Materials
Apple pomace containing apple flesh, skin, seeds, and stems were acquired from Materne-Confilux Company (Namur, Belgium). The samples were well mixed to achieve homogeneity. The acquired samples were subsequently dried in an oven to constant mass at a temperature of 60 °C for 24 h. The dried apple sample was ground to a fine powder and sieved using 0.25-mm Endecott mesh. The samples were then stored in airtight Tedlar bags and preserved in a − 18 °C in freezer. Ethanol (Reagent grade VWR Chemicals, Belgium), acetone (Reagent grade VWR Chemicals, Belgium), methanol (Analytical grade, ProLabo, EEC), Folin–Ciocalteu’s phenol reagent (analytical grade, Chemical Lab, Belgium), 2,2-diphenyl-1picrylhydrazyl radical (DPPH) (Analytical grade, Sigma-Aldrich, St Louis, MO, USA), Gallic acid (Analytical grade, Sigma-Aldrich, St Louis, MO, USA) and sodium carbonate (Reagent grade Merck chemical, Darmstadt, Germany) were utilized as chemical inputs.
Methods
Waste apple pomace (WAP) characterization
Proximate analyses to determine the moisture content, fixed carbon content, volatile matter and ash contents of the WAP sample were undertaken according to the standard methods of the ASTM E1756-08 (ASTM 2015), ASTM method D3172-07 (ASTM 2007), ASTM D3175-11 (ASTM 2011), and ASTM D 2017-98 (ASTM 1998), respectively. The elemental analysis was undertaken using an elemental analyzer (LECO TruSpec CHN, Saint Joseph, Michigan, USA) to determine the carbon, hydrogen, nitrogen, and sulphur contents of the WAP sample. The elemental oxygen content was determined by subtracting the fractional ash, carbon, hydrogen, nitrogen, and sulphur contents from unity (Okoro et al. 2018). The lipid content was determined using the Soxhlet method (Kim et al. 2012). The protein content was determined according to the AOCS official method Ba4e-93 (AOAC 1998). The lignin content was determined using the Klason method (Carrier et al. 2011). The total carbohydrate content was determined by the difference (Okoro et al. 2018).
Extraction procedures of polyphenolic compounds
Solid–liquid extractions using three different solvents of water-only (designated as 100 WA henceforth), 50% ethanol–50% water v/v (designated as 50 ETH henceforth), and 65% acetone–35% water v/v (designated as 65 ACE henceforth) were carried out for the recovery of polyphenolic compounds present in dry AP sample. To this end, the extraction operating parameters were selected based on previous optimization studies for enhanced TPC extraction (Candrawinata et al. 2014; Ibrahim et al. 2019; Zardo et al. 2020). Optimal TPC extraction with 100 WA was attained at solid (dry apple pomace) mass-to-solvent volume ratio of 1:20 g/mL (Candrawinata et al. 2014). The optimal TPC extraction using ethanol has been achieved using 50 ETH and a solid-to-solvent ratio of 1:80 g/mL (Zardo et al. 2020). When 65 ACE was employed, the solid-to-solvent ratio of 1:100 g/mL was imposed (Ibrahim et al. 2019). The experiments were undertaken in a 50 mL laboratory flask with continuous stirring at 200 rpm. For the kinetic studies, when 100 WA was used for extraction, the experiments were run at 85 °C, 60 °C, and 40 °C. For extraction with 50 ETH or 65 ACE, the experiments were carried out at 60 °C, 40 °C, and 20 °C. In all mentioned experimental conditions, extraction times were assessed at 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 40 min. After the extraction, the samples were centrifuged at 6000g for 10 min for extraction with ethanol and acetone, and 9000g for 10 min for water extraction. Finally, the supernatant was carefully collected for TPC determination.
Determination of total polyphenolic content (TPC)
The TPC of the extracts was determined using the Folin–Ciocalteu colorimetric method as described in the literature (Li et al. 2020). Briefly, 50 μL of the extract was mixed with 250 μL Folin–Ciocalteu reagent (2 M) and 3 mL of distilled water and vortexed for 10 s. Then, 1 mL of 15% (w/v) Na2CO3 solution was added, and subsequently, the volume of the mixture was brought up to 5 mL by adding 700 μL of distilled water, vortexed for another 10 s and incubated at 20 °C in dark for 1 h. Finally, the absorbance of the resulting solution was measured using UV–visible spectrophotometer (PerkinElmer Lambda 25, MA, USA) at 765 nm. The TPC was expressed as milligrammes of gallic acid equivalents (determined by a standard curve) per gramme of dry weight basis of AP (mg GAE/g db) as follows:
$$\text{TPC} = \frac{C \times V}{m},$$
(1)
where C denotes the sample concentration (mg/mL) obtained from the standard curve, V is the volume of the solvent used for the extraction, and m represents the weight (g) of the dried AP sample used for the extraction.
Kinetic modelling for the extraction methods
In this study, first-order and second-order kinetic models were employed in modelling the extraction of bioactive polyphenolic compounds from AP by considering solid–liquid extractions using three different solvents of 100 WA, 50 ETH, and 65 ACE. Briefly, the first-order extraction kinetic model as proposed by Harouna-Oumarou et al. (2007) has been assessed such that the rate of leaching (re) is proportional to a driving force (Cs-Ct) and the first-order rate equation is correlated with the idea of a linear driving force as shown in Eq. (2):
$$r_{e} = \frac{{\text{d}C_{t} }}{\text{d}t} = k\left( {C_{s} - C_{t} } \right),$$
(2)
where Ct (mg GAE/g db) is the extraction capacity (concentration of TPC) at a given extraction time t, Cs (mg GAE/g db) is the concentration of TPC at saturation point and k is the first-order extraction rate coefficient (min−1).
A linear equation (Eq. 3) is obtained by the integration of Eq. (2) at the boundary conditions of Ct = 0 at t = 0 and Ct = Ct at t = t, such that plotting ln values against t provides the slope that can be used in the determination of first-order extraction rate constant:
$${\text{ln}}\left[ {\frac{{C_{s} }}{{C_{s} - C_{t} }}} \right] = kt.$$
(3)
In such solid–liquid extractions, the Arrhenius-type equation was proposed to investigate the relation of extraction rate to temperature and so the temperature dependence of the extraction kinetics (Balyan and Sarkar 2016).
Therefore, further determination of the kinetic parameters was based on the Arrhenius-type equation as shown in Eqs. (4) and (5) (Shewale and Rathod 2018). The kinetic parameters (Ae and Ea) are calculated by plotting ln k, against 1/T (Eq. 4) and the slope and the intercept give the Ea and the Ae, respectively:
$$k = A_{e} e^{{ - \left[ {\frac{{E_{a} }}{RT}} \right]}} ,$$
(4)
$$\ln k = \ln A_{e} - \frac{{E_{a} }}{RT}.$$
(5)
In Eqs. (4) and (5), k, Ae, Ea, R, and T represent the approximate overall rate constant, in min−1; pre-exponential constant (Arrhenius constant), in min−1; activation energy, in kJ/kmol; universal gas constant, specified as 8.314 kJ/kmol.K and temperature in K, respectively.
The second-order extraction kinetics is, however, modelled using Eq. (6) (Harouna-Oumarou et al. 2007):
$$r_{e} = \frac{{\text{d}C_{t} }}{\text{d}t} = k\left( {C_{s} - C_{t} } \right)^{2} .$$
(6)
Such that the integration of Eq. (6), using the boundary conditions Ct = 0 at t = 0 and Ct = Ct at t = t is as follows:
$$\frac{1}{{C_{s} - C_{t} }} - \frac{1}{{C_{s} }} = kt,$$
(7)
or
$$C_{t} = \frac{{C_{s}^{2} kt}}{{1 + C_{s} kt}}.$$
(8)
Equation (8) was then rearranged in a linearized form to give Eqs. (9) and (10) as follows:
$$\frac{t}{{C_{t} }} = \frac{t}{{C_{s} }} + \frac{1}{{C_{s}^{2} k}},$$
(9)
$$\frac{t}{{C_{t} }} = \frac{t}{{C_{s} }} + \frac{1}{m},$$
(10)
where m denotes the initial extraction rate coefficient and is equal to kCs2.
The second-order extraction rate coefficient is calculated from the intercept obtained through plotting t/Ct against t using Eq. (10).
The values of the other kinetic parameters (Ae, Ea) were determined using Eqs. (4) and (5), where k and Ae have units of g/(mg min) for the second-order extraction process.
All experiments were conducted in triplicates and data were subsequently analysed.
Determination of the antioxidant activity of apple pomace extracts based on DPPH inhibition activity
The radical scavenging capacity of TPC extracts of AP against 2,2-diphenyl-1 picrylhydrazyl radical (DPPH) was conducted based on the method described in de Torre et al. (2019). The sample extracts of 100 WA, 50 ETH, and 65 ACE containing the highest TPC concentration were selected as representative extracts for the DPPH determination experiments. 150 µL of each extract was mixed with 150 µL of DPPH solution in methanol (0.04 mg/mL) in a 96-well plate. After incubation in dark for 40 min, the absorbance of the sample at 517 nm was recorded using a UV–Vis Spectrophotometer (Microplate spectrophotometer, Epoch-BioTek, Winooski, USA), and the percentage of DPPH inhibition activity of the sample extract was calculated as follows:
$$\% {\text{Inhibition}} = 1 - \frac{{{\text{Abs}}_{s} - {\text{Abs}}_{b} }}{{{\text{Abs}}_{c} - {\text{Abs}}_{b} }} \times 100,$$
(11)
where Abss denotes the absorbance of the sample (sample extract + DPPH solution), Absb denotes the absorbance of the sample blank (sample extract + methanol), and Absc denotes the absorbance of the control (extraction solvent + DPPH solution).