed as mg/100 g -1 mL FW of ascorbic acid, according to Tillman's method (Association of Dfficial Analytical Chemists, 2012); • Carotenoids: estimated as described by Lichtentháler (1987). The samples were read in UV-Vis spectrophotometer (Novainstruments, Serie 1600, Brazil) at wavelengths of 470, 646 and 663 nm, once concentrations of chlorophylls a and b must be used to determine carotenoids. The results were expressed in g.100 g -1 FW of the pulp; • Total phenolic content: measured according to the Folin-Ciocalteau method described by University of California (2012), with modifications. The extracts were prepared from 1 mL of sample, diluted in 50 mL of distilled water and allowed to stand for 30 minutes. 800 μL aliquot of the extract was transferred to test tube containing 1.325 μL of water and 125 μL of the folin-Ciocalteau reagent. The mixture was allowed to stand for 5 minutes and then 250 μL of 20% sodium carbonate was added, followed by stirring and standing in water bath at 40 °C for 30 minutes. The samples were read in UV/Vis spectrophotometer (Novainstruments, Serie 1600, Brazil) at wavelength 765 nm. The results were expressed as mg of gallic acid 100 g −1 FW; • Soluble sugars: estimated as described by Yemm & Willis (1954), with modifications. This variable was determined from 1 mL of juice diluted in 150 mL of distilled water, from this solution, it was taken 100 μL and then it was diluted again in 50 mL of distilled water. From this last dilution, 1.0 mL was used for the dosage of anthrone method. The samples were read in UV-Vis spectrophotometer (Novainstruments, Serie 1600, Brazil) at wavelength 620 nm. The results were expressed as g of glucose 100 g −1 FW.

Statistical analysis
Data were submitted to analysis of variance (ANDVA), and the determination of regression coefficients and response surfaces were performed using the software Statistica 7.0 (StatSoft, 2004).

Results and discussion
The variables of phenolic compounds and total sugars had significant interaction among the factors under study (recoating and storage time), whereas the variables of vitamin C and carotenoid had significant effect among the analyzed factors (recoating and storage time) but in isolated form (Table 1).
Vitamin C content decreased gradually during storage and this reduction was significantly retarded in all coated fruits (T2, T3, T4 and T5) presenting estimated values of 7.8; 8.8; 9.6 and 10.2 mg.100 g -1 , respectively, and control treatment (T1) had the lowest value of 6.5 mg.100 g -1 during the storage period ( Figure 2). Ot is possible that fruit exposure to abiotic conditions (pH, temperature, light, oxygen, etc.) during the conservation period contributed to the reduction of this content (Silva et al., 2019b). The highest retention of vitamin C in Spondias tuberosa coated with Chlorella sp. may occur due to low oxygen penetration in coated fruits, which resulted in inhibition of enzymatic activities and, consequently, in reduction of oxidation of vitamin C (Kumar et al., 2017).
Results showed T5 coating was the most effective to retard the oxidation process in S. tuberosa, maintaining higher levels of vitamin C during storage. Similar results were found by Dnias et al. (2016) and Dliveira et al. (2018), both with 'Tommy Atkins' mango coated with Spirulina platensis and Chlorella sp.
Regardless of the treatment, carotenoid contents in S. tuberosa increased gradually during the storage period (Figure 3), mainly because there was an evolution in maturation and, therefore, color development. Changes in peel color of fruits, with or without coatings, may have been caused by a peak in ethylene production, which promoted increase in the activity of the chlorophyllase enzymes responsible for degradation of chlorophyll, and induced the synthesis of new enzymes responsible for biosynthesis of carotenoids (Forato et al., 2015).
There was no influence of Chlorella sp. concentrations for carotenoid content in "umbu" fruits. When matured, carotenoid  content may vary from 1.5 to 10.0 mg.100 g -1 . Melo & Andrade (2010), evaluating the bioactive compounds in S. tuberosa fruits, reported that carotenoids increase during maturation, finding lower values for semi-mature fruits of 1.70 mg.100 g -1 than mature reaching 3.02 mg.100 g -1 .
The content of phenolic compounds increased throughout storage, peaked after around three days of storage, and then gradually decreased for coated and non-coated samples on the last day of storage (Figure 4). Kumar et al. (2017) reported the same trend regarding phenolic compounds in chitosan-coated plums.
At the end of five days of storage, the reduction in total phenolic compounds was more pronounced in samples coated with T2 and T3, and also in T1 (control). Samples with T4 and T5 coatings presented the highest levels and it may be attributed to delayed senescence of coated fruits. The phenolic reduction after the fourth day may be a result of the decrease in sugar levels, which inhibit the metabolic pathway of their synthesis (Formiga et al., 2019). Melo & Andrade (2010), evaluating the bioactive compounds in "umbuzeiro" fruits, reported that phenolic compounds in semi-mature fruits were 38.03 mg.100 g -1 , while in mature this number was 32.70 mg.100 g -1 , and no significant difference was detected among maturation stages of fruits.
There was initial increase for total sugars in S. tuberosa fruits, which must be related to the increases occurred for carotenoids and also maturation process, followed by a gradual decline. Dn the last day of storage, the reduction in total sugars was more degraded and it must be attributed to the progression of ripening, which soluble sugars started to be metabolized at high rates to support the initial senescence processes in coated fruits (T2, T3, T4, T5) and non-coated fruits (T1) showing estimated values of 3, 8; 3.8; 3.7; 3.5 and 3.8 mg.100 g -1 , respectively ( Figure 5).
There is great energy demand in the cellular system of S. tuberosa fruits to continue the intense metabolic processes during maturation, such as the hydrolysis of starch and other polysaccharides and consequent increase in content of sugars, besides other componentes, as flavor which is responsible for the sweet-acidulated taste of this exotic fruit. Dantas (2008), evaluating fruit quality for 32 genotypes of S. tuberosa from the Northeastern semi-arid region, verified variation of more than double from 3.6 to 8.31 mg.100 g -1 for total sugars, which possibly this amplitude in the content of total sugars must be attributed to the genetic variability of the materials.

Conclusion
Coatings using Chlorella sp. associated with pomegranate seed oil (PSD) had positive effect on the conservation of Spondias tuberosa, prolonging shelf life of fruits. The treatment 2.0% Chlorella sp. associated with PSD was able to preserve the content of vitamin C and phenolic compounds, thus presenting the best results in the maintenance of fruit quality.