Comparison of phenolic compounds of orange juice

Food Chemistry 143 (2014) 354–361

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Comparison of phenolic compounds of orange juice processed by pulsed electric fields (PEF) and conventional thermal pasteurisation E. Agcam a,⇑, A. Akyıldız a, G. Akdemir Evrendilek b a b

Department of Food Engineering, Faculty of Agriculture, Cukurova University, Balcali Adana, Turkey Department of Food Engineering, Faculty of Engineering and Architecture, Abant Izzet Baysal University, Golkoy Campus, Bolu, Turkey

a r t i c l e

i n f o

Article history: Received 24 May 2013 Received in revised form 14 July 2013 Accepted 20 July 2013 Available online 30 July 2013 Keywords: Pulsed electric fields Thermal pasteurisation Orange juice Phenolic compounds Shelf life

a b s t r a c t Processing of orange juice by pulsed electric fields (PEF) and thermal pasteurisation was carried out to compare changes in total phenolic concentration, hydroxybenzoic acid, hydroxycinnamic acids, flavonols, flavones and flavonones before and after being stored at 4 °C for 180 days. Changes in the initial total phenolic concentration of the samples varied depending on the applied electric field intensity and thermal pasteurisation. Hesperidin and chlorogenic acids were detected as the most abounded flavonoid and phenolic acids in the orange juice, respectively. Except for syringic acid and neoeriocitrin, the concentration of the phenolic compounds indentified in the orange juice samples enhanced after the PEF or thermal pasteurisation. The samples treated with PEF had more stable flavonoids and phenolic acids than those treated with the thermal pasteurisation. The PEF-treated samples had higher sensory scores than the heat-treated samples. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Citrus fruits and derived products have been well known to have beneficial effects on human health owing to their high concentration of vitamin C and bioactive compounds such as phenolic acid, flavonoid, limonoid, carotenoid and fibre (Anagnostopoulou, Kefalas, Kokkalou, Assimopoulou, & Papageorgiou, 2005; Bermejo, Llosa, & Cano, 2011; Gorinstein et al., 2001). Epidemiological studies have reported that there is a significant positive association between consumption of polyphenols and reduced risk of chronic diseases such as cancer, cardiovascular disease, diabetes, viral infection, inflammatory activities and Alzheimer’s disease (Galati, Monforte, Kirjavainen, Forestieri, & Tripodo, 1994; Hertog, Feskens, Hollman, Katan, & Kromhout, 1993; Liu, 2003; Scambia et al., 1994; Temple, 2000; Willett, 2002). However, heat (thermal) processing of orange juice may destroy the amount or the bioavailability of these compounds, thus reducing beneficial health effects (Plaza et al., 2006). To overcome such shortcomings of heat processing, different processing technologies including the applications of high pressure and pulsed electric fields (PEF) have been developed as non-thermal emerging technologies for juice processing (SolivaFortuny, Balasa, Knorr, & Martín-Belloso, 2009). A PEF treatment is efficient enough to destroy microorganisms in fruit juices without any significant change in their nutritional

⇑ Corresponding author. Tel.: +90 322 3386537; fax: +90 322 3386614. E-mail address: [email protected] (E. Agcam). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.115

and sensory properties (Min & Zhang, 2003; Yeom, Streaker, Zhang, & Min, 2000). Numerous studies are available about the effect of PEF treatments on orange juice quality including phenolic contents, vitamin C retention, pectin methyl esterase inhibition, microbial inactivation, shelf life extension, and sensory properties (López, Puértolas, Condón, Álvarez, & Raso, 2008; Morales-de la Peña, Salvia-Trujillo, Rojas-Graü, & Martín-Belloso, 2011; Odriozola-Serrano, Soliva-Fortuny, Hernández-Jover, & Martín-Belloso, 2009; Odriozola-Serrano, Soliva-Fortuny, & Martin-Belloso, 2008; Plaza et al., 2006; Puertolas, Saldana, Condon, Alvarez, & Raso, 2009; Puértolas, López, Saldaña, Álvarez, & Raso, 2010; SânchezMoreno et al., 2005). However, studies about the effect of PEF treatments on phenolic contents of orange juice are very limited. Therefore, the objectives of this study are to evaluate and compare the effects of PEF processing and thermal pasteurisation on orange juice phenolic compounds in addition to the effect of storage (4 °C for 180 days) on the concentration of these compounds.

2. Materials and methods 2.1. Orange juice Kozan Yerli variety of oranges (approximate of 500 kg harvested in 2009 season) grown in Cukurova Region (Turkey) were used in the study. Kozan Yerli orange juice had titratable acidity, total soluble solids (TSS), TSS/TA and pH of 1.19% as citric acid equivalent, 11.83%, 9.94% and 3.53%, respectively.

E. Agcam et al. / Food Chemistry 143 (2014) 354–361

After being washed, the oranges were peeled, cut into two pieces, and processed using a bench scale automatic orange squeezing machine (CANCANÒ, Adapazarı, Turkey). Subsequently, the juice sample was passed through 1-mm stainless steel sieves to remove seeds and coarse pulp prior to being immediately processed by PEF and heat pasteurisation processing units. The orange juice samples were stored at 4 °C for 180 days, and analysed at every 60 days.

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conditions causing inactivation of pectinmethylesterase activity by at least 90% were chosen as pasteurisation norms. As a result of the preliminary experiments, heat pasteurisation at both 90 °C for 10 s (HP1) and for 20 s (HP2) was applied. After the treatments, the orange juice samples were immediately cooled down to refrigeration temperatures (+4 °C) and filled into amber colour bottles for further analyses. 2.4. Determination of total phenolic content

2.2. PEF treatments A laboratory-scale PEF OSU-4A system providing square wave bipolar pulses, with 6 co-field flow treatment chambers with 0.23 cm chamber diameter and 0.292 cm gap distance was used for PEF treatments (Fig. 1a). The flow rate of the orange juice was adjusted to 0.633 mL s1 (Evrendilek, Zhang, & Richter, 1999). PEF processing parameters were given in Table 1. The treatment temperature was measured during processing before and after each pair of PEF treatment chamber by K type dual channel digital thermocouples (Fisher Scientific, Pittsburgh, PA, USA). PEF processing was conducted at 35 °C (water bath temperature); however, with increased electric field strength, the application temperature raised up to 58.20 °C during processing. Both control (U) and PEF treated samples (E1, E2, E3, E4, E5, E6, E7 and E8) were filled into amber colour bottles for further analyses.

The amount of the total phenolic compounds was measured using the Folin–Ciocalteu method with some modifications (Abdullakasim et al., 2007). For the analysis, 5 mL of orange juice were mixed with 5 mL of 80% methanol in the Teflon tube, and then, tubes were centrifuged with 4000 rpm for 20 min at 4 °C (Heraeus Bofuge Primo R, Germany). Approximately 100 lL of diluted sample or standard solution at various concentrations were mixed thoroughly with 100 lL Folin–Ciocalteu reagent and 3000 lL deionized water. After incubation for 10 min at room temperature, 100 lL of 20% Na2CO3 solution were added with mixing, and the mixture was further incubated at room temperature for 2 h in the dark. Absorbance measurement of the samples was conducted at 765 nm (Perkin Elmer Lambda 25-UV/VIS, USA). Gallic acid was used as standard, and the total phenolic compounds of the samples were expressed as milligrams per L gallic acid equivalents (mgGAE L1).

2.3. Heat pasteurisation treatment 2.5. Determination of phenolic compounds The bench scale system designed in the Department of Food Engineering of Cukurova University (Adana, Turkey) was used for heat pasteurisation applications (Fig. 1b). Thermal processing

Five mL of orange juice was mixed with 10 mL of 80% methanol in the Teflon tube and sonicated (Bandelin Sonerex, Germany) at

Fig. 1. Schematic diagram of PEF (a) and heat pasteurisation (b) systems (1: stirrer; 2: peristaltic pump; 3: heating coil; 4: cooler).

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1 1 ¼ þ kt C C0

Table 1 PEF processing parameters of orange juice. Treatment E1 E2 E3 E4 E5 E6 E7 E8 a

Electric field strength (kV cm1)

Current (A)

Treatment time (ls)

T (°C)a

Energy (J)

13.82 13.82 17.06 17.06 21.50 21.50 25.26 25.26

2.60 2.60 3.36 3.36 4.54 4.54 5.75 5.75

1033.9 1206.2 1033.9 1206.2 1033.9 1206.2 1033.9 1206.2

31.88 33.25 35.44 35.19 38.52 38.25 38.30 42.60

10.89 12.70 17.37 20.26 29.57 34.50 43.99 51.32

t1=2 ¼

ð2Þ

1 C0k

ð3Þ

where,C is total concentration of phenolics after the certain period of storage time,k is reaction rate constant (L mg1day1), C0 is initial total concentration of phenolics (mg L1) according to the second order kinetics, t is the storage time (day), and t1/2 is half-life time (day) of total phenolics in a certain storage conditions (Arabshahi & Lund, 1985; Johnson, Braddock, & Chen, 1995).

Average temperature in application chambers.

2.7. Sensory analysis room temperature (25 °C) for 15 min. The tubes were centrifuged with 4000 rpm for 10 min at 4 °C (Heraeus Bofuge Primo R, Germany), the supernatant passed through 0.45 lm Teflon membrane filter, and injected to the HPLC (Shimadzu LC-20AT, Japan) system consisting of a quaternary pump, a column temperature control oven (CTO-10AS), an auto sampler unit (SIL-20A), a degasser module (DGU-20A5) and a photodiode array detector (SPD-M20A). Twenty lL of supernatant were injected into the C18 XTerra (Waters, 4.6 250 mm) column at 30 °C, and the flow rate was 0.8 mL min1. Photodiode array detector was set to 280 and 320 nm, and formic acid at 2% (A) and ACN at 100% (B) concentrations were used as mobile phases. According to the preliminary experiments, the best gradient elution was 100% A at 0 min, 95% A + 5% B at 10 min; 90% A + 10% at 25 min, 80% A + 20% B at 55 min, 55% A + 45% B at 70 min, 100% B at 90 min, and 100% A at 95 min. The phenolic compounds were identified by comparing their UV–visible spectra and retention times with that of corresponding standards. Quantification of phenolic compounds was carried out at 280 and 320 nm using external standard method. Calibration curves were obtained using the commercial standards of the concentrations normally present in orange, obtaining regression coefficients (R2) above 0.997 in all cases. 2.6. Degradation kinetics The adjustment obtained by applying the least squares method fits the second order kinetics model for total phenolics degradation. Kinetic equations used for total phenolics degradation in the storage period are shown below:

dc=dt ¼ kC

2

ð1Þ

Sensory analyses of the juice samples were realized using the graphic scale test. Orange juice samples processed with PEF and HP1 treatments of low (E2), medium (E4 and E6) and high (E8) intensity were used for sensory analysis. The panel was composed of thirteen panelists from graduate students and faculty members from the Department of Food Engineering of Cukurova University. Five sensory parameters (turbidity, colour, odour, aroma and general acceptance) were used to test orange juice samples over a 10point hedonic scale. 2.8. Statistical analysis Analysis of variance (ANOVA) and Duncan’s multiple comparison test was performed to determine significant differences between the treatments using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA). Each experiment was repeated at least three times. 3. Results and discussion 3.1. Effect of processing on the total phenolic content The changes in total phenolic compounds after processing with the PEF and heat treatments are shown in Table 2. The highest values of the total phenolic contents were detected as 443.42 and 439.07 mgGAE L1 after the treatments of E6 and HP1, while the lowest value of total phenolics was detected as 350.42 mg GAE L1 after the treatment of E1, respectively. A significant difference was detected between the total phenolic compounds of orange juice samples among the different treatments (p < 0.01). The total phenolic content detected in this study is comparable to previous studies in that total phenolic content in the same

Table 2 Effects of PEF and heat pasteurisation treatments on orange juice total phenolics (mg L1) and total phenolic degradation kinetic data with storage period. Treatment

U E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0. Day

60. Day

abcd

388.97 ± 40.18 a 350.42 ± 54.14db cd 368.11 ± 48.24 c 370.27 ± 38.44cdb 363.84 ± 45.14cda 377.04 ± 45.05bcda 443.42 ± 45.88aa 428.26 ± 59.74aba 416.51 ± 40.00abca 439.07 ± 31.30aa 419.84 ± 49.75abca

– 365.93 ± 26.17bcb 345.29 ± 14.19bcd 418.93 ± 25.03aa 354.63 ± 33.82bca 353.18 ± 25.89bcab 390.68 ± 23.32abb 370.20 ± 15.93bcab 377.38 ± 21.74abca 337.06 ± 16.05cb 344.59 ± 44.65bcab

120. Day

– 426.72 ± 46.47aa 425.61 ± 20.28aa 377.74 ± 33.67bcb 404.67 ± 24.34aba 324.64 ± 24.19db 345.94 ± 28.07cdc 329.80 ± 35.55db 289.08 ± 33.12eb 312.19 ± 15.20dbc 329.41 ± 28.24db

180. Day

– 397.36 ± 22.12ab 393.19 ± 20.05ab 393.97 ± 29.83aab 389.52 ± 32.51aa 309.13 ± 17.14bcb 328.39 ± 13.37bc 320.22 ± 26.79bb 261.98 ± 24.33cb 303.97 ± 25.50bcc 312.59 ± 19.72bcb

Kinetic data⁄⁄⁄

Reaction order 0.

1.

2.

k (Lmg1day1) 106

Half-Life (t1/2, day)

– – – – – 0.985 0.959 0.910 0.963 0.795 0.845

– – – – – 0.990 0.970 0.927 0.965 0.822 0.869

– – – – – 0.993 0.979 0.941 0.964 0.849 0.892

– – – – – 3.44 4.50 4.61 8.59 5.46 4.31

– – – – – 764 501 501 276 417 553

⁄ Superscript letters on the same column show the significant difference between the applications and subscript letters in the same row show the significant difference between storage periods (p < 0.05). ⁄⁄ ± values are standard deviations. ⁄⁄⁄ Kinetic data were calculated according second order kinetics.

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variety orange juice (Turkish cv. Kozan) was reported to be 317.36 mg L1 (Kelebek, Selli, Canbas, & Cabaroglu, 2009). Ramful, Tarnus, Aruoma, Bourdon, and Bahorun (2011) reported that total phenolics in the pulp extracts of different citrus varieties (orange, satsuma, clémentine, mandarin, tangor, bergamot, lemon, kumquat, calamondin and pamplemousses) were determined in the range of 406–1694 mg kg1 GAE1. In general, the orange juice samples processed with the application of high energy had a total phenolic content higher than those processed with the application of low energy. This could be attributed to the fact that extraction of intracellular contents is enhanced by PEF treatment. Permeabilisation of plant cells can be used to increase yield in the production of fruit juices, enhance the extraction of intracellular metabolites, and increase the extraction efficiency of processes (Barsotti & Cheftel, 1999; Brodelius, Funk, & Shillito, 1988; Knorr, Angersbach, Eshtiaghi, Heinz, & Lee, 2001). Pores formed on the cell membrane are most likely to enhance mass transfer out of the cells. Untreated (U) orange juice samples had a shelf life of approximately 10 days, whereas both PEF and heat treated samples had a shelf life of 180 days at 4 °C. During shelf life of the samples, total phenolic compounds started to degrade after processed with high

intensity PEF treatments (E5, E6, E7 and E8) and heat pasteurisation applications (HP1 and HP2) during storage. The total phenolic content of the samples treated with E1, E2, E3 and E4 increased first, and then, decreased with the storage period (Table 2). The degradation kinetics of phenolic compounds were calculated for zero, first and second orders, and obtained coefficients of determination (R2) were given in Table 2. The most suitable degradation kinetics for the total phenolic compounds were determined as the second order during the storage. The calculated k values of the second order degradation determined for E5, E6, E7, E8, HP1 and HP2 were 3.44, 4.50, 4.61, 8.59, 5.46 and 4.31 106 L mg1day1, and half-life time (t1/2) of the same samples was calculated as 764.23, 501.15, 501.32, 275.82, 417.14 and 553.63 days, respectively. Because orange juice processed with E8 treatment had very high intensity of PEF processing conditions, the treatment had the lower k and higher t1/2 values than the other PEF treatments. A comparison of PEF and heat pasteurisation for the total phenolic compounds of juices during certain periods of storage time in the previous studies revealed similar results. PEF processed (35 kV cm1, 1000 ls in bipolar 4 ls pulses) and mild pasteurised

Table 3 Effect of PEF and heat pasteurisation process on hydroxybenzoic and hydroxycinnamic acids in citrus juice (mg L1).

⁄

Samples

Gallic acid

Vanillic acid

PAEE(⁄)

o-Cumaric acid

Ferrulic acid

Caffeic acid

p-Cumaric acid

Chlorojenic acid

Sinapic acid

Syringic acid

0. Day

U E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0.16a 0.18ab 0.17ab 0.19ab 0.22ac 0.19ab 0.14ac 0.16ab 0.20ab 0.19aab 0.13ab

3.83abc 4.52aba 3.67abcb 2.33ca 3.98abca 2.52cb 4.49aba 2.75bca 3.23bcb 5.04aa 2.38ca

0.61a 0.58ac 0.61ac 0.53ac 0.93ac 0.55aa 0.91aa 0.72aa 0.62aa 0.64aa 1.03aa

4.82d 7.15aba 4.96dc 6.49bca 6.98abcab 6.81abcab 6.91abcb 5.73cdb 6.66bca 7.86aba 8.17aa

4.73c 4.82ca 4.45cab 5.27bca 5.85aba 5.24bca 5.38bcab 5.79aba 5.14bca 6.06aba 6.31aa

4.84bc 5.18bca 4.72bcb 4.90bcb 5.62aba 5.08bca 4.89bca 4.62ca 4.63cb 5.14bca 6.06aab

0.27ab 0.19bb 0.22bc 0.45ac 0.31abd 0.30aba 0.22ba 0.19ba 0.17bc 0.24abb 0.32aba

15.22bc 14.37ca 14.00cb 15.45abcb 17.79aba 14.96ca 15.60abca 16.41abca 14.19ca 16.17abca 18.15aa

1.70ab 1.83aba 0.82bc 1.55aba 1.87aa 1.05abb 1.51aba 1.18aba 1.21aba 0.96abb 1.48aba

0.68a 0.54aba 0.50bc 0.51ba 0.48bd 0.42bb 0.42bb 0.56aba 0.47ba 0.47ba 0.47ba

60. Day

E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0.23abb 0.29bab 0.26abb 0.23abc 0.37aab 0.22abb 0.26abb 0.20cb 0.22abab 0.20ca

4.36ca 5.75aa 2.36ca 2.42cb 2.64bcb 2.67bcb 2.11cb 2.00cb 5.51aa 3.31bca

1.87ab 1.59abb 1.74abb 1.50abb 1.09bca 0.97bca 0.70ca 0.67ca 1.40abca 1.03bca

4.78deb 5.11dec 7.91aa 7.97aa 6.15bcdab 5.21cdec 7.83aa 6.52bca 4.59ec 6.63abb

4.98aa 5.15aa 5.30ba 5.16bab 5.55aa 5.19ab 6.05aa 5.42aa 4.70ab 5.82ab

4.04ba 5.75aba 5.37aba 4.61abb 5.47aba 5.02aba 4.99aba 4.71abb 5.80aba 6.23aa

0.32bb 0.34bc 0.44bc 3.08ac 0.28ba 0.31ba 0.23ba 0.20bbc 0.32bab 0.28ba

15.19bca 16.89aa 16.92aa 16.54abb 16.50aba 15.93aba 16.05aba 14.53aba 16.03ca 17.34aab

1.84aba 0.97cbc 1.55abca 2.04aa 2.15aa 2.04aa 1.55abca 1.66abca 1.13bcb 1.34abca

0.55ba 0.63abbc 0.52ba 0.68abc 0.50ba 0.49ba 0.52ba 0.48ba 1.38aa 0.56ba

120. Day

E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0.24ab 0.32aa 0.21ab 0.31ab 0.61aab 0.25ab 0.19ab 0.37ab 0.32aa 0.23aa

2.52bb 3.97abab 2.79ba 2.69bb 3.18bab 5.05aa 2.58ba 2.60bb 3.31bb 2.56ba

3.21aa 3.63aa 3.35aa 3.03aa 1.32ba 1.25ba 1.16ba 1.02ba 1.51ba 0.99ba

4.91cb 5.67abcb 5.82abca 7.26aa 5.65abcb 5.40bcc 6.26abcb 5.36bca 5.60abcbc 7.05abab

4.83ba 5.77aa 4.65bb 5.00abab 5.56aba 5.72aa 5.66aab 5.39aba 5.10abb 5.43abc

5.22aa 5.71aa 5.02aab 3.82ac 5.44aa 5.45aa 5.29aa 4.89ab 5.24aa 5.32abc

2.25ca 2.95bb 1.92cb 5.68ab 0.43da 0.31da 0.26da 0.30da 0.47da 0.41da

13.67ca 16.80aa 15.58abb 15.37abc 15.72aba 16.19aba 16.54aa 14.91bca 14.96bcb 15.96abb

1.67aba 1.53aba 1.67aba 1.69aba 1.59abab 1.84aba 1.61aba 1.25ba 2.39aa 0.96ba

0.68ba 0.74bab 0.58ca 0.83ab 0.56ca 0.51cda 0.51cda 0.54cda 0.51cda 0.44dab

180. Day

E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0.49ba 0.32bca 0.45ba 0.35bca 0.82aa 0.38bca 0.94aa 1.10aa 0.14bcb 0.09cb

3.36bcab 3.61bcb 3.38bca 2.65bcb 3.60bca 3.98ba 2.50cab 5.99aa 3.00bcb 2.73bca

3.18aa 2.96aba 3.47aa 3.16aa 1.25cda 1.44cda 1.10da 1.06da 2.10bca 1.49cda

6.73abca 7.26aba 5.64ca 5.63cb 7.68aa 7.51aba 5.85cb 5.37ca 6.42abcb 6.21bcb

4.02ba 4.33bb 5.07abab 4.63abb 5.55aba 5.76aa 5.30abb 5.60aba 4.94abb 4.81abd

4.90abca 4.57abcb 4.89abcb 3.15cd 5.34aba 5.06abca 4.85abca 6.15aa 4.12bca 4.52abcc

2.19ca 4.20aba 3.35aba 7.31aa 0.36da 0.34da 0.23da 0.27dab 0.19db 0.20da

13.75fa 14.98deb 15.75bcab 14.40ed 15.53cda 16.20aba 14.47ea 16.55aa 14.63eb 12.89gc

1.53aa 1.48aab 2.16aa 1.52aa 1.69aa 1.78aa 1.41aa 1.51aa 1.26ba 1.00ba

0.65aba 0.78aba 0.69aba 0.96aba 0.55aba 0.32bb 0.48aba 0.52aba 1.10aa 0.33bb

Superscript letters on the same column show the significant difference between the applications and subscript letters in the same row show the significant difference between storage periods (p < 0.05). ⁄⁄ Protocatechuic acid ethyl ester.

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Table 4 Effect of PEF and heat pasteurisation process on flavonones in citrus juice (mg L1). Samples

Naringenin a

Naringin de

Neohesperidin cd

Hesperidin bcd

Neoeriocitrin a

Eriocitrin

0. Day

U E1 E2 E3 E4 E5 E6 E7 E8 HP1 HP2

0.76 0.78ab 0.66ba 0.69ba 0.69bc 0.68ba 0.74aba 0.69ba 0.72aba 0.68ba 0.79aa

10.59 10.99dea 10.13dea 12.48bcda 11.97cdea 11.37cdea 12.23bcdea 14.09abca 9.53ea 14.77aba 15.44aa

0.42 0.51bca 0.39db 0.50bca 0.55bb 0.66aa 0.48bcda 0.52bca 0.43cda 0.50bca 0.57aba

233.07 221.30cda 195.02db 228.56bcdb 227.76cda 219.81cda 235.32bca 257.83abca 237.68bca 265.78aba 276.91aa

0.95 0.63bca 0.29da 0.38cdb 0.52cda 0.36cda 0.59bca 0.47cdc 0.45cda 0.87aba 0.49cda