# A Preliminary Study on the Potential of the New Red-Fleshed 'JB' Clone Apple for Cider Production
## 2 Materials and Methods  
The study used fruit from the red-fleshed apple clone 'JB', bred by Prof. [PERSON]. Plants were grown in the Warsaw University of Life Sciences Experimental Orchard in Wilanow [18]. The trees of the 'JB' clone were planted in 2018, marking the first experimental planting of this cultivar in Poland. The first fruit was harvested two years later. This explains the limited number of publications on this cultivated apple clone and the preliminary nature of our study. A unique feature of these apples is their red flesh (Figure 1). The high content of anthocyanins, which enhance the fruit's health-promoting properties, is responsible for the red color of the flesh. Another essential feature of the 'JB' clone is its resistance to apple scab, the most common disease in orchards [14]. The apples were harvested in early September 2023. A starch test was conducted to determine their harvest readiness, yielding an index value of 4 to 5 on a 10-point scale. The fruit was then briefly stored in a cold chamber to facilitate the juice-pressing process and further steps in cider production. The fruit was pressed using a Bucher piston press (HPL14, BUCHER Unipectin AG, Niederweningen, Switzerland) immediately before the physicochemical analysis of the obtained must. The color was measured in reflected light using a CR-300 chromateter (Konica Minolta, Osaka, Japan). The device was calibrated against standard colors in the Commission Internationale de l'Eclairage (CIE) Lab* system. The results were presented as lightness (L*), green/red value (a*), and blue/yellow value (b*). The analysis was performed at least ten times on randomly selected juice samples [19]. Images of the apple and juice surface were captured using a Samsung S630 digital camera (Seoul, Republic of Korea). The must extract was determined by the areometric method using a Balling areometer with a range of 0 to 15 \({}^{\circ}\)Blg, and the active acidity was measured using a CP-505 pH meter (Elmetron, Zabrze, Poland). The sucrose, D-fructose, and D-glucose contents of the apple must were tested using a specialized set of reagents from Megazzyme, according to the manufacturer's instructions [20]. The reagent kit consisted of six sealed vials with the following reagents: No. 1--buffer; No. 2--NADP+/ATP; No. 3--HK/G6P-DH; No. 4--PGI; No. 5--D-glucose and D-fructose standard; No. 6--\(\beta\)-fructosidase. All the absorbance values were read using a V-1200 visible light spectrophotometer VWR (Leuven, Belgium) at 340 nm and then used to calculate the content of sucrose, D-glucose, and D-fructose in g/L using the Excel calculator in Microsoft Office 365 (access provided by Megazyme). The total sugar content was obtained by summing the average results. The total acidity of the must was determined by potentiometric titration with a 0.1 M sodium hydroxide solution to pH 7 using a CP-505 pH meter [21]. The volume of 0.1 M sodium hydroxide solution used to titrate 10 mL of juice (in mL) was converted to malic acid according to the formula K = 0.67 \(\times\) n, where n is the volume of 0.1 M sodium hydroxide. The polyphenol content of the must was examined using the Folin-Ciocalteu method [22]. The determination was performed on a 96-well plate. An automatic pipette was used to add 0.02 mL of the sample, 0.04 mL of [PERSON]'s reagent, and 0.25 mL of sodium carbonate solution. Distilled water was used instead of juice for the control sample. The plate was set aside in a dark place for one hour. The absorbance at 750 nm was then read using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The resulting absorbance values (y) were inserted into the formula x = 512.77 \(\times\) y \(-\) 0.9798 (\(R\) = 0.98), obtaining the polyphenol content expressed as chlorogenic acid [ug/mL]. The calibration curve equation covered the range of 0-100 ug/mL chlorogenic acid. The results are given in mg/L [23].