Removal of phenolic, turbidity and color in sugarcane juice by electrocoagulation as a sulfur-free process

Elsevier Editorial System(tm) for Food Research International Manuscript Draft Manuscript Number: FOODRES-D-18-03744R1 Title: Removal of phenolic, turbidity and color in sugarcane juice by electrocoagulation as a sulfur-free process Article Type: Research Articles Keywords: sugarcane juice; sulfur-free clarification; electrocoagulation; CIELAB. Corresponding Author: Mr. Felipe Iwagaki Braga Ogando, MSc Corresponding Author's Institution: Luiz de Queiroz College of Agriculture - University of São Paulo First Author: Felipe Iwagaki Braga Ogando, MSc Order of Authors: Felipe Iwagaki Braga Ogando, MSc; Claudio Lima de Aguiar; João V Napolitano Viotto; Francisco J Heredia; Dolores Hernanz Abstract: This work analyzed the use of electrocoagulation as substitute for sugarcane clarification process using sulfitation. It was evaluated technological parameters (Icumsa color and turbidity), phenolic compounds content and CIELAB color parameters. Four kinetics of reduction color from sugarcane juice were carried out. The essays were divided according to the voltage applied: 35, 45, 55 and 65 V (also based on previous tests). Higher voltage treatments achieved greater reduction of Icumsa color, turbidity and total phenolic compounds. However, none of treatments impacted simple phenolic content analyzed in this work. Tristimulus analysis presented some pattern that went beyond technological analysis, including that 65 V essay changed the pigmentation of sugarcane juice and had an early stabilization on chroma. This kind of results could be useful for industry, once they could correlate quality with different color parameters and finally improve the clarification in general with finer settings of technique according to different situations. 1 Removal of phenolic, turbidity and color in sugarcane juice by electrocoagulation as a sulfur-free process Felipe Iwagaki Braga Ogando¹†, Claudio Lima de Aguiar¹, João Vitor Napolitano Viotto¹, Francisco José Heredia2, Dolores Hernanz3 ¹ Hugot Sugar Technology Laboratory, Luiz de Queiroz College of Agriculture, University of São Paulo, 13418-900, Piracicaba, Brazil ² Food Colour & Quality Laboratory, Área de Nutrición y Bromatología, Universidad de Sevilla, Facultad de Farmacia, 41012, Sevilla, Spain ³ Department of Analytical Chemistry, Universidad de Sevilla, Facultad de Farmacia, 41012, Sevilla, Spain † To whom correspondence should be addressed. E-mail: [email protected] Telephone: +55 19 34478680 Running Title: Removal of color in sugarcane juice by electrocoagulation Abstract This work analyzed the use of electrocoagulation as substitute for sugarcane clarification process using sulfitation. It was evaluated technological parameters (Icumsa color and turbidity), phenolic compounds content and CIELAB color parameters. Four kinetics of reduction color from sugarcane juice were carried out. The essays were divided according to the voltage applied: 35, 45, 55 and 65 V (also based on previous tests). Higher voltage treatments achieved greater reduction of Icumsa color, turbidity and total phenolic compounds. However, none of treatments impacted simple phenolic content analyzed in this work. Tristimulus analysis presented some pattern that went beyond technological analysis, including that 65 V essay changed the pigmentation of sugarcane juice and had an early stabilization on chroma. This kind of results could be useful for industry, once they could correlate quality with different color parameters and finally improve the clarification in general with finer settings of technique according to different situations. Keywords: sugarcane juice, sulfur-free clarification, electrocoagulation, CIELAB. 2 1. Introduction Sugarcane juice is an important Brazilian feedstock for sugar, ethanol and bioenergy. Sugarcane is a complex material and its composition and characteristic are influenced by climate, phytosanitary, soil, harvest process, age and cultivars. Among the parameters measure on sugarcane juice, color stands out as the most important given the difficulty of its removal, leading to an increase in process cost of crystal sugar (Nguyen et al. 2015; Sartori et al., 2017a; Sartori et al., 2015b; Sartori et al., 2017c). Usually, the low color in crystal sugar means that its value will be high (Clarke and Godshall, 1988). There are four color-causing agents in cane sugar processes: plant pigments (chlorophylls, xanthophylls and anthocyanins), Maillard´s reaction, caramelization and alkaline fructose degradation products (Rein, 2007). In general, the sugarcane color is related to enzymatic reactions with phenolic compounds naturally in sugarcane (Nguyen and Doherty, 2012). Phenolic compounds have a hydroxyl group attached to an aromatic ring and are classified as: simple phenols or polyphenols, according to number of phenol unities. In the sugarcane juice, they are in a form of phenolic acids as hydroxycinamic acids (for example caffeic and sinapinic acids) and flavonoids (for example, vitexin and diosmetin-8-C-glycoside) (Abbas et al., 2014; Duarte-Almeida et al., 2006; Colombo et al., 2006), however, pigmentation was more related to polymerized phenolic compounds than simple phenolic compounds with low molecular weight (Clarke and Godshall, 1988). In Brazilian sugarcane mills (world's largest producer of sugar), sulfitation is the technique widely used for color reduction during the white crystal sugar manufacturing, even though it has many operational limitations, such as environmental problems (for example, atmospheric pollution and acid rain), workplace security, difficulty of process adjustment and sulfur residues in sugar above European, Canadian and North American market legislation. In this way, there are barriers to commercialization of Brazilian crystal white sugar, then Brazil has been exporting basically VHP sugar (very high polarization), which is a raw sugar with low added value (Morilla et al., 2015; Morilla et al., 2016; Hugot, 1986; Rein, 2007). Our group has been evaluating some alternative techniques to replace sulfur 3 as clarifying agent in the sugarcane juice process, like ozone (Sartori et al., 2017), hydrogen peroxide (Sartori et al., 2017; Mandro et al., 2017) and ionizing radiation (Lima et al., 2016). Electrocoagulation emerges as a possible substitute for sulfitation due to its success in potable water and wastewater treatment (tannery, electroplating, dairy, textile and oil industries) (Chen, 2004; Tak et al., 2015). The process is based on the production of coagulating agents in situ by electrical charge; such coagulants can aggregate impurities in the solution and remove them by flotation and/or sedimentation. The kind of coagulants depends on the type of electrode (Garcia-Segura et al., 2017). Aluminum is widely used once it has a low acquisition price, high availability, presents higher effectiveness to remove organic carbon and greater durability than iron (another typical electrode material) (Asha and Kumar, 2016; Souza et al., 2016; Emamjomeh and Sivakumar, 2009). Electrocoagulation with aluminum electrode presents the half-reactions represented by Equations 1, 2 and 3. Al → Al3+ + 3 e- (1) 2 H2O + 2e- → H2 (g) + 2 OH- (2) Al3+(aq) + 3 H2O → Al(OH)3 + 3 H+(aq) (3) The half-reaction of Equation 1 happens at the anode when as electrical charge is applied. Meanwhile, Equation 2 occurs at the cathode. The Al3+ generated in the solution spontaneously hydrolyses and forms monomeric species (Al(OH)3). Eventually, Al(OH)3 can polymerize in Al2(OH)24+, Al6(OH)153+, Al7(OH)174+, Al8(OH)204+, Al13O4(OH)247+ and Al13(OH)345+, although the main coagulant is Al(OH)3 (Garcia-Segura et al., 2017; Gurses et al., 2002). Electrocoagulation has four most important mechanisms to remove organic material: a) entrapment (trapping impurities with coagulant), b) adsorption (similar to entrapment, however, there are physico-chemical interactions), c) charge neutralization (coagulant blocks the free charges of impurities) and d) complexation (coagulant binds to functional groups of impurities, evolving the molecule) (Nassef, 2014; Garcia-Segura et al., 2017). Those mechanisms normally happen simultaneously and they are efficient to remove impurities 4 including phenolic compounds as showed by Fajardo et al. (2014) who treated a synthetic mixture of gallic and 4-hydroxybenzoic acids. Another sugarcane sector lag is the juice color monitoring. In sugarcane mills, the methodology is based on laboratory analysis established by Icumsa (International Commission for Uniform Methods of Sugar Analysis), where absorbance at 420 nm variation is measured (Icumsa, 2011). However, the colorant in sugarcane is very heterogeneous and it is not indicated to be monitored using one wavelength. For example, the reaction of complexation of cathecol and iron that happens in sugarcane juice is not sensitive at 420 nm (Clarke and Godshall, 1988). Icumsa also had an official measurement at 520 nm and there are some works that used this methodology (Chai et al., 2015, Pacheco et al., 2012). Still, there are some recommendations of analysis at 280 nm, once most colorants have their peak at this wavelength (Clarke and Godshall, 1988). Despite their usage, all absorbances mentioned above have problems with influence of pH in the sugarcane juice color and depending on the acidity the solution can be lighter or darker. To date, the use of chromatic variations has been scarcely applied to evaluate sugarcane juice clarification, especially for alternative processes such as electrocoagulation. Considering the demand to replace the current sugarcane juice clarification process (sulfitation) and the need to evolve the color monitoring system during the white crystal sugar manufacturing, this work aimed to evaluate the efficiency of electrocoagulation as an alternative clarification process focusing on the technological parameters normally evaluated in the industry (Icumsa color and turbidity), phenolic compounds content and CIELAB color parameters. Then it was aimed to determine which color analyses are fundamental and critical for monitoring sugarcane changes. 2. Material and Methods 2.1 Sugarcane samples and juice extraction Sugarcane plants were harvested randomly from Fazenda Areão located in Piracicaba/SP, Brazil (22°43'30" S, 47°38'56" W). Sugarcane juice was obtained from healthy sugarcane plants. They 5 were disintegrated in forage and pressed (1 min at 250 kgf cm-2) to obtain raw sugarcane juice, which was sifted against sieves at 200 mesh to remove insoluble impurities, sand and fibers. The juice was immediately stored at -18 °C until the electrocoagulation assays. 2.2 Treatment of sugarcane juice by electrocoagulation Electrocoagulation process was carried out in a 400 mL glass reactor with a jacket. It was coupled to a thermostated water-bath, which kept the reaction temperature constant. A Pump-King (HP 1/30, 1550 rpm, 60 cycles) transferred water from the water-bath to the reactor. The electrodes were two aluminum plates (15 × 3.5 × 2 cm) and were disposed in vertical position with 80% of its area emerged in sugarcane juice. A transformer (Superior Electric, Connecticut, USA) was used to supply energy to electrodes (0-120 V; 15 A) (Figure 1). Four kinetics of color reduction from sugarcane juice were carried out with 300 mL of it. The parameter conditions were set according to preliminary tests and closer to the sugarcane mills’. Temperature, pH, distance between electrodes and soluble solids (Brix) were fixed at 40 ºC (sugarcane juice temperature before entering the treatment during crystal sugar manufacturing), 5.5 (sugarcane juice normal pH), 1 cm (determined in previous tests) and 18º Brix (similar to sugarcane juice). The essays were divided according to the voltage applied: 35, 45, 55 and 65 V (also based on previous tests). The process had a duration of 1 h and 5 mL samples of clarified sugarcane juice were collected every 10 min with a syringe (the process did not need to stop). After that they were centrifuged to remove the supernatant and sludge formed during electrocoagulation. Then the clarified sugarcane juice samples were stored at -18 °C until the analyses of turbidity, Icumsa color, total and simple phenolic compounds and tristimulus colorimetry. 2.3 Technological parameters Icumsa color and turbidity are parameters normally monitored in the sugarcane mill and both were analyzed as part of technological parameters. To measure Icumsa color, sugarcane juice was diluted to 1.25º Brix. The samples were filtered through PTFE membrane (0.45 µm) and the pH was adjusted to 7.0 using NaOH (0.05 mol L-1). Then the absorbance was measured at 420 nm using 6 spectrophotometer (UVmini 1240, Shimadzu Co., Japan). The final result was expressed as dimensionless Icumsa color using Equation 4, where: ρ is the samples’ density (determined for Equation 5); Brix0 is the initial soluble solids content; Brixc is the soluble solids content of diluted samples and Abs420nm is absorbance at 420 nm (ICUMSA, 2011). Icumsa color = [(Abs420nm × 1000) / ( × (Brixc/100)] (4)  = 1 + [((Brix0 × (200 + Brix0)) / 54000) × (Brixc/Brix0)] (5) Turbidity was measured directly in turbidimiter TB-10000 (Tecnopom Co., Brazil) and the equipment expressed the results in nephelometric turbidity units (NTU). Icumsa color and turbidity of the four treatments (35, 45, 55 and 65 V) were discussed in percentage of reduction. The initial values (at 0 min) were considered 0% of reduction, and then the evolution of both parameters was in function of the beginning. 2.4 Phenolic compounds by UHPLC 2.4.1 Extraction procedure 1 mL of sugarcane juice was acidified to pH 2 with HCl (37% w/v) and the extraction of phenolic compounds was carried out with solid-phase cartridges. It was used cartridges with polypropylene filling (XAD-2 Purified Clean - SPE Tube; Supelco) and methanol as eluent. After that, the material was dried in rotary evaporator. It was added 500 µL of ultrapure water and 500 µL of ethyl acetate. The material was homogenized in ultrasound and centrifuged for 5 min at 17.970 × g to collect the supernatants. The procedure was repeated three times. Then, the supernatant was dried in rotary evaporator and the extract was suspended with 200 µL of methanol and filtered at 0.45 µm prior to their injections in the Ultra-High Performance Liquid Chromatography (UHPLC) system. 2.4.2 UHPLC-DAD Chromatography analyses were performed in duplicate using UHPLC Agilent 1290 system with diode-array detector (DAD), which was set to scan from 200 to 770 nm. The methodology covered 7 flavonoids and phenolic acids with low molecular weight. The separation was performed using C18 Eclipse Plus 120 column (1.8 μm, 50 × 2.1 mm) at 20°C. The mobile phase profile was: formic acid/water at 0.01% proportion (A) and acetonitrile (B). The linear gradient was: 0-5 min: 100% A; 5- 20 min: 95% A−5% B; 20−22 min: 50% A−50% B; 22 min: washing and re-equilibration of the column. The injection volume and flow rate were 1 µL and 0.70 mL min-1, respectively. The UV-Vis detector was monitoring 280 nm with the software Open Lab ChemStation. The identification of compounds was executed by external calibration from the areas of the chromatographic peaks obtained by DAD detection at 280 nm (Coyago-Cruz et al., 2018). 2.4.3 Total phenolic compounds by spectrophotometric Folin-Ciocalteu method Concentration of the total phenolic content was determined by Folin-Ciocalteu reagent according to Singleton and Rossi (1965). Briefly, sugarcane juice collected from the treatment (0.25 mL), Folin-Ciocalteu reagent (1.25 mL) and sodium carbonate 20% (m/v; 3.75 mL) were mixed with distilled water to a total volume of 25 mL. The solution was homogenized and left settling during two hours for reaction and stabilization. Then absorbance was measured at 795 nm in spectrophotometer Hewlett–Packard UV-Vis HP8453 (Palo Alto, CA, USA), using 10 mm path length glass cells and distilled water as reference. The standard curve was made with gallic acid and the results were expressed in mg GAE L-1. As was done previously with turbidity and Icumsa color, the results of total phenolic compounds was discussed in percentage of reduction. 2.5. CIELAB color parameters The kinetics samples were scanned by CAS-140 spectroradiometer covering visible wavelength of light (380−770 nm). The standard illuminant and observer was D65 and 10°, respectively. A plastic cuvette (45 × 12.5 mm) was used against a reference white background (distilled water). Each sample was read three times consecutively, and then colorimetric measurements were the average. The color parameters of space CIELAB (L* - lightness, a* - green-red axis, b* - blue-yellow 8 axis, Cab* - chroma and hab - hue) were obtained directly from the spectroradiometer as described by Gordillo et al. (2015). Moreover, euclidean distance between two points in three-dimensional space defined by L*, a* and b* were calculated to define the color differences (Equation 6). ΔE*ab = [(ΔL*)² + (Δa*)² + (Δb*)²]½ (6) 2.6. Statistical analysis Statistica version 8.0 software (Statsoft, 2007) was used for statistical analysis. Univariate analyses (F test, Tukey test and Pearson correlation coefficient) were used to discriminate means of turbidity, Icumsa color, total phenolic compounds and colorimetric parameters in each treatment. 3. Results and discussions 3.1. Turbidity and Icumsa color monitoring Each result of the four electrocoagulation essays (35, 45, 55 and 65 V essays) was compared. The analysis of variance (ANOVA) was executed to verify the existence of significant difference of Icumsa color and turbidity means between the four essays. Since the F-test was significant, it was carried out a comparison of means test (Tukey test) to identify which means of treatment differ. Each voltage tested (35, 45, 55 and 65 V essay) reduced turbidity after one hour (Table 1). The turbidity reduction was directly proportional to the tension applied. At the end of 35 V essay, the turbidity reduction was 11.9%. This was the only treatment that did not present significance turbidity reduction (p < 0.05) throughout the process. 45, 55 and 65 V essays had significance results and presented 44.6, 83.0 and 99.7% turbidity reduction, respectively. Another tendency was the higher voltage treatments (55 and 65 V) decreased the turbidity faster than lower voltage treatments. 55 and 65 V essays in the first 10 min presented a reduction of 81.3 and 44.8% (both significant at (p < 0.05) against 2.4 and 1.1% from 45 and 35 V essays (none of them significant at (p < 0.05). Furthermore, 45 V essay presented a significant reduction in the turbidity, only after 40 min of electrocoagulation. 15 4. Conclusion The electrocoagulation treatment significantly reduced Icumsa color, turbidity and total phenolic compounds. Higher voltages applied accomplished better results of sugarcane juice clarification. However, all treatments did not influence in simple phenolic content analyzed in this work. 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Time (min) Turbidity Icumsa color 35 V 45 V 55 V 65 V 35 V 45 V 55 V 65 V 0 0.0% ± 0.438%a 0.0% ± 0.516%a 0.0% ± 0.397%a 0.0% ± 0.749%a 0.0% ± 1.1%AB 0.0% ± 1.1%AB 0.0% ± 1.1%AB 0.0% ± 1.3%AB 10 1.1% ± 0.130%a 2.4% ± 0.348%a 44.8% ± 0.102%bcde 81.3% ± 0.045%de 2.8% ± 11.2%ABC -3.0% ± 10.3%A 8.8% ± 3.3%ABCDE 10.3% ± 4.6%ABCD 20 3.5% ± 0.085%ab 35.3% ± 0.050%abcd 51.5% ± 0.102%bcde 78.9% ± 0.000%de 0.0% ± 5.9%A -10.0% ± 3.6%A 52.2% ± 12.2%EFG 39.3% ± 8.3%BCDEFG 30 6.4% ± 0.130%abc 44.9% ± 0.217%bcde 48.7% ± 0.088%bcde 75.7% ± 0.278%de -8.0% ± 7.2%A 6.9% ± 4.3%ABCD 51.7% ± 1.8%EFG 47.1% ± 10.8%DEFG 40 9.3% ± 0.130%abc 51.5% ± 0.050%bcde 54.9% ± 0.102%cde 99.9% ± 0.001%e -5.0% ± 4.5%A 16.5% ± 11.5%ABCDEF 39.7% ± 7.2%CDEFG 70.4% ± 1.3%G 50 12.0% ± 0.345%abc 49.3% ± 0.537%bcde 71.4% ± 0.134%de 99.9% ± 0.002%e -9.0% ± 13.3%A 18.1% ± 3.0%ABCDEF 50.9% ± 12.6%EFG 69.8% ± 5.9%G 60 11.9% ± 0.385%abc 44.6% ± 0.086%bcde 83.0% ± 0.051%de 99.7% ± 0.008%e -3.0% ± 2.1%A 17.5% ± 40.8%ABCDEF 52.3% ± 14.4%EFG 58.7% ± 1.7%FG Turbidity values and standard deviation with different lower case are statistically different by Tukey test ( < 0.05). Icumsa color values and standard deviation with different capital letter are statistically different by Tukey test ( < 0.05). 23 Table 2. Concentration and standard deviation of individual phenolic compounds, benzoic acids, hydroxycinnamic acids, flavonas and simple phenolic expressed in mg L-1 identified during the electrocoagulation treatments (35, 45, 55 and 65 V). Phen. Comp mg L-1 Treatments 35 V 45 V 55 V 65 V 0 min 30 min 60 min 0 min 30 min 60 min 0 min 30 min 60 min 0 min 30 min 60 min Gallic acid 1.05± 0.00 1.58± 0.06 1.60± 0.03 1.03± 0.03 0.94± 0.03 1.66± 0.00 0.59± 0.03 0.77± 0.03 0.57± 0.06 0.88± 0.00 0.39± 0.12 1.23± 0.12 p- Hydroxyben zoic acid 1.91± 0.00 1.43± 0.04 1.62± 0.08 1.51± 0.16 1.37± 0.04 1.03± 0.04 1.34± 0.00 1.03± 0.03 0.43± 0.00 1.57± 0.00 1.20± 0.04 0.43± 0.00 Syringic acid 8.06± 0.13 7.08± 0.10 8.35± 0.04 6.13± 0.03 7.24± 0.00 6.36± 0.80 6.07± 0.06 6.93± 1.22 7.26± 0.16 7.83± 0.06 8.35± 0.16 9.89± 0.03 Vanillic acid 3.47± 0.03 2.09± 0.43 2.76± 0.00 1.91± 0.09 1.38± 0.39 1.78± 0.09 1.54± 0.26 0.74± 0.00 5.68± 0.30 1.84± 0.17 0.65± 0.04 2.70± 0.09 ∑Benz acid 14.5± 0.16 12.2± 0.51 14.3± 0.07 10.6± 0.29 10.9± 0.46 10.8± 0.93 9.6± 0.33 9.5± 1.28 13.9± 0.52 12.1± 0.23 10.6± 0.20 14.3± 0.24 p-Coumaric acid 9.56± 0.06 8.15± 0.04 10.8± 0.11 6.75± 0.18 8.40± 0.05 7.95± 0.37 7.53± 0.18 7.41± 0.56 7.19± 0.11 8.03± 0.00 8.63± 0.20 8.95± 0.06 Ferulic acid 1.87± 0.57 1.26± 0.14 1.06± 0.14 1.87± 0.14 0.00± 0.00 0.00± 0.00 ND ND ND 2.42± 0.21 1.16± 0.00 2.63± 0.07 ∑Hydroxyc. acid 11.4± 0.62 9.4± 0.18 11.9± 0.25 8.6± 0.33 8.4± 0.05 7.9± 0.37 7.5± 0.18 7.4± 0.56 7.2± 0.11 10.5± 0.21 9.8± 0.20 11.6± 0.13 Apigenina 0.85± 0.00 0.00± 0.00 0.00± 0.00 0.00± 0.00 0.32± 0.04 0.00± 0.00 0.73± 0.06 0.41± 0.13 0.00± 0.00 1.64± 0.04 1.03± 0.00 0.00± 0.00 Naringenina 2.35± 0.00 2.31± 0.11 1.36± 1.40 2.37± 0.03 2.45± 0.03 2.29± 0.03 2.37± 0.03 2.5± 0.11 2.45± 0.08 2.99± 0.06 3.14± 0.17 3.01± 0.08 Quercetin- o-glucoside ND ND ND ND ND ND ND ND ND 0.80± 0.02 0.00± 0.00 1.07± 0.00 ∑Flav. 3.2± 0.00 2.3± 0.00 1.4± 1.4 2.4± 0.03 2.8± 0.07 2.3± 0.03 3.1± 0.08 2.9± 0.24 2.5± 0.08 5.4± 0.12 4.2± 0.17 4.1± 0.08 Simple Phen. 29.1± 0.78 23.9± 0.57 27.5± 0.18 21.6± 0.64 22.1± 0.58 21.1± 0.93 22.0± 0.74 19.8± 2.08 23.6± 0.71 28.0± 0.56 24.5± 0.57 29.9± 0.45 ND - not detected. ∑Benz. acid - sum of concentration of benzoic acids (gallic, p-hydroxybenzoic, vanillic and syringic acids) ∑Hydroxyc. acid - sum of concentration of hydroxycinnamic acids (caffeic, p-Coumaric and ferulic acids) ∑Flav. - sum of concentration of flavonas (apigenina, naringenina and quercetin-o-glucoside) ∑Simple phen - sum of concentration of all individual phenolic compounds identified 24 Table 3. Concentration and standard deviation of total phenolic compounds in percentage of reduction (%) during electrocoagulation treatment. Time (min) Total Phenolic Compounds 35 V 45 V 55 V 65 V 0 0.0% ±1.2%AB 0.0% ± 0.6%AB 0.0% ± 0.1%AB 0.0% ± 0.1%AB 10 9.1% ± 1.0%ABCD -15% ± 2.4%A 11.9% ± 1.8%BCDE 19.2% ± 0.4%BCDEF 20 17.0% ± 0.9%BCDEF 5.5% ± 1.9%ABCD 20.6% ± 1.5%BCDEFG 17.0% ± 2.4%BCDEF 30 12.4% ± 0.6%BCDE 9.7% ± 2.1%BCDE 15.8% ± 1.4%BCDEF 30.1% ± 1.2%EFGH 40 5.5% ± 0.3%ABCD 20.0% ± 2.4%BCDEFG 24.4% ± 2.2%CDEFGH 45.7% ± 1.5%H 50 4.0% ± 0.3%ABC 24.1% ± 0.5%CDEFGH 26.2% ± 0.6%CDEFGH 42.2% ± 2.1%GH 60 12.7% ± 0.6%BCDE 19.4% ± 4.9%BCDEF 27.5% ± 0.5%DEFGH 35.2% ± 1.5%FGH Total phenolic compounds values and standard deviation with different capital letter are statistically different by Tukey test ( < 0.05).