Characterization and Stability of ‘de Russas’ Orange Juice From Organic Cultivation

through a Master’s scholarship for S.M.B.A. (financial code 001) and by


Introduction
According to the Food and Agriculture Organization of the United Nations (FAO), Brazil leads the orange production world ranking (Citrus × sinensis (L.) Osbeck (Rutaceae), as specified by the Global Biodiversity Information Facility-GBIF), and oranges occupy the 6th position of the country's top ten commodities, with a total production of 16,707,897 tons in 2020 (FAO, 2021). Oranges give Brazil not only the title of the world's largest producer of the fruit, but also the one of the largest producer and exporter of frozen concentrated orange juice 65 °Brix (FCOJ) (Kist et al., 2021).
As an example of these characteristics, aroma and flavor are very important quality parameters. They appeal to consumers, especially in the case of juice, because they are closely related to the aroma of the fruit in natura, as it is the case of orange juice (Fan et al., 2009).
The constitution of the fruit aroma is derived from the release of volatile compounds of low molecular weight, which provides odors that are captured by the human olfactory sensory system. The aroma, associated with color, texture and size parameters of a fruit, contributes to the consumer's propensity to purchase it, and it also allows differentiating varieties of the same species and characterizing their quality (Mariano et al., 2022).
For these reasons, aroma has become an essential characteristic for the food industry. Given its properties of interest, new alternatives have been studied to retain the volatile compounds while maintaining the aromatic stability of the food (Belmeskine & Kaced, 2018;Vukoja et al., 2020).
The frequent consumption of fruits and vegetables helps to reduce the risk of cancer, cardiovascular diseases, hypertension, and stroke. Citrus fruit juice is an important source of bioactive compounds that provide health benefits due to the high content of vitamins, carotenoids, and phenolic compounds (Igual et al., 2021).
Orange has a high content of vitamin C, folic acid, potassium and pectin, and high concentrations of phytochemical substances, which contribute to the antioxidant action. During juice processing, heat treatment is applied with the purpose of increasing its shelf life, ensuring food safety, at a low cost. However, changes also occur in the nutritional and sensory properties and freshness of the product (Lee et al., 2021).
In this perspective, it is observed the increasing search of consumers for whole fruit juice (without added sugar and in its natural concentration), because, despite the short storage period compared to the commercial one, the substances are more easily preserved during its obtaining, and its acceptance is superior to pasteurized juice, such as orange juice (Ephrem et al., 2018;Olmedilla-Alonso et al., 2022;Wang & Xu, 2022).
Therefore, the aim of this study was to elaborate a whole orange juice from the orange tree 'de Russas' varieties (BRS 'Russas 01' and BRS 'Russas 02') with organic certification, as an incremental innovation, and to analyze its quality characteristics, correlating the results between the varieties to ascertain potential uses.
This study evaluated the compliance of the juice's physicochemical characteristics with the Brazilian current legislation; it also analyzed the maintenance of the ascorbic acid in the food during the consumption period, since its commercialization occurs at domestic level; and it characterized the juice's volatile fraction in order to convey the information to the academic community and to the industry, contributing to the dissemination of the culture's knowledge, to local sustainability, and to the market supply of this by-product.
For this purpose, the physicochemical characteristics of the juice were evaluated. The stability of the ascorbic acid present in the juice was analyzed during three days of storage after extraction, through high performance liquid chromatography with ultraviolet detection. Method validation was performed to verify its performance, correlating with the results obtained among the varieties. The volatile organic compounds were extracted and characterized by gas chromatography coupled to mass spectrometry.

Material and Methods
The research was carried out at the Laboratório de Físico

Obtaining 'de Russas' Oranges
The fruits of the orange tree 'de Russas' which belong to the varieties of greater cultivation in region (BRS 'Russas 01' and BRS 'Russas 02') ( Figure 1), grafted on clove lemon (according to the GBIF -Citrus limonia Osbeck) (Rutaceae), were acquired through a partnership with a family farmer from Sítio Pocinhos, in the rural area of Limoeiro do Norte-CE, located at 5°4′50.46″S latitude, 38°2′8.45″W longitude and 21 meters above sea level. Each orchard consists of 128 (0.32 ha) citrus plants, with 5 × 5 m spacing in double rows in a micro-sprinkler irrigation system, with two emitters per plant with a blade of 9.6 mm, twice a week, and a flow rate of 60 L/h.

Prep
The secon ascorbic ac of 1000 m concentrat light.

Valid Detection
The linearity was verified from the linear regression equation of the calibration curve, constructed from the peak area of the compound under study versus the concentration of the analyte, using five concentrations of the standard solution.
(3) Precision Precision was evaluated in terms of repeatability (same analyst, same instrumentation and in a single run), and intermediate precision (analyst and different days, same instrumentation). The results were expressed as coefficient of variation (CV%).
(4) Accuracy The accuracy was determined through the recovery of the analyte by its quantification in the matrix under study. The samples were fortified at a concentration of 250 mg L -1 of the ascorbic acid standard. The results were expressed in percentage (%).
(5) Limits of detection (LOD) and quantification (LOQ) The sensitivity of the method was defined through the limits of detection and quantification, based on the parameters of the analytical curve (Equations 1 and 2).

LOD = 3.3 × s/S
(1) Where, 's' is the linear coefficient of the equation and 'S' is the angular coefficient of the analytical curve.

Sample Preparation and Injection
Before the sample conditioning step, which was protected from light, the whole orange juices from the BRS 'Russas 01' and 'Russas 02' varieties were diluted in Milli-Q ® (Millipore ® , USA), water for ascorbic acid (1:2) and citric acid (1:10) quantification and filtered in black-band filter paper (Quanty ® , Germany, 12.5 cm, 28 μm of porosity) to remove impurities.
Next, the solid-phase extraction (SPE) cartridge (Supelco ® , USA, ENVI-18, 6 mL), stationary phase C18, was treated with a solution of Acetonitrile (Sigma-Aldrich®, USA): Water Milli-Q ® (Millipore ® , USA) (1:1). 10 mL of the sample was added to the cartridge, then the liquid was removed with Manifold (Supelco ® , USA) and 5 mL was discarded. The remaining 5 mL was transferred to a 10 mL test tube, and it was filtered through a Millex membrane (Millipore ® , Brazil, 0.45 μm pore size, 13 mm) to remove impurities.
After the conditioning step, the samples were placed in the vials of the automatic injector and analyzed by HPLC-UV, in duplicate, in isocratic elution mode, with a flow rate of 0.8 mL min -1 , detector wavelength of 254 nm, in the UV region, for ascorbic acid, and 214 nm for citric acid, ambient column temperature, and a 10-minute run.

Analysis of Volatile Organic Compounds (VOCs) by Gas Chromatography Coupled to Mass Spectrometry (GC-MS)
The analysis of volatile organic compounds was performed in gas chromatography coupled to mass spectrometry (GC-MS), using the methodology adapted from Biasoto et al. (2015) and Mirhosseini et al. (2007).

Extraction and Injection of the Analytes into the Chromatographic System
The analytes were extracted using the headspace solid-phase microextraction (HS-SPME) technique. In appropriate 40 mL flasks, aliquots of 10 mL of the juice and 3 g of sodium chloride were added, followed by their vortexing for 1 minute and then their extraction in a thermostatic bath. As extraction conditions, it was used fiber for SPME of polyacrylate (Supelco ® , USA, polar, 85 μm of film thickness), time (30 minutes) and extraction temperature (65 °C).
The extracted volatile organic compounds were analysed on a GC-MS (Thermo Scientific TM , USA, Focus GC, DSQII) equipped with a Quadrupole-type mass analyzer and a column: DB5-ms (Agilent Technologies J&W ® , USA, 30 m length × 0.25 mm internal diameter × 0.25 μm film thickness). The injection temperature was 260 °C and the fiber was exposed in splitless mode. The helium 5.0 (99.999%) carrier gas flow rate was 1.0 mL min -1 . The initial temperature programming was 60 °C held for 1 minute and at a rate of 5 °C.min -1 until 210 °C, then 15 °C.min -1 until 300 °C, held for 1 minute (total of 38 minutes). The temperature of the transfer line (interface) was maintained at 300 °C. The mass spectrometer was operated in electron impact (EI) mode with electron energy of 70 eV at 250 °C. Full scan mass spectra were acquired in the mass range 50 to 400 (m/z). For data analysis and processing: the software Xcalibur TM 2.0.7 was used (Thermo Scientific TM , USA).

Identification of Volatile Compounds
The majority of the volatile organic compounds (VOCs) were identified by comparing their mass spectra with the mass spectra of the standards contained in the library of the National Institute of Standards and Technology (NIST, 2022). The other compounds present in the volatile matrix of the orange juice 'de Russas' were not identified due to the low similarity with the mass spectra listed in the library.

Statistical Analysis
A randomized block design was used in which the treatments were composed of the varieties BRS 'Russas 01' and BRS 'Russas 02' with 4 repetitions of 5 fruits per experimental unit (plot), which comprised 20 fruits of each variety (experimental sample).
The major volatile organic compounds identified in the juices of the BRS 'Russas 01' and BRS 'Russas 02' varieties were evaluated by principal component analysis (PCA) to verify differences between the varieties, as well as to obtain the varieties' profiles.
The data from the analyses are presented as average±standard deviation. They were evaluated by analysis of variance (ANOVA) followed by Student's t test at 5% significance level. STATISTICA TM version 10 (Statsoft, 2011) was the software which was used.

Physicochemical Characterization
The pH values of the orange juices of the studied 'Russas 01' and 'Russas 02' varieties presented an average 4.15a±0.02 and 4.06b±0.03 respectively, with a significant difference among the varieties (Table 1). The pH for orange juice is not established by the legislation (Brasil, 2018). Note. Means±standard deviation followed by the same letters in the column did not differ significantly (p ≥ 0.05) by Student's t test.
Source: Research data. Sombra et al. (2018b) carried out the physicochemical characterization of the fruits of the orange tree 'de Russas' in the city of Limoeiro do Norte (Ceará/Brazil), comparing them with fruits of the variety BRS 'Russas 02' grafted on citrandarin San Diego (according to the GBIF-Citrus sunki hort. ex Tanaka × Poncirus trifoliata (L.) Raf.) (Rutaceae), obtaining average pH value of 4.40 for the orange juice of the variety BRS 'Russas 02'. It is worth mentioning that the varieties analyzed in this study were grafted on clove lemon (according to the GBIF-C. limonia Osbeck), which can influence the differences in pH (among other factors, such as luminosity, irrigation, soil), even though they are from the same variety.
In Brazil, the varieties destined for direct consumption and juice production are: 'Pera', 'Valencia', 'Natal' and 'Folha Murcha' (C. × sinensis) (Bastos et al., 2014). The results obtained in this study for the pH in the juice from the fruits BRS 'Russas 01' and BRS 'Russas 02' varieties (ranging from 4.06 to 4.15) were similar to those reported by Spira et al. (2018) in non-processed juice (4.06) of the fruits of the 'Pera' orange (C. × sinensis) grown in Bauru, São Paulo, Brazil.
The results obtained in this study for the pH of the fruit juice of the BRS 'Russas 01' and BRS 'Russas 02' varieties (4.06 to 4.15) were higher than those reported by Habibi et al. (2021) in juices from four blood orange cultivars (3.00 to 3. 40) ('Moro', 'Tarocco', 'Sanguinello' and 'Sanguine') (C. × sinensis), grafted onto the 'C-35' The soluble solids of the orange juices of the studied 'Russas 01' and 'Russas 02' varieties presented an average 10.05a±0.09 and 10.73a±0.46 respectively; furthermore, it was not observed a difference among the varieties (Table 1). The values of soluble solids complied with that established by the legislation (Brasil, 2018) for orange juice (minimum 10 ºBrix). Sombra et al. (2018b) obtained an average content of 12.02 ºBrix for the juice of the BRS 'Russas 02' variety in their study. The results obtained in this work for the soluble were similar to those reported by Coelho et al. (2021), when they evaluated 22 citrus fruits from the Brazilian semiarid region, obtaining levels of 11.6, 13.3, 12.8, 10.7 and 10.1 ºBrix for the varieties 'Pera D12', 'Pera C21', 'Pera D09', 'Natal 112' and 'Valencia Tuxpan' (C. × sinensis), respectively.
The results obtained in this study for the soluble solids are also similar to those reported by Akinola et al. (2018), when they evaluated the chemical, microbiological and sensory characteristics of orange juice (C. × sinensis) samples chemically preserved in Akure (Nigeria), obtaining 9.40 ºBrix for the control sample (without preservatives) on the first day of analysis.
The titratable acidity of the orange juices of the studied 'Russas 01' and 'Russas 02' varieties presented an average 0.42a±0.02 and 0.40a±0.01 respectively; once more, it was not observed a significant difference among the varieties (Table 1). A value for titratable acidity in orange juice (Brasil, 2018) has not been established.
The results obtained in this study for the citric acid in the juice of the fruits of the BRS 'Russas 01' and BRS 'Russas 02' varieties (ranging from 0.40 to 0.42 g 100 g -1 ) were similar to those reported by Sombra et al. (2018b) in the juice of the fruits of the variety BRS 'Russas 02' (0.46 g 100 g -1 ). Coelho et al. (2019)  The results obtained in this study for the citric acid were similar to those reported by Hayatullah et al. (2021) in whole tangerine juice (0.65) (Citrus reticulata Blanco) (Rutaceae) (according to the GBIF), manufactured in Peshawar (Pakistan).
Some studies have shown that the variation in the chemical composition of citrus fruits is associated with edaphoclimatic factors, maturation, and agricultural practices, even when they belong to the same variety, intervening in the quality and acceptance of the fruits. However, more studies are needed to evaluate the influence of the cultivation system, both organic and conventional, on the physical, chemical and sensorial characteristics of citrus fruits and their derivatives (Petry et al., 2015).
The soluble solids/titulable acidity ratio (Ratio) of the orange juices of the studied 'Russas 01' and 'Russas 02' varieties presented an average 23.81a±1.34 and 26.72a±1.50 respectively; there was not a significant difference among the varieties (Table 1). The values of Ratio complied with what is established by the legislation (Brasil, 2018) for orange juice (minimum 7).
It is noteworthy that the official standard used for comparison of the physicochemical analyses is for processed orange juice (Brasil, 2018). It is also worth noting that this is a local study, with the data compared to the Brazilian specifications for this type of product; nevertheless, these standards may differ according to the country in which the study is conducted.
The Ratio is the balance between sweet or acidic taste, which is between the sugars and the organic acids present in the fruit. When the sugar content increases and the acid content reduces, the Ratio also increases (Sampaio et al., 2019). The results of this parameter indicate that the orange juice from variety BRS 'Russas 02' (26.72) presents a higher degree of sweetness than the orange juice from variety BRS 'Russas 01' (23.81) ( These results are associated to this study since the varieties from 'de Russas' oranges which were studied are cultivated in an organic system and presented a high soluble solids/titulable acidity relation. The results obtained in this study for the soluble solids/titratable acidity ratio in the juice of the BRS 'Russas 01' and BRS 'Russas 02' varieties were higher than those reported by Kenge et al. (2015), who evaluated the quality Therefore, other swee variety als sweeter th

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Valida Detection
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The results obtained in this study for the validation of the method for the determination of ascorbic acid in the juices of the BRS 'Russas 01' and BRS 'Russas 02' varieties were similar to those presented by Klimczak and Gliszczyńska-Świgło (2015), with emphasis on the repeatability and intermediate precision of the present study (2.86 and 3.14%). The authors validated two chromatographic methods (ultra-performance liquid chromatography-UPLC and HPLC) for the determination of ascorbic acid contents in fruit juices and beverages and in pharmaceutical preparations. The methods were validated in terms of linearity (0.9990 for both methods), limits of detection (0.049 and 0.024 mg L -1 ) and quantification (0.149 and 0.073 mg L -1 ), repeatability (2.20 and 1.70%), intermediate precision (2.40% and 1.90%), and recovery (99.8 and 99.9%), for HPLC and UPLC, respectively.
The results found in this study also are similar to those described by Hasan et al. (2021). A method was validated for the quantification of ascorbic acid through the coefficient of determination of the calibration curve (0.9988), recovery (95.71%), and limits of detection (1.032 mg L -1 ) and quantification (3.103 mg L -1 ), in fruit pulp juice of Citrus macroptera Montrouz. (Rutaceae) (according to the GBIF), a semi-wild citrus species native to Malaysia, Melanesia, and Bangladesh, by high-performance liquid chromatography with variable wavelength detector, in Sylhet (Bangladesh).

Evaluation of Ascorbic Acid Stability by High Performance Liquid Chromatography With Ultraviolet Detection (HPLC-UV)
The whole orange juice from the BRS 'Russas 01' variety presented an average ascorbic acid content varying from 55.16 to 43.76 mg 100 mL -1 during the four days of analysis, with a significant difference between the storage times. The juice from the BRS 'Russas 02' variety showed a decrease in ascorbic acid content from 57.07 to 49.31 mg 100 mL -1 , differing significantly between the days of analysis, except at T1 and T2, which were statistically equal (p ≥ 0.05). The two varieties studied showed a decrease in ascorbic acid contents during storage (Table 4). Note. Means±standard deviation followed by the same lowercase letters on the same line did not differ significantly between days of storage (p ≥ 0.05) by Student's t test.
Means±standard deviation followed by equal capital letters in the same column did not differ significantly between varieties (p ≥ 0.05) by Student's t test.
Source: Research data.
While evaluating the vitamin C stability in the orange juices between the varieties BRS 'Russas 01' and BRS 'Russas 02', it was observed that only on the day of preparation (T0) there was not significant difference between them (p ≥ 0.05). From the first day of storage (T1), a significant difference between the varieties was observed, showing that the ascorbic acid maintenance in the samples was influenced by the 'de Russas' orange variety (Table 4).
The percentage of ascorbic acid degradation in the orange juices from the BRS 'Russas 01' and BRS 'Russas 02' varieties is presented in Figure 7.   The orange juice obtained from the BRS 'Russas 02' variety has a higher Ratio, proving a higher degree of sweetness; moreover, it keeps a higher vitamin C content during storage and presents a higher number of major volatile compounds, compared to the BRS 'Russas 01' variety. Thus, among the studied varieties, the BRS 'Russas 02' variety presents a higher potential for the production of whole fruit juice.
For future approaches, it is suggested to evaluate the microbiological quality of juices, observing the compliance with the current legislation and the hygienic-sanitary quality of the processing, and identify quantitatively the volatile compounds present in each variety. Other proposals would analyze the nutritional and sensorial profile of the juices in order to identify the nutritional quality and the consumer's behavior towards the product, and to verify the potential of commercialization of a derivative of the orange 'de Russas' in the market and extract the essential oil from 'de Russas' orange peel, as a way to use the residues.
For a more comprehensive study on the possible use of this orange cultivar, it is recommended that the parameters are assessed for a longer storage period, besides the consumption in natura (proposal of this study) and that the refrigeration technique may be compared with other non-conventional conservation methods.