multivariate analysis as a reliable tool for …

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MITT EILUNGEN KLOSTERNEUBURG 69 (2019): 244-257 MILOSEVIĆ et al.

MULTIVARIATE ANALYSIS AS A RELIABLE TOOL FOR SEGREGATING STRA WBERRY CULTIVARS WITH THE BEST ANTIOXIDANT CAPACITYTomo Milošević1, Nebojša Milošević2 and Jelena Mladenović3

1 Department of Fruit Growing and Viticulture, Faculty of Agronomy, University of Kragujevac, RS-32000 Čačak, Cara Dušana 342 Department of Pomology and Fruit Breeding, Fruit Research Institute Čačak RS-32000 Čačak, Kralja Petra I/93 Department of Chemistry and Chemical Engineering, Faculty of Agronomy, University of Kragujevac RS-32000 Čačak, Cara Dušana 34 E-Mail: [email protected]

During 2018 and 2019 we ad hoc investigated fruit weight, chemical composition and antioxidant activity of fi ve strawberry cultivars ('Irma' as a day-neutral cultivar, and 'Clery', 'Roxana' 'Asia' and an 'Unknown' genotype as short-day strawberries). By ANOVA and multivariate analysis (PCA), diff erences were observed between cultivars based on the fruit weight, contents of soluble solids, acidity, maturity indexes, main phenolic compounds and antioxidant activity. Results showed that 'Roxana' had the largest fruit at the same growing and environmental conditions. Th e highest soluble solids content (SSC), acidity and ripening and taste indexes, phenolic compounds had 'Clery' and 'Asia', respectively. 'Asia' is the cultivar with the highest antioxidant capacity. Th e poorest evaluated properties were shown by the 'Unknown' genotype, followed by 'Irma'. Season (year) had more infl uence on fruit weight, acidity, phe-nolic compounds and antioxidant activity whereas eff ect on other properties was not signifi cant. Interaction cultivar x year had signifi cant eff ect on all evaluated properties with exception of fruit weight. Correlation matrix indicated signifi cant positive or negative correlations between most primary and secondary metabolites in fruit of strawberries. Principal component analysis (PCA) can be a very good tool for segregating strawberry genotypes with the best nu-tritional value and health promoting compounds.Keywords: antioxidant capacity, phenolic compounds, principal component analysis, soluble solids, acidity, strawberry fruit

Multivariate Analyse als zuverlässiges Instrument zur Diff erenzierung von Erdbeersorten mit der besten anti-oxidativen Kapazität. In den Jahren 2018 und 2019 wurden das Fruchtgewicht, die chemische Zusammensetzung und die antioxidative Aktivität von fünf Erdbeersorten ('Irma' als tagesneutrale Sorte und 'Clery', 'Roxana', 'Asia' und ein unbekannter Genotyp als Kurztagssorten) untersucht. Durch ANOVA- und multivariate Analyse (PCA) wurden Unterschiede zwischen Sorten basierend auf Fruchtgewicht, Gehalt an löslichen Feststoff en, Säuregrad, Reifeindex, den wichtigsten phenolischen Verbindungen und der antioxidativen Aktivität untersucht. Die Ergebnisse zeigten, dass 'Roxana' bei gleichen Anbau- und Umweltbedingungen die größten Früchte aufwies. Die höchsten Werte für lös-liche Feststoff e (SSC), Säuregehalt und Reife- und Geschmacksindizes und phenolische Substanzen zeigten 'Clery' bzw. 'Asia'. 'Asia' ist die Sorte mit der höchsten antioxidativen Kapazität. Die am schlechtesten bewerteten Eigenschaf-ten zeigten der unbekannte Genotyp gefolgt von 'Irma'. Die Jahreszeit ( Jahr) hatt e einen stärkeren Einfl uss auf das Fruchtgewicht, den Säuregehalt, die Phenolverbindungen und die antioxidative Aktivität, wohingegen der Einfl uss auf andere Eigenschaft en nicht signifi kant war. Die Interaktion Sorte x Jahr hatt e einen signifi kanten Einfl uss auf alle bewerteten Eigenschaft en mit Ausnahme des Fruchtgewichts. Die Korrelationsmatrix zeigte signifi kante positive oder negative Korrelationen zwischen den meisten primären und sekundären Metaboliten in Erdbeeren. Die Haupt-komponentenanalyse (PCA) kann ein sehr gutes Instrument zur Diff erenzierung von Erdbeergenotypen mit dem besten Nährwert und den besten gesundheitsfördernden Verbindungen sein.Schlagwörter: antioxidative Kapazität, phenolische Verbindungen, Hauptkomponentenanalyse, lösliche Feststoff e, Säuregehalt, Erdbeere

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MITT EILUNGEN KLOSTERNEUBURG 69(2019): 244-257 MILOSEVIĆ et al.

Small or berry fruits production is positioned aft er banana, citruse and apple production, aft er these fruit species (FAOSTAT, 2019). According to the same source, among berry fruits, strawberry cultivation domi-nated worldwide with a production of 9.223.815 tons from 395.844 ha harvesting area in 2017. Strawberry (Fragaria × ananassa Duch.) is very desirable and appreciated due to its unique pleasant fl avor and taste being one of the most popular fruit types in the world. Alongside with sweet cherry, strawberries are the fi rst fruit from the open fi eld. Th eir fruit are not only consu-med as fresh fruit but also an important component of processed food such as juice, syrup, ice cream, yogurt, jam, a traditional product called "slatko" in some coun-tries such as Serbia which is served only to guests, added to pies and cakes, etc. (Rangana, 1986; López-Valen-cia et al., 2018).In ancient time, it was praised as a medicinal plant. Many centuries later, thanks to the types of large-frui-ted strawberries brought from South America and bree-ding programmes worldwide in which new cultivars of strawberries were created, its nutritional value was recognized. Today, similar to ancient times, the health value of strawberries is more appreciated than the nu-tritious value, because emerging research provides sub-stantial evidence to classify strawberries as a functional food with several preventive and therapeutic health be-nefi ts (Basu et al., 2014; Taghavi et al., 2019). Fruit of strawberry are a rich source of primary (soluble so-lids, sugars, dietary fi ber, cellulose, pectin, protopectin, proteins, organic acids, minerals) (Djilas et al., 2011) and many bioactive compounds, also called secondary metabolites benefi cial to the human body, such as ascor-bic acid, anthocyanins, phenolic acids (ellagic, couma-ric and p-hydroxybenzoic acid), fl avonoids (quercetin, kaempferol, myricetin, catechins) etc. (Halvorsen et al., 2002). Hence, strawberries have been showed to possess high antioxidant activity (Milošević et al., 2016).It should, however, be noted that these bioactive com-pounds can be signifi cantly aff ected by diff erences in strawberry cultivars, pedo-climatic conditions, cultu-ral practices, ripening season, storage, pre-harvest and post-harvest factors, shelf-life and processing methods (Wozniak et al., 1997; Scalzo et al., 2005; Weissin-

ger et al., 2011; Tulipani et al., 2011; Basu et al., 2014; Asadpoor and Tavallali, 2015; Moshiur Rahman et al., 2015; Koch et al., 2018).Cultivar per se (genotype) is the major factor in deter-mining fruit nutritional quality and metabolites benefi -cial to the human body and health (Voća et al., 2008; Capocasa et al., 2008; Aaby et al., 2012; Milošević et al., 2016). Today's strawberry comprises about 500 commercial cultivars grown worldwide (Hancock, 1999). However, newly bred cultivars from day to day present a great challenge for growers and consumers over traditional ones. At the same time, the choice of new cultivars for cultivation without a prior scientifi c evaluation is not recommended.Consumers prefer strawberries with a wide range of sen-sory, external and internal fruit quality att ributes. Th e evidence-based nutritional and health benefi ts of fruit consumption provide a means of promoting strawber-ries (Schöpplein et al., 2002). Th erefore, the proper choice of a cultivar is of paramount importance for suc-cessful strawberry cultivation (Moshiur Rahman et al., 2015).With this background, we have studied the eff ect of culti-var and season on the main physico-chemical properties and antioxidant activity of fi ve strawberry cultivars and selected superior ones with multivariate analysis usage.

MATERIALS AND METHODS

PLANT MATERIAL AND ORCHARD MANAGEMENT

Th e strawberry plantation is located in Prislonica vil-lage (43°33'N and 16°21'E), near Čačak city (western Serbia) at 280 m above mean sea level. Strawberry fruit weight and nutritional quality were analysed in plants cultivated in open fi eld experimental trials with plots for cultivars. All cultivars ('Unknown', 'Clery', 'Roxana', 'Asia' and 'Irma') were grown in a complete randomized block, with four replicates of 50 plants for each plot du-ring 2018 and 2019, i. e. in the 1st and 2nd year aft er plan-ting. 'Irma' is a day-neutral cultivar, whereas the other cultivars belong to the group of short-day strawberries. Plantation was established in summer (mid-August) 2017 with "frigo" seedlings in simple rows at 80 x 25

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cm spacing. Th e space between rows was mulched with crushed straw. Standard cultural practices were used, except irrigation. Th e fertilization of strawberries was done with multi-nutrient fertiliser commercially named "Multi Comp Base” (NPK + MgO + microelements + humic acids) in amounts of 1 to 1.5 g per plant about 5 times on average during plant and fruit development.

QUALITY PARA METERS

Soluble solids content, acidity, phenolic compounds and antioxidant capacity were studied with undamaged fruit samples (≈ 500 g) in the middle of the harvest pe-riod (Krüger et al., 2012). For fruit weight, 25 fresh strawberry fruit in four replicates for each plot (n = 100) at commercial maturity stage (¾ of the fruit has de-veloped red colour (Rangana, 1986)) have been used for each cultivar. Fruit of 'Irma' as a day-neutral cultivar were harvested in the fi rst fruiting period. All cultivars matured between mid-May and June, 10th, on average in both years with 4 to 5 harvests.Fruit weight (FW; in g) was measured immediately af-ter harvest on a digital balance FCB 6K (Kern & Sohn GmbH, Belingen, Germany).For chemical analyses, the collected fruit were washed in water, frozen, and stored at -35 °C until the analysis. Some fruit of each sample were sliced and then homo-genized in a blender, for testing ascorbic acid (vitamin C) immediately. For other analyses, frozen fruit samples were homogenized using a kitchen blender with solvent solution (ethanol/water, 80:20 v/v) and extracts as pre-viously described (Scalzo et al., 2005) were utilized to assay the non-nutritional parameters by the following methods.

SOLUBLE SOLIDS CONTENT, ACIDITY, RIPENING AND TASTE INDEXES

Th e level of SSC was measured as °Brix by a digital hand-held refractometer, model 32-G 110d (Carl Zeiss, Jena, Germany) at room temperature (20 °C). TA was calcu-lated as percentage of citric acid by titrating 10 ml of the strawberry juice with a solution of NaOH (0.1N) till pH 8.1. Th e RI was calculated as SSC/TA ratio. A taste in-dex (TI) was calculated using the equation proposed by Navez et al. (1999).

Equation 1: T1 = ° Brix 20 x Acidity

VITAMIN C, ANTHOCYANIN AND PHENOLIC COMPOUNDS CONTENT AND ANTIOXIDANT ACTIVITY

Ascorbic acid (vitamin C) was determined by the 2,6-dichloroindophenol method (Arya et al., 2000). Data are given as milligrams per 100 g fresh weight (mg/100 g FW). Total anthocyanin content (TAc) was determined according to the method proposed by Giusti and Wrolstad (2001) based on the pH-dif-ferential method previously described by Fuleki and Francis (1968). Content was expressed as milligram of malvidin-3-o-glucoside equivalents per 100 g FW (mg ME/100 g FW). Th e TPC was quantifi ed accor-ding to the Folin-Ciocalteu method (Gutfinger, 1981). Values were expressed as milligram of gallic acid equivalent (GAE) per 100 g FW (mg GAE/100 g FW). Th e TFC was determined according to the method proposed by Brighente et al. (2007), and expressed as milligram of quercetin equivalent (QE) per 100 g FW (mg QE/100 g FW). Th e TAC was evaluated by the phosphor-molybdenum method (Prieto et al., 1999). Ascorbic acid (AA) was used as standard and the TAC is expressed as microgram of AA per gram FW (μg AA/g FW).Total anthocyanin content (TAc), total phenolic con-tent (TPC), total fl avonoid content (TFC) and total antioxidant capacity (TAC) were analyzed using a UV-visible spectrophotometer MA9523-SPEKOL 211 (Iskra, Horjul, Slovenia). Contents of all bioactive compounds are expressed as means ± SE of triplicate analyses per each cultivar in both years.

CHEMICALS

All chemicals and reagents were of analytical grade and were purchased from Sigma Chemical Co. (St Louis, USA), Aldrich Chemical Co. (Steinheim, Germany) and Alfa Aesar (Karlsruhe, Germany).

+ Acidity

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STATISTICAL ANALYSES

All results were tested by two-way analysis of variance (ANOVA), model 5 × 2 using Offi ce Excel soft ware (Microsoft Corp., Redmond, USA). Source of variations was cultivar (A) and year (B). Means were compared by LSD test at P ≤ 0.05. Correlations between the evaluated parameters were analyzed using Pearson's correlations (α = 0.05). Factor analysis, using principal components (PCA) as the factor cultivar, was used to summarize the information in a reduced number of factors by using an XL-STAT procedure of computer statistical package (XL-STAT v 7.5, Addinsoft , Paris, France).

RESULTS AND DISCUSSION

FRUIT WEIGHT

Table 1 shows the quality characteristics of the fi ve strawberry cultivars during two consecutive years. Th ese quality characteristics include FW, SSC, TA, SSC/TA ratio (RI) and TI, which can infl uence the consumer's purchase intention and acceptance.Th e FW of single fruit is signifi cantly diff erent between the fi ve cultivars which is in agreement with other rese-arches (Bogunovic et al., 2015; Moshuir Rahman et al., 2015; Liu et al., 2016). 'Roxana' is the cultivar with the largest single fruit of the fi ve genotypes. Kiprija-novski et al. (2010) also reported that this cultivar had the highest fruit weight in comparison with others. 'Asia' and 'Irma' had similar and lower FW than 'Roxana' with no signifi cant diff erences between them, whereas 'Clery' and 'Unknown' genotype had the lowest and statistically similar FW.Our FW values for 'Asia' and 'Clery' were higher than those reported by Radunić et al. (2014), Boguno-vic et al. (2015) and Kikas et al. (2016) for the same cultivars or Skender et al. (2016) for 'Asia'. In a study of Weissinger et al. (2010) and Dobričević et al. (2014) 'Asia' also had signifi cantly higher single fruit weights than other cultivars, including 'Clery', which confi rmed our results.

Fruit weights of all cultivars were higher in 2019 than in 2018 probably due to the slightly bett er weather condi-tions during harvest period (data not shown). On the other hand, the interaction between the two main fac-tors (cultivar × year) was not signifi cant for FW.FW depends on endogenous factors such as the culti-var's genetics (Milošević et al., 2016), and exogenous factors such as climate, soil and cultural management (López-Valencia et al., 2018). As the climate, soil and the cultural management are the same for all cultivars in our study it is likely that the genotype factor was one of the most infl uential elements in the FW diff erences (Ra-dunić et al., 2014; Dobričević et al., 2014; Liu et al., 2016). Th is view was confi rmed by our results. Namely, although both single factors, i. e. cultivar and year, had statistically signifi cant impact on FW, the sources of va-riations obtained by ANOVA showed that infl uence of cultivar was eight times stronger than infl uence of year in our study.

SOLUBLE SOLIDS CONTENT, ACIDITY AND QUALITY INDEXES

Soluble solids content, acids and their ratio (RI) are most commonly associated with the taste of fruits, in-cluding strawberries, and are measured through SSC and TA. Th ey are used as harvest indices in strawberries with ¾ of fruit surface showing pink or red color (Ranga-na, 1986). In our study, SSC, TA, RI and TI signifi cantly varied between cultivars (Table 1). Year plays a signifi -cant role only for TA, whereas its impact on other traits is random. In general, vital impact of diff erent seasons on fruit phytochemicals accumulation was previously determined (Kiprijanovski et al., 2010; Krüger et al., 2012; Kikas et al., 2016).'Clery' is the cultivar with the highest values of all evalua-ted traits. Other cultivars had lower or similar SSC than 'Clery'. Similar tendency was reported by Kikas et al. (2016) for this cultivar, but their SSC value is much higher than ours. Weissinger et al. (2010) also repor-ted that 'Clery' fruit are richer in SSC than 'Asia'. SSC of 'Irma' and 'Roxana' fruit in our work was higher than

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pendent on environmental conditions during producti-on than on the genetic make-up of the plant. Th is could explain the discrepancies found in the SSC by diff erent authors. In general, SSC in strawberries varied between 4.6 and 11.9 °Brix (Kader, 1999). In our earlier study, SSC of strawberries varied between 9.7 and 10.40 °Brix (Milošević et al., 2016). Th e present SSC results are within these limits.Th e lowest TA and RI values were determined in fruit of 'Unknown' genotype and 'Asia', respectively (Table 1). Taste index (TI) was the lowest and statistically si-milar in 'Unknown' genotype, 'Roxana' and 'Irma', res-pectively. Our range of TA values for 'Irma' and 'Roxana' were lower in comparison to the results of Capocasa et al. (2008) and Kiprijanovski et al. (2010) for the same cultivars or 1.75-fold lower for 'Clery' than the value reported by Kikas et al. (2016). In contrary, our TA value for 'Clery' was 1.3-fold higher than those previ-ously reported by Milivojević et al. (2009) under con-ditions like ours. Data from literature revealed that TA

those found by Capocasa et al. (2008) for the same cultivars, but 'Roxana' in a study of Kiprijanovski et al. (2010) had more SSC than our sample. Strawberries which are of excellent fl avor, for instance, would measu-re 8 % SSC or above (Kader, 1999). In the present stu-dy only 'Clery' fulfi lls these criteria although there are other criteria reported in available literature. As known, soluble solids, which include mainly sugars (approxi-mately >65 %) and smaller amounts of organic acids, vitamins, proteins, free amino acids, essential oils, salts, and glucosides, are good indicators of the sugar content of strawberry and presumably of sweetness (Wang and Lin, 2002).Exogenous factors such as climate, soil and cultural management (López-Valencia et al., 2018; Koch et al., 2018), cultivation system and harvest time (Voća et al., 2007) can modify chemical composition of strawberry fruit, including SSC. For example, Watson (2002) reported that SSC in strawberry was more de-

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A modern fully ripe strawberry fruit is characterized by its large size, vibrant red color, reduced fi rmness, distinct aroma, and sweet fruity fl avor or taste (Schwieterman et al., 2014). For the most of consumers, taste is a very important internal quality att ribute. Usually, the taste of the fruit is evaluated by panelists or a group of them. So, taste is subjective, as the subject which is tasting deter-mines the outcomes, which is why taste is highly perso-nal and driven by personal preference. For these reasons, some researchers (Navez et al., 1999) recommended a mathematical model to calculate taste indices to avoid subjectivity in taste evaluation using modifi ed relations-hip between SSC and TA (Equation 1). We found the highest TI value in 'Clery' and the lowest in 'Roxana', 'Irma' and 'Unknown' genotype, respectively, with not signifi cant diff erences between them. When using data presented in Table 1 for TI, it can be said that fruit of all strawberry cultivars analyzed are tasty due to values around and/or higher than 1.2, as previously reported (Nielsen, 2003). If the value of the taste index is lower than 1.2, the strawberry is considered as having litt le tas-te.Th e eff ects of interaction cultivar × year were signifi cant for all evaluated traits. It shows that some cultivars beha-ved diff erently at diff erent years. From this point of view, the accumulation of SSC and TA as well as their ratio in strawberry fruit has a very complex physiological nature (López-Valencia et al., 2018). However, in these inter-action relationships, ANOVA showed that cultivar as a source of variation is a more infl uential factor than year, although bad weather conditions immediately before and during the harvest period can drastically worsen these fruit properties (Milošević et al., 2016).

PHENOLIC COMPOUNDS AND ANTIOXIDANT CAPACITY

Phytochemicals, also called secondary metabolites, are compounds synthesized by plants in response to specifi c environmental stimuli, pathogen att acks, or nutrient de-privation (Kennedy and Wightman, 2011).Results in Table 2 show that fruit of strawberry are a rich source of main non-nutrient compounds with high hu-man health benefi t. Th e eff ect of genotype and cultivar conditions (years i. e. season) on fruit phenolic com-

and organic acids content are genetically determined and varied signifi cantly between the genotypes, while less infl uenced by environment (Shaw, 1988). Howe-ver, it has been known that site (Krüger et al., 2012), cultivation system and harvest date (Kafkas et al., 2007; Voća et al., 2007) play important roles in the accumulation of SSC and TA in strawberry fruit. For example, strawberry fruit that are grown at northern latitudes contain more SSC and TA than fruit grown at southern sites (Krüger et al., 2012). Our data for TA are in agreement with results of Kader (1991) who reported that their amount in strawberries varied from 0.50 to 1.87 %. Th e same author also reported that the main organic acid in strawberry fruit is citrate (0.42 to 1.24 %), followed by malic acid as the second most prominent organic acid (0.09 to 0.68 %). Th e evaluation and determination of the malic acid level in the fruit of strawberry was not reliable due to the co-elution with fructose. Citric acid contributes signi-fi cantly to fruit titratable acidity (about 88 %), which declines gradually during fruit development (Per-kins-Veazie, 1995). Th e same author also reported that TA content in strawberries varied more strongly due to fruit maturity, genotype and nutrition than ecologic factors. Kader (1999) noted that minimum SSC of 7 % and maximum TA of 0.8 % are accepted as a minimum standard for timing of strawberry har-vest. Otherwise, strawberries with higher acidity are more suitable for processing than for fresh consump-tion (Taghavi et al., 2019) whereas consumers prefer sweet fresh strawberries (Shaw, 1987).It has been long known that SSC/TA ratio (RI) is a major parameter of strawberry taste (Perkins-Vea-zie, 1995) and oft en used as a measure of sweet-ness, as a good indicator of organoleptic evaluation for strawberries (Liu et al., 2016). For example, a strawberry with very low sugar and acid content tastes fl at (Wang and Lin, 2002). Th e fruit defi ned as sweet, the sugar-acid ratio was 7:1, and in the fruit defi ned as acid, this ratio was 6:1 (Wozniak et al., 1997). In the present study, fruit of 'Clery' had the highest RI value, whereas the lowest was registered in 'Asia' (Ta-ble 1). Generally, our range values of RI were lower than those obtained by Liu et al. (2016), higher than the ranges noted by Voća et al. (2009) or Kikas et al. (2016) and basically similar to data reported by Kaf-kas et al. (2007).

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accounted for 19 %, 62 % and 18 % of the total variance for TPC, respectively. However, for TAC, cultivar had 62 % of the total variance, followed by year with 36 % of the total variance and interaction cultivar × year with only 3 % of the total variance. Gündüz and Özdemir (2014) showed that genotype had 71 % and 72 % of the total variance for total anthocyanin contents, whereas it had only 12 % and 13 % of the variance for TPC in the fi rst and second year of the trial. Year-by-year variations in phenolic compounds were previously described by other researches (Capocasa et al., 2008). So, Park et al. (2017) reported that diff erences in sunlight and temperature between years and also plant age might be a cause for the contradictory observations for the same cultivar.Among vitamins, vitamin C is the predominant. It has been proved that vitamin C and phenolic compounds contribute to antioxidant capacity of fruits, including strawberry (Liu et al., 2016). Th e content of vitamin

pounds and antioxidant capacity was tested by compa-ring the non-nutrient parameters of fi ve commercial cul-tivars, grown for two consecutive open fi eld production cycles.Th e ANOVA evidences a signifi cant eff ect of both key factors (cultivar and years alone) on content of vitamin C, TPC, TFC, TAc and TAC during two cultivation cy-cles with exception of vitamin C because its content was statistically similar in both years (Table 2). Th e content of other compounds which were evaluated was higher in the fi rst season, i. e. in 2018. Th e interaction between the two main factors (cultivar × year) was also signifi cant for all compounds evaluated. Similar impacts of these fac-tors have been previously determined (Capocasa et al., 2008; Moshiur Rahman et al., 2015; Bogunovic et al., 2015). In addition, ANOVA also showed that the strongest infl uence on the content of the most com-pounds evaluated (TPC, TFC, TAc) was performed by season, followed by cultivar and interaction cultivar × year. For example, variations of the factors A, B and A x B

μ

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termination. In addition, our data for 'Clery' and 'Asia' were also lower than those previously described by Dob-ričević et al. (2014). Th e results reported by diff erent authors indicate that cultivation method, cultivar, region and climatic conditions of the growing area signifi cant-ly infl uenced the secondary metabolites of strawberries. In addition, the mean content of the total phenols con-sistently decreased with increasing stages of ripeness as previously reported (Tulipani et al., 2011).Th e major phenolic compounds in strawberries, among others, include anthocyanins, accounting for 75.3 % of the total phenolics. Th e color of strawberry skin and fl esh depends mainly on the presence of water solub-le anthocyanin pigments. Among fruit types species, strawberries have more TAc (from 2- to 11-fold) than ap-ples, peaches, pears, grapes, tomatoes, oranges or kiwif-ruit (Scalzo et al., 2005). In the present study, 'Clery' showed the highest TAc, followed by 'Asia', 'Roxana' and 'Irma'. Th e lowest TAc was determined in fruit of 'Unk-nown' genotype. Th ere was a 1.4-fold diff erence between the highest and the lowest TAc i. e. between 'Clery' and 'Unknown' genotype. Our data are in agreement with results of Dobričević et al. (2014) who reported that fruit of 'Clery' are a bett er source of total anthocyanins than 'Asia'. Beside cultivar and year (season), ripening stage also plays an important role in anthocyanins cont-ent (Wang and Lin 2000).Th e strawberry fruit tested in Čačak region showed good antioxidant activity (Table 2). Th e TAC in strawberry extracts from whole fruit varied considerably between cultivars as previously reported (Scalzo et al., 2005; Milošević et al., 2016). Th e best antioxidant capacity was found in fruit of 'Asia' and the poorest in day-neutral (also called ever-bearing or summer strawberry) 'Irma' strawberry. 'Clery' and 'Roxana' also had good antioxi-dant capacity. Th ese data are not in agreement with re-sults of Park et al. (2017) who reported that summer strawberries such as, for example, 'Irma' in our study, had bett er antioxidant capacity than June-bearing (winter) genotypes under South Korean conditions. Th is obser-vation may not be consistent with other reports from European countries because of the regional diff eren-ces between these countries, specifi cally diff erences in weather and temperature conditions. Additionally, the-se fi ndings confi rmed a relevant genotype-dependent response to environmental conditions, which may in

C of 'Clery' and 'Asia' cultivars was similar and signifi -cantly higher than others (Table 2). Th e lowest content of this compound was registered in 'Unknown' genoty-pe and 'Roxana' but with no signifi cant diff erences bet-ween them. Our result for vitamin C content in 'Clery' is in agreement with previous work on the same cultivar (Voća et al., 2008), but lower in comparison with re-sults of Kikas et al. (2016). For 'Roxana', we obtained similar data as Kiprijanovski et al. (2010), but much higher than those found by Dobričević et al. (2014). As known, the cultivar type can be defi ned as an im-portant factor aff ecting ascorbic content of strawberry (Scalzo et al., 2005; Weissinger et al. 2010, 2011; Taghavi et al., 2019). But climatic and soil conditions, geographic position, cultural practice, maturity stage, cultivation system, season etc. also play signifi cant ro-les in accumulation of vitamin C in strawberries (Kays, 1999; Liu et al., 2016).Regarding the TPC, 'Asia' showed a very high mean value, 'Irma' a very low one, in both cases with signifi -cant diff erence to all other cultivars (Table 2). Kader (1991) reported that strawberries contained between 58 and 210 mg TPC per 100 g FW. In the present study, the 'Unknown' genotype and 'Irma' had a lower level of TPC than the limits mentioned. More authors evalua-ted total phenols in diff erent strawberries, and obtained values were very non-uniform and diff erent, depending on the cultivars, climatic conditions, cultivation sys-tems and harvest time (Scalzo et al., 2005). Our data are partially in agreement with results of Capocasa et al. (2008) who reported that fruit of 'Irma' were poorer in TPC than 'Roxana' but without signifi cant diff eren-ces between them. From the above mentioned, the fi ve strawberry cultivars in our study might be considered as a relative low source of TPC.As known, fl avonoids are a group of phenolic com-pounds naturally present in most edible fruit and vege-table plants and, among others, constitute most of the yellow, red and blue colours in the fruit.In the present study, quantity of TFC was diff erent de-pending on the cultivar. Fruit of both 'Clery' and 'Asia' cultivars contained the highest and statistically simi-lar TFC, whereas the lowest was shown by 'Unknown' genotype (Table 2). Our data were much lower than those obtained by Djilas et al. (2011) and Voća et al. (2008) for 'Clery', although a second group of authors used other units, methods and equipment for TFC de-

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TI, indicated that increasing of SSC improved TI as pre-viously reported by Navez et al. (1999). Correlations between SSC and phenolic compounds were weak and not signifi cant. Th ese data revealed that fruit of strawber-ries with high sugar levels had a low phenolic content and small antioxidant capacity, as previously reported (Milošević et al., 2016).A high and signifi cant correlation was observed between TA and vitamin C, TFC and TAc, respectively. Th ese correlations indicated that fruit of strawberries with high TA posses higher levels of these compounds, i. e. antioxidant activity (Oliveira et al., 2015). Th e weak and negative correlation was found between RI vs. other phenolic compounds and antioxidant capacity. Vitamin C strongly correlated with phenolic compounds and TAC which is in agreement with results of other rese-arch (Wang and Lin, 2000), whereas TAc signifi cantly correlated with vitamin C, TPC and TFC, respectively. Th ese correlations in our study were expected due to the fact that anthocyanins are the major known polypheno-lic compounds, as previously reported by Scalzo et al. (2005). Other researches also reported that TAc highly correlated with antioxidant activity (Oliveira et al., 2015). Th e strong correlation between TPC and TAC confi rmed that phenolic compounds are the major sour-ce of antioxidants in most fruit and berry species (Mi-lošević et al., 2016).

part explain the changes in the antioxidant and pheno-lic properties of strawberry fruit in diff erent regions and years (Tulipani et al., 2011). In our earlier study on strawberry, TAC value varied between cultivars from 51.37 to 122.80 mg AA/g on dry weight basis, being hig-her in cultivated vs. woodland forms (Milošević et al., 2016).Signifi cant interaction cultivar × year in the present stu-dy indicated that some cultivars had inconsistent beha-vior in diff erent years, i. e. they are more susceptible to environmental factors (Kader, 1991; Tulipani et al., 2011).

CORRELATIONS BETWEEN EVALUATED VARIABLES

Table 3 shows the correlation matrix between the vari-ables studied. Th e FW negatively correlated with SSC, RI and TI, indicated the largest fruit gave lower values of these chemical properties. Although these correlations were not signifi cant, except FW vs. RI, which was nega-tive, they are a signal that new breeding programs based more on agronomic traits (fruit yield and size) than nu-tritional and health values, a fact that confi rms previous reports by (Capocasa et al., 2008). A strong signifi cant and positive correlation was observed between SSC and

α

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Also, 'Asia' and 'Roxana' stand out as very promising cultivars for commercial production under conditions like ours. 'Irma' and 'Unknown' genotype may be recom-mended for limited cultivation. Correlation matrix and PCA can be important models in order to segregate stra-wberries with the best pomological and chemical pro-perties for growers and consumers. Finally, the results obtained in this study will be a useful reference for future strawberry-breeding programs.

PRINCIPAL COMPONENT ANALYSIS (PCA)

As it is known, PCA is a multivariate statistical analysis method used to reduce the number of variables and was applied to identify which volatile compounds provided the best ability to diff erentiate the cultivars. Th e PCA result shows that the fi rst three principal components have a cumulative reliability of 100 % (Table 4). Th e ei-genvalues of the covariance matrix showed that the set of the two principal components (PCs) accounted for 94.40 % of the total variance in the dataset with respect to cultivar. PC1 explained 62.69 % of the variance in the dataset, whereas PC2 and PC3 explained an additional 31.71 % and 5.60 % of the variance, respectively. PC1

CONCLUSION

Th e comparison of four short-day and one day-neutral strawberry cultivars showed diff erences in terms of fruit weight, primary metabolites, main phenolic compounds and antioxidant activity, especially in the content of so-luble solids, titratable acidity, vitamin C, total anthocya-nin and antioxidant capacity as one of the most im-portant parameters defi ning fruit quality, i. e. harmony of taste and health promoting compounds. Among the cultivars evaluated, the cultivar 'Clery' which has been produced in Serbian plantations for many years, stands out, with already recognized quality for consumers.

represents mainly TA, TI, vitamin C, TPC, TFC, TAc and TAC, whereas PC2 explains fruit weight SSC and RI. Figure 1 represents PC1 and PC2 plott ed on a bidi-mensional plane. Positive values for PC1 indicate culti-vars with highest contents TA, TI, vitamin C, TPC, TFC, TAc and TAC. Positive values for PC1 indicate cultivars with highest acidity and largest contents of vitamin C, TPC, TFC, TAc and taste index values and antioxidant activity. 'Clery' and 'Asia' belong to this group. Th e hig-hest PC2 values correspond to cultivars with the largest fruit, SSC and RI values. Positive PC2 values denoted that high SSC and RI were found in 'Unknown' genoty-pe. Th e largest negative PC2 value showed that 'Roxana' and 'Irma' were strawberries with the largest fruit.

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Fig. 1: Segregation of fi ve strawberry genotypes according to their quality characteristics determined by principal component analysis (PCA); for abbreviations see section Materials and methods

CONFLICT OF INTEREST

Th e authors report no confl icts of interest. Th e authors alone are responsible for the content and writing of this article.

ORCIDS:

T. Milošević: htt ps://orcid.org/0000-0002-0402-3395

N. Milošević: htt ps://orcid.org/0000-0002-8779-4354

J. Mladenivić: htt ps://orcid.org/0000-0002-0522-2214

Biplot (axes PC1 and PC2: 94.40%)

Irma

Asia

Clery

Unknown

Roxana

TA C

TA c

TF CTP CVit a m in C

TI

R I

TA

S S C

F W

-5

-2.5

0

2.5

5

-10 -7.5 -5 -2.5 0 2.5 5 7.5 10

PC1 (62.69%)

PC2

(31.

71%

)

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