Optimization of Ultrasound Assisted Extraction of Bioactive Compounds from Apple Pomace

molecules Article Optimization of Ultrasound Assisted Extraction of Bioactive Compounds from Apple Pomace Itziar Egüés1,2, Fabio Hernandez-Ramos 2, Iván Rivilla 1,3 and Jalel Labidi 1,2,*   Citation: Egüés, I.; Hernandez-Ramos, F.; Rivilla, I.; Labidi, J. Optimization of Ultrasound Assisted Extraction of Bioactive Compounds from Apple Pomace. Molecules 2021,26, 3783. https:// doi.org/10.3390/molecules26133783 Academic Editors: Francesco Cacciola and Piotr Paweł Wieczorek Received: 2 May 2021 Accepted: 18 June 2021 Published: 22 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Ma+D-Waste Recovery Engineering, Edificio Joxe Mari Korta—Avda Tolosa 72, 20018 Donostia-San Sebastián, Spain; itziar[email protected] (I.E.); [email protected] (I.R.) 2 Biorefinery Process Research Group Chemical and Environmental Engineering Department, University of the Basque Country (UPV/EHU), Plaza Europa, 1, 20018 Donostia-San Sebastian, Spain; [email protected] 3IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain *Correspondence: [email protected] Abstract: In the present work, the optimization of the extraction of antioxidant compounds from apple pomace using ultrasound technology as an environmentally friendly and intensification process was developed. Different sonication powers, extraction temperatures and extraction times were studied and their influence on extraction yield and characteristics of the extracted samples (total phenolic compounds, flavonoid content and antioxidant capacity) are presented. The elaborated experimental design and the analysis of Pareto and response surface diagrams allowed us to determine the optimal extraction conditions. The conditions that allow the maximum extraction of phenolic compounds were found at 20 min, 90 ◦ C and 50% ultrasound amplitude. Nevertheless, at these conditions, the antioxidant capacity measured by DPPH decreased in the extracted samples. Keywords: ultrasound; optimization; apple pomace; antioxidant capacity 1. Introduction Waste generation and management is one of the main concerns in the industry. The waste generated in the food sector is generally rich in water and organic compounds and therefore these by-products can cause environmental problems. The manufacturers face additional costs for the adequate disposal or treatment of their residues. However, food waste could be a potential raw material for the extraction of valuable products. It is cheap and produced in large quantities as by-products. In this aspect, more comprehensive studies about its valorizations could lead to economic and environmental benefits [1]. Cider is an alcoholic beverage produced by the total or partial fermentation of apple juice. The manufacture of cider is carried out annually and Spain is the second largest producer in the European Union after the United Kingdom [ 2 ]. In 2017, the cider industry used more than 1,000,000 tons of apples during its production [ 2 ]. To produce cider, apples are harvested after the summer and then are pressed for several days. After pressing, apple pomace waste is generated, which is approximately 25–35% of the initial weight of the apples [ 3 , 4 ]. Then, the juice obtained is transferred to a vat where it undergoes spontaneous fermentation. As it can be seen, apple pomace is a waste generated in large volume during the cider manufacturing process. Therefore, new processes for its valorization are currently being investigated [4]. Apple pomace is composed of a mixture of pulp, epidermis, seeds and stems [ 5 ]. Besides water, this mixture contains mainly carbohydrates, phenolic compounds, proteins, fat, ash and crude fiber [ 6 ]. However, its composition varies according to the origin and the degree of maturation [ 7 ]. In the literature, the composition of the apple pomace covers a wide range of values (% on a dry basis): cellulose 7.2–43.6% [ 8 ], hemicellulose 4.26–29.90% [ 7 , 8 ], lignin 15.3–23.5%, pectins 3.5–14.32% [ 8 ], proteins 2.9–5.7% [ 8 ], lipids Molecules 2021,26, 3783. https://doi.org/10.3390/molecules26133783 https://www.mdpi.com/journal/molecules Molecules 2021,26, 3783 2 of 14 1.20–3.9% [ 8 ] and ashes 1.47–1.66% [ 7 ]. Therefore, apple pomace is considered as a good source of fiber, pectin and phenolic compounds [9]. Some studies have used apple pomace as a source of high value-added products [ 4 , 9 ] for production of lactic acid and citric acid [ 8 ] and the production of sustainable biomaterials [ 10 ], among others. The extraction of bioactive compounds could be another interesting route. In fact, some studies indicated a possible adverse effect of the consumption of synthetic antioxidants; therefore, the extraction of antioxidant compounds from natural resources could be a promising route [ 11 ]. Recently, the potential application of apple pomace for antioxidant extraction was reported in several works [6,12–15]. Phenolic compounds like quercetin, catechin, phloridzin, gallic acid and chlorogenic acid can reduce chronic disease risk [ 13 ]. Leyva-Corral et al. (2016) [ 6 ] reported the presence of chlorogenic acid, epicatechin, rutin and phloridzin as phenolic compounds in apple pomace and their antioxidant capacity was assessed. Pingret et al. (2012) [ 12 ] also reported the chemical composition of polyphenols extracted from apple pomace, where catechin monomers and phenolic acids with antioxidant capacity were found. Ferrentino et al. (2018) [ 14 ] also reported the presence of phloridzin, epicatechin and quercetin and their antioxidant capacity was evaluated. In this context, the use of apple pomace as a source of phenolic compounds with antioxidant activity, particularly flavonoids, could be a promising route for its valorization, as they remain in the pulp during the cider production process [ 16 , 17 ]. In the literature, the extraction of phenols from apple pomace has been performed by different technologies. Among conventional methodologies, solid-liquid extraction, stirring and shaking [ 18 ] and the use of different organic solvents, namely methanol, acetone and ethanol [ 19 , 20 ] can be found. The optimization and intensification process are crucial steps for the development of a feasible process at industrial scale. In addition, the implementation of green technologies and the use of more environmentally friendly solvents are required. In this context, previous studies have shown that water could be a suitable extraction solvent able to extract phenolic compounds from apple pomace [17]. As a new environmentally friendly and intensification process, sonication-assisted extraction has gained attention. Ultrasound-assisted extraction is an effective and environmentally friendly method for bioactive compound extraction [ 21 , 22 ]. Its application could be an efficient alternative to traditional techniques, since it can improve the productivity, yield (as it increases the contact area between the solid and liquid phase) and selectivity of the process, reducing the cost of extraction operations [ 23 , 24 ]. Ultrasonic probes can be used for the application of ultrasound, where they can be immersed in the solution containing the sample for the extraction of the desired component. Ultrasonic probes have some advantages over an ultrasonic bath, because the ultrasound probe produces more energy than a conventional ultrasound bath. This technology can accelerate chemical reactions [ 25 ]. In recent studies, ultrasound technology has also gained attention for antioxidant extraction from apple pomace [12]. Therefore, the objective of this work was the optimization of the ultrasound-assisted extraction of antioxidant compounds from apple pomace using water as a solvent. The aim was to find the optimum conditions that allow a better extraction of valuable compounds such as antioxidants using environmentally friendly and intensification technologies. For that, we show the effect of time, temperature and sonication power on the extraction yield and the characteristics of the extracted samples (total phenolic compounds, flavonoids content and antioxidant capacity). For a better correlation and interpretation of the obtained results, different analyses and representations of Pareto and surface response diagrams were performed. 2. Results 2.1. Composition of Apple Pomace The apple pomace we used had the following chemical composition (% on a dry basis): α -cellulose: 21 ± 0.5, hemicelluloses: 23 ± 0.5, klason lignin: 19 ± 0.4, pectins: 15.0 ± 0.5, proteins: 5.00 ±0.3, lipids: 5.55 ±0.3, and ashes content: 1.7 ±0.1. Molecules 2021,26, 3783 3 of 14 As described in the introduction section, the composition of the apple pomace is highly variable (% on a dry basis): cellulose 7.2–43.6% [ 8 ], hemicellulose 4.26–29.90% [ 7 , 8 ], lignin 15.3–23.5%, pectins 3.5–14.32% [ 8 ], proteins 2.9–5.7% [ 8 ], lipids 1.20–3.9% [ 8 ] and ashes 1.47–1.66% [ 7 ]. The characterization techniques used, as well as the origin of the apple pomace, may explain the variability of the results. In this work, the percentages found were in the range seen in the literature. Therefore, the apple pomace we used demonstrates its suitability for the extraction of biocomponents. 2.2. Effect of Extraction Time and Ultrasound Amplitude on TPC, TFC and AC Table 1summarizes the content of polyphenols (TPC), total flavonoids (TFC) and the antioxidant capacity (AC) per gram of dried apple pomace, extracted at 40 ◦ C at different times and at different ultrasound amplitudes (50% and 70%). Table 1. Content of phenolic compounds (TPC), total flavonoids (TFC) and antioxidant capacity (using DPPH assays) for different extraction times and ultrasound power. Amplitude (%) Time (min) TPC (mg GAE/g *) TFC (mg CE/g *) AC (DPPH) (mg TE/g *) 50 5 3.07 ±0.16 2.20 ±1.12 1.29 ±0.32 10 3.51 ±0.22 1.66 ±0.21 1.39 ±0.13 15 3.10 ±0.70 1.92 ±0.12 1.36 ±0.27 20 3.29 ±0.43 1.88 ±0.15 1.36 ±0.13 70 5 2.88 ±0.14 1.60 ±0.30 1.09 ±0.27 10 3.00 ±0.30 1.76 ±0.18 1.29 ±0.20 15 3.54 ±0.33 2.37 ±0.31 1.11 ±0.14 20 3.61 ±0.57 2.34 ±0.17 0.92 ±0.23 * Values presented are the average of triplicate measurements. As it can be seen, the TPC varies from 2.88 to 3.61 mg GAE/g and TFC ranges between 1.60–2.37 mg CE/g. The antioxidant capacity measured in the extracted liquors ranged from 0.92 to 1.39 mg TE/g. Çam et al. (2010) [ 26 ] showed total phenolic levels between 2.23 and 6.18 mg GAE/g using ultrasound extractions and water as solvent. However, Candrawinata et al. (2015) [ 17 ] showed lower values of TPC between 0.83 and 1.26 mg GAE/g and antioxidant activity between 1.00 and 1.65 mg TE/g (by DPPH). In this work, the obtained experimental values showed acceptable levels. On the other hand, Hernández-Carranza et al. (2016) [ 27 ] obtained a flavonoid content between 4.55 and 6.63 mg of CE/g of apple pomace, using conventional extraction methods (magnetic stirred hot plate and constant agitation of 3–12 h). The values obtained in this study (1.60 to 2.34 CE/g) were lower than those reported in literature. These differences could be affected by the extraction technique used, the extraction time, and the origin of the raw material used in the different studies. The adequacy of the results was verified by analysis of variance (ANOVA). The Pareto diagrams obtained in the ANOVA analysis are shown in Figure 1. As it can be noted, the effect of the amplitude affects the antioxidant activity measured by the DPPH assay. On the other hand, it should be noted that the TPC and TFC were not significantly affected by any of the variables studied. This behavior was verified by studying the adequacy of the model through the study of the F-ratio whose significance can be evaluated using the p-value establishing a confidence level of 95%. Thus, a p-value under 0.05 implies that the studied effect significantly affects the response. The values of the F-ratio and p-values obtained using the Statgraphics software are shown in Table 2. As can be seen, only the independent variable amplitude had a significant effect on the antioxidant activity (DPPH), confirming the aforementioned. Molecules 2021,26, 3783 4 of 14 Molecules 2021, 26, x FOR PEER REVIEW 4 of 15 Figure 1. Standardized Pareto diagram of the screening for (a) TPC, (b) TFC, and (c) DPPH. This behavior was verified by studying the adequacy of the model through the study of the F-ratio whose significance can be evaluated using the p-value establishing a confidence level of 95%. Thus, a p-value under 0.05 implies that the studied effect significantly affects the response. The values of the F-ratio and P-values obtained using the Statgraphics software are shown in Table 2. As can be seen, only the independent variable amplitude had a significant effect on the antioxidant activity (DPPH), confirming the aforementioned. Table 2. Adequacy of the model through the analysis of F-ratio and p-value. TPC TFC DPPH F-Ratio p-Value F-Ratio p-Value F-Ratio p-Value Amplitude (A) 0.01 0.9303 0.35 0.5951 19.72 0.0212 Time (B) 4.45 0.1255 1.9 0.2622 1.05 0.3816 AB 3.08 0.1775 5.21 0.1067 3.05 0.1793 BB 0.23 0.6674 0.2 0.6844 4.83 0.1154 Furthermore, Figure S1 (supplementary data) shows the impact of variables on different responses. According to the figures it can be concluded that the longer the extraction time, the more the responses (TPC, TFC, DPPH) increased, while increasing the amplitude decreased the properties of the extracts. Considering these results, we established an amplitude of 50% to carry out further experimental design, as shown in Table 3. Figure 1. Standardized Pareto diagram of the screening for (a) TPC, (b) TFC, and (c) DPPH. Table 2. Adequacy of the model through the analysis of F-ratio and p-value. TPC TFC DPPH F-Ratio p-Value F-Ratio p-Value F-Ratio p-Value Amplitude (A) 0.01 0.9303 0.35 0.5951 19.72 0.0212 Time (B) 4.45 0.1255 1.9 0.2622 1.05 0.3816 AB 3.08 0.1775 5.21 0.1067 3.05 0.1793 BB 0.23 0.6674 0.2 0.6844 4.83 0.1154 Furthermore, Figure S1 (supplementary data) shows the impact of variables on different responses. According to the figures it can be concluded that the longer the extraction time, the more the responses (TPC, TFC, DPPH) increased, while increasing the amplitude decreased the properties of the extracts. Considering these results, we established an amplitude of 50% to carry out further experimental design, as shown in Table 3. Molecules 2021,26, 3783 5 of 14 Table 3. Experimental variables of the experimental design. Variable Designation Units Nomenclature Value Fixed Amplitude (%) 50 Solid-liquid ratio (w/v) 1:40 Solvent water Particle size µm <250 Independent Temperature (◦C) Temp 65–90 Time (min) t 10–20 Dependent Total phenolic content (mg GAE/g) TPC YTPC Total flavonoid content (mg CE/g) TFC YTFC Antioxidant capacity (mg TE/g) DPPH YDPPH 2.3. Optimisation of the Extraction Conditions Table 4summarizes in detail the set of experiments obtained using the Statgraphic software. In addition, it also includes the experimental values corresponding to the dependent variables YTPC, YTFC, and YDPPH, and the extraction yield. Table 4. Set of experiments and experimental results. Exp. Independent Variables Normalized Variables Dependent Variables Yield (%) Temperature(◦C) Time (min) X1X2YTPC (mg GAE/g *) YTFC (mg CE/g *) YDPPH (mg TE/g *) 1 40 10 −1−1 3.51 ±0.22 1.66 ±0.21 1.39 ±0.13 24.8 ±0.07 2 40 15 −1 0 3.10 ±0.70 1.92 ±0.12 1.36 ±0.27 28.6 ±0.10 3 40 20 −1 1 3.29 ±0.44 1.88 ±0.15 1.36 ±0.13 29.5 ±1.10 4 65 10 0 −1 4.68 ±0.36 3.16 ±1.08 0.69 ±0.38 39.5 ±0.05 5 65 15 0 0 4.74 ±1.84 4.30 ±1.05 0.65 ±0.27 55.1 ±0.10 6 65 20 0 1 5.50 ±0.95 4.00 ±1.87 0.71 ±0.16 50.6 ±1.03 7 90 10 1 −1 5.75 ±0.72 4.59 ±0.23 0.61 ±0.11 50.6 ±0.05 8 90 15 1 0 6.02 ±0.28 4.40 ±0.72 0.53 ±0.17 58.4 ±1.15 9 90 20 1 1 6.02 ±0.29 3.53 ±0.82 0.68 ±0.14 38.7 ±1.50 * Values presented are the average of triplicate measurements. Regression coefficients together with their corresponding significance levels according to Student’s t-test, as well as the determination coefficient (R 2 ), fitted R 2 and statistical significance (Fisher’s F test) are summarized in Table 5. Table 5. Regression coefficients and statistical parameters of the experimental design. Regresion Coefficients YTPC YTFC YDPPH b04.86 a3.83 a0.64 a b11.31 a1.18 a−0.38 a b20.15 0.00 0.01 b12 0.12 −0.32 0.02 b11 −0.36 −0.67 0.31 a b22 0.17 −0.25 0.06 b R20.9731 0.9400 0.9900 R2fitted 0.9284 0.8600 0.9900 F-exp 21.7720 10.8585 187.0543 F-critical 0.0145 0.0387 0.0006 Significance level 98.54 96.12 99.94 a Significant coefficients at the 99% confidence level. b Significant coefficients from the 90% to 95% confidence level. As shown in Table 5, the determination coefficients R 2 obtained for each of the studied dependent variables Y TPC , Y TFC , and Y DPPH were 0.97, 0.94 and 0.99, respectively, indi- Molecules 2021,26, 3783 6 of 14 cating the validity of the design. These high determination coefficient values indicate that a small number of the total variations remain unexplained, concretely 0.03%, 0.06%, and 0.01% for Y TPC , Y TFC , and Y DPPH , respectively. Such values, together with the high F values, indicate that the selected model is adequate to represent the selected variables and the model result is statistically representative. According to the regression coefficients reported in Table 5, as well as in the Pareto diagrams presented in Figure 2, temperature (X 1 ) is the most independent variable that influences obtaining the phenolics, flavonoids and antioxidant capacity. On the other hand, it should be noted that the independent variable X 2 (time) did not exhibit a significant influence over any of the dependent variables. Regarding the quadratics effects (X 12 and X 22 ), it should be noted that they only showed significant influence on the antioxidant capacity of the obtained extracts. Molecules 2021, 26, x FOR PEER REVIEW 6 of 15 a Significant coefficients at the 99% confidence level. b Significant coefficients from the 90% to 95% confidence level. As shown in Table 5, the determination coefficients R 2 obtained for each of the studied dependent variables Y TPC , Y TFC , and Y DPPH were 0.97, 0.94 and 0.99, respectively, indicating the validity of the design. These high determination coefficient values indicate that a small number of the total variations remain unexplained, concretely 0.03%, 0.06%, and 0.01% for Y TPC , Y TFC , and Y DPPH , respectively. Such values, together with the high F values, indicate that the selected model is adequate to represent the selected variables and the model result is statistically representative. According to the regression coefficients reported in Table 5, as well as in the Pareto diagrams presented in Figure 2, temperature (X 1 ) is the most independent variable that influences obtaining the phenolics, flavonoids and antioxidant capacity. On the other hand, it should be noted that the independent variable X 2 (time) did not exhibit a significant influence over any of the dependent variables. Regarding the quadratics effects (X 12 and X 22 ), it should be noted that they only showed significant influence on the antioxidant capacity of the obtained extracts. F-critical 0.0145 0.0387 0.0006 Significance level 98.54 96.12 99.94 Figure 2. Standardized Pareto diagram for (a) TPC, (b) TFC, and (c) DPPH. In addition, the extraction yield (Table 4) increased as the temperature increased, obtaining the maximum yield of 58.44% at a temperature of 90 ◦ C, while the minimum yield of 24.8% was obtained at the lowest temperature (40 ◦C). 2.3.1. Influence of the Studied Variables The interaction of the independent variables and their influence on the TPC, TFC, and DPPH are shown in Figure 3. Molecules 2021,26, 3783 7 of 14 Molecules 2021, 26, x FOR PEER REVIEW 7 of 15 Figure 2. Standardized Pareto diagram for (a) TPC, (b) TFC, and (c) DPPH. In addition, the extraction yield (Table 4) increased as the temperature increased, obtaining the maximum yield of 58.44% at a temperature of 90 °C, while the minimum yield of 24.8% was obtained at the lowest temperature (40 °C). 2.3.1. Influence of the Studied Variables The interaction of the independent variables and their influence on the TPC, TFC, and DPPH are shown in Figure 3. Figure 3. Response surface diagrams showing optimal extraction time conditions and temperature against (a) TPC, (b) TFC, and (c) antioxidant properties. Total Phenolic Content (TPC) Different research indicated that the extraction of phenols from food waste was improved with the temperature and time [12,27]; however, heat degradation may occur at high temperatures. In this study, the TPC was strongly affected by the effect of the temperature, while time exhibited a low effect as indicated by the regression coefficient corresponding to the Y TPC dependent variable (Table 5). Figure 3a shows the response surface corresponding to the TPC as a function of the independent variables X 1 and X 2 . According to this figure, it can be appreciated that as the temperature (X 1 ) increases, the TPC increases independently of the extraction time used. Figure 3. Response surface diagrams showing optimal extraction time conditions and temperature against ( a ) TPC, ( b ) TFC, and (c) antioxidant properties. Total Phenolic Content (TPC) Different research indicated that the extraction of phenols from food waste was improved with the temperature and time [ 12 , 27 ]; however, heat degradation may occur at high temperatures. In this study, the TPC was strongly affected by the effect of the temperature, while time exhibited a low effect as indicated by the regression coefficient corresponding to the YTPC dependent variable (Table 5). Figure 3a shows the response surface corresponding to the TPC as a function of the independent variables X 1 and X 2 . According to this figure, it can be appreciated that as the temperature (X 1 ) increases, the TPC increases independently of the extraction time used. It is reported in the literature that the length of time for the extraction was also an important factor when the extraction is performed below the critical temperature [ 17 ]. In this work, the range studied (10, 15, 20 min) did not have strong influence. The TPC content of the experiments were in the range of 3.10 to 6.02 (mg GAE/g) as shown in Table 4. The lowest value corresponded to the experiment 2 (40 ◦ C and 15 min) while the highest value was obtained for the highest temperature and maximum extraction time in experiment 9 (90 ◦ C, 20 min). Although time has little influence, it can be observed that at the maximum temperature of 90 ◦ C and the longer extraction time, there was a slight improvement in the TPC content. This result is in agreement with Candrawinata et al. (2015) [17], who concluded that the TPC values increased with longer extraction times. Molecules 2021,26, 3783 8 of 14 Total Flavonoid Content (TFC) Similarly to TPC, the flavonoid content is mainly affected by the extraction temperature, while time has a low influence due to the low significance of its regression coefficient (Table 5). However, in this case, unlike TPC, the highest TFC value (4.59 mg CE/g) was obtained when the extraction time was 10 min (experiment 7). On the other hand, the lowest value was observed in experiment 1, at 40 ◦ C and 10 min, as can be shown in Table 4 and Figure 3b. Antioxidant Capacity (DPPH) In Figure 3c, it is shown how temperature (X 1 ) and time (X 2 ) variables affect the antioxidant capacity of the extracts. As in the previous cases, time has no significant influence, and the antioxidant capacity is only affected by the effect of temperature. However, in this case, temperature has a negative effect on the obtained response, i.e., at low temperatures, the antioxidant capacity was higher. Thus, the highest antioxidant capacity is obtained for experiment 1 (1.39 mg TE/g), while at 90 ◦ C and 15 min (experiment 8), the capacity is minimal (0.53 mg TE/g). As it can be seen, the temperature significantly improved all responses except the DPPH values, i.e., at higher extraction temperature, higher TPC was obtained; however, the antioxidant capacity measured by DPPH was lower. These conclusions may be contradictory, since some studies have shown a correlation between the content of phenolic compounds and the antioxidant capacity [ 17 , 28 ]. Nevertheless, it is important to highlight that the determination of the total polyphenol content by the Folin–Ciocalteu method can generate an overestimation of the measurements since it can react with other reducing compounds, as sugars [ 29 ]. On the other hand, some studies have reported that the antioxidant capacity decreased with temperature, because exposure of phenolic compounds to high temperature can cause their degradation [30]. 2.3.2. Optimization of the Extraction Conditions and Model Validation The objective of the optimization was to determine the best extraction conditions to provide the highest values of phenolic and flavonoid compounds and the greatest antioxidant capacity. For this purpose, the desirability function was used, employing the Statgraphics Centurion XVI software: TPC = 4.86117 + 1.31392T+ 0.1457t−0.36155T2+ 0.12155Tt + 0.1697t2(1) TFC = 3.82718 + 1.17562T−0.0005333t−0.666217T2−0.32105Tt −0.245167t2(2) DPPH = 0.64333 −0.381667T+ 0.01Tt + 0.305T2+ 0.025Tt −0.06t2(3) The optimum conditions for the studied independent variables were 90 ◦ C and the extraction time of 18.43 min. Under the cited optimum conditions, the estimated values were: TPC = 6.07 mg GAE/g, TFC = 4.00 mg CE/g and DPPH = 0.62 mg TE/g. 2.4. Relation between TPC and Antioxidant Capacity As commented before, there are studies that suggest the existence of a relationship between the total content of polyphenols and the antioxidant activity of the sample [ 17 , 28 ]. In Figure 4, the relationship between the content of phenolic compounds (Figure 4a) and flavonoids (Figure 4b) against antioxidant capacity determined by the DPPH method is shown. It can be observed that at higher concentrations of polyphenols and flavonoids, the antioxidant capacity measured by DPPH remained stable or decreased. Molecules 2021,26, 3783 9 of 14 Molecules 2021, 26, x FOR PEER REVIEW 9 of 15 The optimum conditions for the studied independent variables were 90 °C and the extraction time of 18.43 min. Under the cited optimum conditions, the estimated values were: TPC = 6.07 mg GAE/g, TFC = 4.00 mg CE/g and DPPH = 0.62 mg TE/g. 2.4. Relation between TPC and Antioxidant Capacity As commented before, there are studies that suggest the existence of a relationship between the total content of polyphenols and the antioxidant activity of the sample [17,28]. In Figure 4, the relationship between the content of phenolic compounds (Figure 4a) and flavonoids (Figure 4b) against antioxidant capacity determined by the DPPH method is shown. It can be observed that at higher concentrations of polyphenols and flavonoids, the antioxidant capacity measured by DPPH remained stable or decreased. Figure 4. Correlation between (a) TPC and (b) flavonoid content with antioxidant activity tested by DPPH assay. These results may be due to the fact that the quality of the extracted phenolics and flavonoids could be worse with the temperature and consequently, they could provide worse antioxidant capacity measured by DPPH. In addition, other reducing compounds could interfere in the Folin–Ciocalteu [29] and flavonoid tests. In order to observe the quality of the obtained samples, the antioxidant capacity of the samples is divided by the total phenols, and the results are shown in Table 6 (represented as Trolox equivalent/GAE). On the other hand, the radical inhibition capacity of the samples is also demonstrated. Table 6. The quality (represented as Trolox equivalent/GAE) and % of inhibition capacity of apple pomace extracted samples against the radical DPPH (after 15 min) and commercial antioxidant Trolox at 0.05 g/L of concentration. Time (min) Temperature (°C) Quality (TE/GAE) Inhibition (%) 10 0.40 43.84 15 40 0.44 42.94 20 0.41 42.99 10 0.15 22.62 15 65 0.14 21.22 20 0.13 23.35 10 0.11 20.52 15 90 0.09 17.70 20 0.011 22.97 Trolox 65.27 Figure 4. Correlation between (a) TPC and (b) flavonoid content with antioxidant activity tested by DPPH assay. These results may be due to the fact that the quality of the extracted phenolics and flavonoids could be worse with the temperature and consequently, they could provide worse antioxidant capacity measured by DPPH. In addition, other reducing compounds could interfere in the Folin–Ciocalteu [ 29 ] and flavonoid tests. In order to observe the quality of the obtained samples, the antioxidant capacity of the samples is divided by the total phenols, and the results are shown in Table 6(represented as Trolox equivalent/GAE). On the other hand, the radical inhibition capacity of the samples is also demonstrated. Table 6. The quality (represented as Trolox equivalent/GAE) and % of inhibition capacity of apple pomace extracted samples against the radical DPPH (after 15 min) and commercial antioxidant Trolox at 0.05 g/L of concentration. Time (min) Temperature (◦C) Quality (TE/GAE) Inhibition (%) 10 0.40 43.84 15 40 0.44 42.94 20 0.41 42.99 10 0.15 22.62 15 65 0.14 21.22 20 0.13 23.35 10 0.11 20.52 15 90 0.09 17.70 20 0.011 22.97 Trolox 65.27 As it can be seen, the reduction capacity shown by the compounds extracted from apple pomace at 40 ◦ C and 50% amplitude was around 40%, where this value decreased until 23% at 65 ◦ C and 90 ◦ C extraction temperature. These values indicate that the highest percentage of reduction was registered in the samples extracted at lower temperature, which is in agreement with the previous obtained results. On the other hand, the antioxidant capacity of the commercial antioxidant compound Trolox was slightly higher, about 65%. 2.5. Sugars Characterization of the Extracted Samples In Table 7, the sugar analysis and the obtained by-products in the extracted samples at different temperatures and extraction times is shown.