Characterization and antioxidant activity of pectin from lemon peels

2.1 Raw materials

The lemon peels were harvested from two farms, FBP and PP, for 2 consecutive years (2023 and 2024). Commercial pectin (Commercial P) was obtained from Sigma-Aldrich (Pty) Ltd. and Merck (Pty) Ltd. in Johannesburg, South Africa, via Shalom Laboratories and Supplies, South Africa, an authorized local distributor.

2.2 Extraction of pectin

The pectin extraction process was done using a conventional method proposed by Zhang et al. [20] (Figure 1). Lemon peels (albedo) were blanched in boiling water to deactivate the enzymes, dried in a tray dryer, and later ground into a powder. A 1 g:30 mL ratio of lemon peel powder and water was added in a conical flask and mixed. The pH was lowered to 2.0 by adding 2 M HCl, and the mixture was heated in a water bath at 70°C for 45 min. The acidified extract was then filtered through a cheesecloth, and pectin was precipitated by the addition of 99.1% ethanol. The mixture was left at 4°C for 3 h, after which the coagulated material was collected by filtration, rinsed with a 1:1 mixture of 75% ethanol, and later dried and ground into powder.

Figure 1

Extraction of pectin.

2.3 Differential scanning calorimetry (DSC) analysis

DSC Q200 V24.11 Build 124 was used to study the thermal characteristics of the extracted pectin according to the method of Wani and Uppaluri [18] with minor modifications. A sample of 2.41 mg of dried pectin was placed in a standard aluminium crucible and properly sealed. The heating range on a crucible was set from 25 to 315°C at a rate of 10°C/min in a dynamic inert nitrogen atmosphere (50 mL/min). Additionally, an empty standard aluminium crucible was used as a reference during the whole process. TA universal Analysis software was used to determine the enthalpy of melting (ΔH _m), melting temperature (T _m), enthalpy of degradation (ΔH _d), and degradation temperature (T _d).

2.4 Colour analysis of pectin

The colour and colour difference of pectin were determined using a chroma meter (CR-400 Narich) based on the CIE-Lab system with L* (lightness), a* (redness), and b* (yellowness) values [19].

2.5 Energy-dispersive X-ray (EDX) spectroscopy analysis of pectin

Elemental analysis was performed using an EDX spectrometer, Thermo Electron Corporation, NORAN System Six, model 6733B-IUUS-SN, Madison, WI, USA.

2.6 1D ¹³C cross-polarization (CP) and non-quadrature spectroscopy (NQS) magic angle spinning nuclear magnetic resonance (MAS NMR) analyses

1D ¹³C CP and NQS MAS experiments were performed on Bruker Avance III HD and Avance 500 MHz (¹³C, 125 MHz) spectrometers equipped with a 4.0 mm double resonance MAS probe. Samples were packed into 4 mm (outer diameter) zirconia rotors. ¹³C π/2 pulse lengths were 3.5 μs. The CP contact time was 2.0 ms. Heteronuclear decoupling using SPINAL64 with a ¹H pulse duration of 6 μs was applied for an acquisition time of 27 ms. A total of 4,096 transients were co-added using relaxation delay times of 2 and 10 s for the CP/MAS and NQS/MAS experiments, respectively. A dephasing delay of 26 μs was additionally applied for the NQS experiments.

2.7 Reducing power of pectin

The reducing power of pectin was evaluated following the method of Zhang et al. [20]. About 2.5 mL of phosphate buffer (200 mM, pH 6.6), 2.5 mL of 1% potassium ferricyanide, and 1 mL of pectin solution (ranging from 0 to 2,500 mg/mL) were incubated at 50°C for 20 min. After cooling, 2.5 mL of 10% (w/v) trichloroacetic acid (TCA) was added to the mixture. Subsequently, 2.5 mL of the supernatant was collected and mixed with 2.4 mL of distilled water followed by 1.0 mL of 0.1% (w/v) FeCl₃ in a test tube and allowed to react for 10 min. The same protocol was followed by ascorbic acid (AA) which was used as a positive control. The absorbance of all samples was measured at 700 nm using a DR6000 UV-Vis spectrophotometer.

2.8 Statistical analysis

Each pectin experiment was conducted in triplicate. IBM SPSS was used to do statistical analysis (version 29.0). To ascertain the statistical difference between the samples, analysis of variance and the Duncan multiple range test (DMRT) were employed. At p < 0.05, the difference was deemed statistically significant. The DMRT was used to compare the findings presented as mean ± SD.

3.1 DSC analysis

The area under this first endothermic peak is known as ΔH _m, which represents the heat absorbed during the evaporation of moisture from the pectin sample. TA Universal Analysis software was used to determine the ΔH _m and T _m. Both an endothermic and an exothermic peak were seen in the DSC thermograms (Figure 2) of the pectin sample. The initial endothermic peak observed between 72.03 and 94.85°C is primarily attributed to water evaporation [21,22]. This peak provides important information about the moisture content, hydration properties, and potential gel formation tendencies of pectin in different applications [23,24,25]. The second peak, an exothermic peak occurring between 249 and 255.5°C, indicates the thermal degradation of pectin [26]. This peak marks the onset of decomposition and heat release during this process. The area under this degradation peak is called the ΔH _d, reflecting the heat emitted during the thermal breakdown of pectin. This parameter provides insights into the thermal stability and behaviour of pectin at high temperatures [27].

Figure 2

DSC thermograms of pectin samples.

The TA Universal Analysis software was also used to determine the ΔH _d and T _d. As shown in Table 1, the commercial P sample exhibited the highest ΔH _m (400.2 J/g), indicating strong intermolecular interactions. This suggested that it was more stable and required more energy to transition from solid to liquid. The FBP 2023 and FBP 2024 samples displayed moderate ΔH _m’s (248.2 and 318.0 J/g, respectively). Meanwhile, the PP 2023 and PP 2024 samples showed the lowest ΔH _m’s (106.4 and 147.4 J/g, respectively), suggesting that they were more amorphous. The T _m’s varied across the samples, with commercial P having the highest T _m (94.85°C). This aligned with its high ΔH _m, indicating it remained stable at higher temperatures before melting. The FBP 2023 sample had the lowest T _m (73.11°C), which might suggest it was less thermally stable and could melt at lower processing temperatures. FBP 2024 recorded a T _m of 75.00°C. The PP samples (2023 and 2024) exhibited T _m’s around 72.03 and 75.5°C, respectively, indicating they were also less stable than commercial P but slightly more stable than FBP 2023. The ΔH _d is an important parameter that indicated the energy required for the degradation of the pectin samples. Commercial P again showed the highest ΔH _d (79.12 J/g), suggesting it had a more robust structure that required more energy to break down. The FBP 2023 and FBP 2024 samples had similar ΔH _d’s (72.08 and 61.58 J/g), respectively, indicating that they might degrade more easily than commercial P. In contrast, the PP samples (2023 and 2024) showed the lowest ΔH _d’s (52.93 and 45.16 J/g), respectively, suggesting that they are more susceptible to thermal degradation, which could limit their application in high-temperature processes. The T _d’s were relatively close across the samples, with FBP 2024 having the highest T _d (255.63°C), indicating that it might have better thermal stability than the others. The PP 2023 sample had the lowest T _d (248.86°C), which aligned with its lower ΔH _d, suggesting that it might start to degrade at lower temperatures. Therefore, commercial P, followed by FBP 2024, would be the most preferred samples in the food and pharmaceutical industries due to their superior thermal stability, enabling them to withstand high processing temperatures without degrading [28].

3.2 Colour analysis

The colour of pectin is essential because it affects the visual appeal of the resulting solution or gel, which influences the overall look of the food product in which it is used, so some food industries have special colour preferences [29]. The L* values ranged from 61.03 (PP 2024) to 69.96 (commercial P), with higher values of L* indicating lighter colours and lower values suggesting darker colours. According to Table 2, the commercial P sample had the highest lightness, indicating that it was the lightest in colour, which is preferred in industries that want lighter pectin. In contrast, the PP 2024 sample had the lowest lightness, indicating a darker colour, which could be attributed to the presence of polyphenols [30]. The a* values ranged from 0.53 (PP 2023) to 2.21 (commercial P), with positive values indicating a tendency towards red and negative values towards green. The commercial P sample had the highest a* value, which suggests a slight red hue that could enhance its visual appeal. On the other hand, the PP 2023 sample had the lowest a* value, indicating a more neutral colour with less red influence.

The b* values ranged from 20.27 (PP 2023) to 24.41 (FBP 2023), with positive values indicating yellow hues and negative values indicating blue. All samples exhibited positive b* values. The FBP 2023 sample had the highest b* value, suggesting a more pronounced yellow colour. The presence of a striking yellow hue in all pectin samples was possibly due to the presence of carotenoids [31]. The C* values ranged from 20.28 (PP 2023) to 24.50 (FBP 2023), with higher values indicating more vivid colours. The FBP 2023 sample had the highest chroma, making it the most saturated and visually striking, which could be appealing in food applications. Conversely, the PP 2023 sample had the lowest chroma, suggesting a more muted colour that might be less visually appealing. More comparable results were also reported by La Cava et al. [32], when a conventional heating method was employed to extract pectin from white grapefruits. The significant difference in the extracted pectin samples (PP 2023, PP 2024, FBP 2023, and FBP 2024) is due to differences in climate conditions (in 2023 and 2024, potentially affecting pigmentation) and soil composition (variations in nutrients and soil type) that can influence crop growth and pigmentation, considering the two different farms, FBP and PP. Hence, our pectin samples had different colour properties. Consequently, the difference in commercial pectin might be attributed to the source in which the pectin was extracted.

3.3 EDX analysis

The elemental composition of the pectin samples is presented in Figure 3. Commercial P had a composition of carbon (C): 36.54%, oxygen (O): 56.02%, sodium (Na): 1.18%, aluminium (Al): 0.99%, potassium (K): 0.91%, calcium (Ca): 1.15%, manganese (Mn): 0.62%, and silicon (Si): 2.59%. It was noted that the high percentages of carbon and oxygen indicated a polysaccharide composition typical of pectin. The presence of Na, K, Ca, and Al in this pectin may have originated from a source rich in these minerals [33].

Figure 3

EDX spectra of pectin samples. (a) Pectin extracted from FBP lemons in 2023, (b) pectin extracted from PP lemons in 2023, (c) pectin extracted from FBP lemons in 2024, (d) pectin extracted from PP lemons in 2024, and (e) commercial pectin.

FBP 2023 exhibited the composition of 37.89% C and 62.11% O, indicating a higher oxygen content compared to commercial P. This difference may imply a distinct structural composition or hydration level. Conversely, FBP 2024 had a composition of 36.08% C and 63.92% O, showing a similar high oxygen content but with a lower carbon percentage, potentially indicating variations in molecular structure.

Then PP 2023, its elemental composition included 41.45% C, 53.91% O, and 4.64% Cl. The higher carbon content suggested a carbon-rich structure, possibly due to different sourcing or processing methods. The presence of Cl is attributed to HCl, which was used during extraction.

Finally, PP 2024 had a composition of 37.17% C and 62.83% O, resembling the high oxygen content seen in FBP samples. The absence of additional elements indicated a relatively pure form of pectin. Our results regarding the composition range of carbon and oxygen in all pectin samples align with the findings of Arora et al. when the microwave green extraction method was employed to extract pectin from Citrus maxima.

3.4 NMR results

The peaks observed in the ¹³C NMR spectra of pectin samples recorded using CP and NQS MAS techniques can be attributed to specific structural features of pectin. Figure 4 shows peaks at 52–70 ppm which suggest methoxyl groups (–OCH₃) attached to carboxyl groups of galacturonic acid. The presence of these signals suggests a degree of methylation, which is crucial for the gelling properties of pectin [35]. The signal at 110–112 ppm in the ¹³C spectrum is associated with arabinose anomeric resonances [36]. The signal at 170 ppm in all pectin samples is attributed to C-6 of the carboxyl group of the galacturonic acid units in esterified form [37]. Furthermore, the consistency in chemical shifts across the samples suggests they may exhibit similar chemical characteristics. However, the slight variations in chemical shifts could also indicate differences in the degree of esterification and the presence of side chains.

Figure 4

Stacked NQS NMR spectra of commercial pectin and four isolated pectin samples.

3.5 Reducing power determination

The higher absorbance in the reducing power assay signifies more potent reducing power, leading to enhanced antioxidant activity. Consequently, evaluating the reducing capacity of pectin can indirectly assess its antioxidant capability [38]. Figure 5 illustrates a comparison of the reducing capacity of pectin samples with AA. The results indicate that AA exhibits the highest reducing capacity, followed by PP 2023, PP 2024, FBP 2024, and FBP 2023, which showed the lowest reducing capacity among the pectin samples. The experimental principle involves reducing Fe³⁺ in potassium ferricyanide to Fe²⁺ and subsequent reaction with ferric chloride to produce Prussian blue [39]. Reductones present in the samples are known to disrupt free radical chains by donating hydrogen atoms, which play a crucial role in preventing peroxide formation [40]. Thus, these findings suggest that pectin possesses antioxidant properties due to the presence of reductones.

Figure 5

Reducing power of pectin samples in comparison with AA.