Impact of storage conditions on the composition and antioxidant activity of peanut skin phenolic-based extract (2025)

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International Journal of Food Science and Technology, Volume 57, Issue 10, October 2022, Pages 6471–6479, https://doi.org/10.1111/ijfs.15964

Article history

Abstract

Introduction

Oxidation is an important concern for production, storage and sale of many types of foods. Oxidation reactions not only cause loss of nutrients, vitamins and phytochemicals (essential fatty acids, tocopherols, pigments, sterols) but also produce rancid flavours, discoloration and other forms of spoilage (Frankel, 2005). To overcome these problems, the use of antioxidants (AOs) is one of the most effective, convenient and economical strategy. The food industry uses mostly synthetic AOs (butylhydroxytoluene, t-butylhydroxyquinone, alkyl gallates) even though these compounds are being questioned due to possible harmful effects on human health (Wang et al., 2021).

Since the turn of the century, there has been a great interest in replacing synthetic food AOs by natural ones. Screening of natural sources of AOs has recently focused on agricultural by-products (Jiménez-López et al., 2020; Panzella et al., 2020). Among them, the peanut skin (PS) – i.e. the tegument that surrounds the kernel – constitutes an abundant source of phenolic compounds having remarkable bioactive properties (Bodoira et al., 2022a). Strikingly, it is still considered an industrial waste; most of the PS arising from peanut processing industries is discarded or used in energy production. Considering the global peanut production (about 50 million metric tons in 2021, USDA, accessed on June 2022) and the average shelling percentage (75%), and bearing in mind that the PS represents about 3% of the total kernel weight (Lorenzo et al., 2018), more than 1 million metric tons of PS could be produced annually worldwide.

The antioxidant capacities of PS phenolics (PSP) in in vitro systems as well as in biological systems have been largely demonstrated (Bodoira et al., 2022a). Several studies report antioxidant protection of PSP in different types of food systems such as bulk oils (Larrauri et al., 2016; Franco et al., 2018), emulsion systems (Bodoira et al., 2022b), bakery (Costa de Camargo et al., 2014) and meat products (Munekata et al., 2016; Lorenzo et al., 2018). According to studies by Wang et al. (2007), Larrauri et al. (2016), Oldoni et al. (2016) and Bodoira et al. (2017) the scavenging activity of PSP against peroxyl and others common free radicals (DPPH•, HO•, O₂•-) could be higher than those from synthetic AOs.

In the search for a safe and effective procedure for extraction of PSP, a method-based exclusively on GRAS (Generally Recognised as Safe) solvents (water, ethanol) has been developed (Bodoira et al., 2017). By doing so, a purified, pollution-free peanut skin extract (PSE) with good antioxidant properties was obtained. Besides in vitro antioxidant capacities (Bodoira et al., 2017, 2022b), the PSE showed cytoprotective effects—by decreasing reactive oxygen species and superoxide dismutase activity—against oxidative stress, and did not present cytotoxicity against normal cells in the concentration ranges with maximum free radical scavenger activity (Rossi et al., 2020). Therefore, it could be safely used as additive in food formulations.

An important issue related to the use of natural phenolic compounds as food additives has to do with their chemical stability, possible degradation and/or loss of bioactivity over time. Phenolic compounds are susceptible to oxidative and other chemical reactions that could lead to bioactive properties impairment. Degradation of phenolics should be an important part of the testing program for phenolic extracts intended for food antioxidants. However, there is not much information on this subject. Studies conducted by Srivastava et al. (2007) and Del Toro et al. (2015) showed that temperature and storage time significantly affect the polyphenol content and biological activities of blueberry and herb extracts. Sharma et al. (2016) reported anthocyanin degradation in Indian blackberry pulp extracts after one-year storage period, even under refrigerated conditions. Chaaban et al. (2017) also found severe degradation of several flavonoids when exposed to light and oxygen, though the extent of degradation varied according to structural characteristics of the compounds tested. Likewise, Zorić et al. (2017) reported that anthocyanins were more susceptible to high temperatures than flavonol glycosides and phenolic acids. This latter study and other related (Zorić et al., 2016) stressed the importance of the storage temperature and light- and oxygen-protective packaging to maintain polyphenols stability of sour cherry (Prunus cerasus) products. All of the above-mentioned studies indicate that natural phenolic compounds may be unstable; so bioactivity of phenolic extracts may be lost during storing. Despite growing interest in the use of peanut skin phenolics as antioxidant food additives, there are no studies providing information on the fate of these compounds during storage and how that affects their antioxidant capacities.

Our general interest is to contribute information to thoroughly characterise a PSE that could be used as food antioxidant additive. The present study was aimed to assess the composition, stability and antioxidant efficacy of the PSE stored during a long time period under different temperature and light conditions. Because of the chemical diversity of compounds present in the PSE and their cooperative action to the total antioxidant power (Larrauri et al., 2016; Oldoni et al., 2016; Bodoira et al., 2017, 2022a), we carried out several assays based on radical scavenging activities, reducing power, and antioxidant efficacy in a highly unsaturated lipid system. The research was driven on the hypothesis that the composition and selected antioxidant capacities of the PSE can be fully preserved when stored in darkness and refrigerated conditions.

Materials and methods

Obtaining the peanut skin phenolic extract

The peanut skin (PS) was obtained from Runner-type peanuts using a typical blanching process (90 °C, 10 min). Skins were cleaned to remove small pieces of kernel, and then milled (cutting mill, Moulinex, France) and sieved to get uniform particle sizes (0.5 mm). This material was extracted by means of subcritical fluids following the experimental setup and conditions reported elsewhere (Bodoira et al., 2017). A mixture of distilled water and absolute ethanol (40:60, v/v) was used as solvent. The extraction temperature, mass flow and pressure were fixed at 220 °C, 7 g/min and 7 MPa, respectively. After the extraction process, the solution was centrifuged (9000 rpm, 20 min) and the liquid phase was concentrated under vacuum (40 °C). The remaining residue was lyophilised (Pfeiffer, Model DUO 5 M, Germany) and the obtained dry extract (henceforth the PSE) was maintained in an amber glass container (−20 °C, nitrogen atmosphere) until use.

Storage assay

Prior to the start of the storage test, the composition and selected antioxidant properties were analysed in three independent samples of the PSE (control treatment, t0). For the storage test, aliquots (100 mg) of PSE were put into amber glass 5 mL-vials sealed with screw caps, and stored in thermostatic chambers either at refrigerated condition (4 ± 1 °C, henceforth ReTD) or at room temperature condition (25 ± 1 °C, RoTD). For each condition, a total of 18 samples were used (the number required to perform an analysis every 2 months in triplicate for 1 year). On the other hand, six transparent 5 mL-vials, each containing 100 mg of PSE, were stored under light (800 Lux) at 25 ± 1 °C (RoTL), and sampled at 6 and 12 months.

Impact of storage conditions on individual phenolic compounds

The PSE composition from all storage treatments were analysed at sixth and twelfth months. Samples of PSE, previously diluted in methanol:water (1:1 v/v), were filtered (0.45 μm nylon membrane) and then submitted to HPLC–DAD–ESI–MS/MS. An Agilent 1200 (Agilent Technologies, USA) HPLC system was used under analytical conditions reported previously (Bodoira et al., 2022b). The HPLC system was connected to a photodiode array detector (Agilent G1315 C Starlight DAD), and then to a QTOF mass spectrometer (micrOTOF-Q11 Series, Bruker) equipped with electrospray ionisation (ESI) interface. Identification of phenolic compounds was based on their retention times, elution order, u.v.–Vis spectra and MS fragmentation data from available standards (caffeic acid, quinic acid, kaempferol, catequin, quercetin and rutin) and bibliographical data (Larrauri et al., 2016; Munekata et al., 2016;Bodoira et al., 2017, 2022b ). Quantification was based on external calibration curves of the standards at concentrations between 0.025 and 56 μg/mL. The linear analytical range was between 0.025 and 7.0 μg/mL (R² > 0.98). Coefficient of variation was below 10%. Limit of detection (LOD) ranged between 0.003 and 0.050 mg/L, whereas limit of quantification (LOQ) ranged from 0.009 to 0.185 mg/L.

Antioxidant capacities

At each of the scheduled sampling dates, three vials from each of the storage treatments were withdrawn from the thermostatic chambers. From each vial, a 10-mg aliquot of PSE was taken and solubilised in 10 mL methanol for analysis as indicated below.

Radical scavenging capacity

Radical scavenging capacity (RSC) was analysed by means of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals assays (ABTS, DPPH and HO, respectively). Analytical determinations were performed essentially as described in previous studies (Bodoira et al., 2017, 2022b). Calibration curves were run with Trolox as standard in concentrations ranging between 1.16 and6.94, 0.30 and3.75 and 0.74 and 5.15 μg/mL for ABTS, DPPH and HO assays, respectively (R² ≥ 0.95 in all cases). Results were expressed as mmol Trolox equivalents/g PSE.

Reducing power determinations

Ferric reducing antioxidant power (FRAP) determinations were carried out according to Munekata et al. (2016). A working solution was made by mixing 5 mL 10 mM TPTZ (2,4,6-tri-pyridyl-s-triazine) prepared in 40 mM HCl, 50 mL of 300 mM sodium acetate buffer pH 3.6, and 5 mL 20 mM FeCl₃ 6H₂O. A 3 mL-aliquot of this solution was added to 10 μL of methanol PSE solution. The reaction mixture was homogenised and incubated at 37 °C. Finally, the absorbance of the solution was measured at 593 nm, when it reached the plateau (45 min). Calibration curves were run with Trolox as standard in concentrations ranging from 0.39 to 5.86 μg/mL (R² ≥ 0.99). Results were expressed as mmol Trolox equivalents/g PSE.

Total phenol content (TPC) was measured by means of the Folin–Ciocalteu assay as previously described (Bodoira et al., 2017). Calibration curves were run with gallic acid (GA) as standard in concentrations ranging from 0.25 to 2.50 μg/mL (R² ≥ 0.99). Results were expressed as mg GA equivalents (GAE)/g PSE.

Antioxidant activity of PSE in bulk chia oil

Chia oil was used as lipid system to evaluate the antioxidant efficacy of the stored PSE. Working samples were prepared using chia oil added with PSE, previously diluted in methanol, to reach concentrations of 3 mg PSE/g oil. Samples were submitted to an accelerated oxidation test (Rancimat, Metrohm, Switzerland) under conditions used previously (100 °C, airflow rate 20 L/h, Bodoira et al., 2022b). The oxidative stability of chia oil with the added PSE was expressed as the induction time (IT, hours) and corresponded to the break point in the plotted curve. A negative control containing 150 μL of methanol but without PSE was also tested.

Statistical analyses

All the results reported are an average of triplicate analysis ± standard deviation. Statistical analyses were carried out using the software Infostat (2018, National University of Córdoba, Argentina). Differences between average values were determined by means of one-way (Table 2) and two-way (Table 3) ANOVA at the 1% level (P < 0.01) of significance. Whenever ANOVA indicated a significant difference, a pair wise comparison of mean by least significant difference (LSD) was carried out. In order to explore the influence of storage time, temperature and light condition, multivariate analysis of variance (MANOVA) was performed and marginal means were compared by means of the Hotelling's test.

Results and discussion

Effect of storage conditions on PSE composition

Table 1 presents the concentrations of phenolic compounds from the PSE stored at different conditions. By considering the LOD above indicated, the initial composition of the extract (t0) was found to show a total of twenty-eight phenolic compounds. Most of the identified compounds were condensed flavonoids (procyanidin and proanthocyanidin dimers and trimers), monomeric flavonoids and derivatives. These latter comprised compounds having the basic structure of flavanols (compounds 4 and 6), flavonols (compounds 14, 20, 21 and 23), flavones (compounds 12, 15–18, 22, 24, 26 and 28) and flavanones (compounds 13, 25 and 27). Two hydroxycinnamic acids (compounds 8 and 10) and one stilbene hydroxylated derivative (compound 19) were also detected. The described chemical profile is in general agreement with those of other peanut skin extracts obtained previously (Bodoira et al., 2017, 2022b). Altogether, procyanidins and proanthocyanidins accounted for about 42% of the total phenolic compounds quantified; flavanols represented 6.1%, flavonols 17.5%, flavones 7.2%, flavanones 4%, hydroxycinnamic acids 11.1%, and the stilbenoid trans-piceatannol 12.2%.

In order to analyse the influence of the storage conditions on the composition of the extracts, concentration losses were estimated only for those compounds that showed statistically significant differences (P < 0.01) with respect to the control treatment (t0). Concentration losses were assumed as degradation of the compounds and were reported in parentheses in Table 1. The examined storage conditions affected differently the PSE composition. The PSE stored in darkness at refrigerated conditions (ReTD treatment) showed the lowest degradation rate. With the exception of the flavones hydroxyapigenin and dihydroxymethoxyflavone (compounds 12 and 17), which showed degradation rates of around 40%, the rest of the extract components did not show significant losses in concentration at the final time of the storage period (Table 1).

Differently, the extracts stored at room temperature experienced meaningful losses in concentration for most of the compounds analysed (Table 1). Concentration losses were higher under light conditions. Flavanols, flavones and flavanones showed higher degradation rates relative to other phenolic compounds. Notably, the flavanols catechin and epicatechin—which showed relatively high concentrations in the original extract (t0)—were absent in the PSE stored under light (RoTL). At this condition, the flavone hydroxyapigenin and the flavanone sakuranetin were also fully degraded (Table 1).

Taken as a whole, the concentration of the different procyanidin and proanthocyanidin oligomers showed no changes at 6 months of storage under RoTD treatment, when compared with t0; but a moderate decrease in concentration (about 16%) was observed at 12 months. Under RoTL conditions, the loss rate reached 15.3% at 6 months, and increased to 36% at 12 months of storage (Table 1).

Room temperature and light conditions had not effects on hydroxycinnamic acid concentration. In all treatments evaluated, at the final storage, the concentrations of both isomers identified remained practically unchanged as compared with t0. Similarly, trans-piceatannol concentration was fully retained, except for the RoTL treatment where it was decreased 22% (12 months storage) relative to t0 (Table 1).

As previously stated, flavonoid-based compounds were more degraded when compared with other PSE components. The marked loss of (epi)-catechin concentrations during storage at room temperature is consistent with that found in phenolic extracts from different sources. For instance, severe catechin degradation has been reported in blueberry liquid extracts stored for 60 days, even at mild temperatures (Srivastava et al., 2007). Partial degradation of (epi)-catechin and derivatives has been observed in dry red wine extracts after 70 days of storage at 28 °C (Galmarini et al., 2013). Temperature was identified as a key factor for degradation of (epi)-catechin derivatives present in green tea extracts (Li et al., 2011). In our study, the storage temperature had a strong impact on the concentrations of most of the flavonoids present in the PSE, as indicated by the data obtained at 4 and 25 °C. Besides temperature, the storage time also affected the stability of these compounds, as can be seen by analysing the concentration losses at 6 and 12 months of storage at room temperature, both in darkness and light conditions (Table 1). Degradation of flavonoids—including catechin and derivatives—attributable to the storage time has been noted in tea leaves and several fruit and herbal extracts (Friedman et al., 2009; Koyu & Haznedaroglu, 2015; Sharma et al., 2016; Zorić et al., 2017). Unlike the flavonoids, the hydroxycinnamic acids and piceatannol showed remarkable stability in all treatments we tested. Consistent with these results, the study by Galmarini et al. (2013) also showed that the stilbenoid resveratrol and several phenolic acids in the red wine extracts remained virtually unchanged during storage.

Impact of storage conditions on PSE antioxidant properties

Several assays based on reducing power (TPC and FRAP) and radical scavenging activities (DPPH, ABTS and HO) were carried out to check possible effects of storage treatments on antioxidant activity (AA) of PSE. Table 2 resumes data from t0, and extracts stored at 6 and 12 months. Firstly, it should be noted that the activities measured in PSE from both ReTD and RoTD treatments did not differ significantly from those of t0; as a result, AA retention was virtually complete at the final storage time (Fig. 1). Differently, the PSE exposed to light underwent significant losses in all the activities tested; the lowest retention percentages were observed for DPPH, TPC and FRAP (Fig. 1). The different PSE showed substantial differences in AA, measured as the induction time (IT) of chia oil subjected to accelerated oxidation conditions. While this activity was fully retained in PSE from ReTD, the extracts from both RoTD and RoTL had significantly lower activity than PSE at t0 (Table 2, Fig. 1).

The preservation of antioxidant properties of natural phenolic extracts has been addressed in several studies. Most of them have stressed the effects of temperature, light, pH and oxygen concentration as the main factors that affect the total AA (Koyu & Haznedaroglu, 2015; Zorić et al., 2017; Ali et al., 2018). Moreover, such effects have generally been more noticeable in liquid extracts. For instance, remarkable effects of temperature have been noted in blueberry liquid extracts where the total AA decreased strongly at 6 °C and upper temperatures (Srivastava et al., 2007). Likewise, strong impacts of light and pH have been reported in pomegranate peel and Eugenia jambolana fruit pulp extracts (Sharma et al., 2016).

Under darkness storage conditions (Table 3), no significant differences were observed between PSE kept at 4 and 25 °C, except for the AA in chia oil which was slightly decreased in the RoTD extract at 12 months. Reducing power and RSC of PSE stored in darkness were virtually retained; irrespective of the temperature condition, the parameters analysed remained almost constant over all the storage time (Table 3). These results highlight the importance of the dark condition as a key factor for the preservation of the examined AA. The AA retention may also be favoured by the dry condition of the PSE used. Such a condition combined with dark storage has been effective in decreasing hydrolysis and oxidation reactions that cause losses of antioxidant activity in natural phenolic extracts (Koyu & Haznedaroglu, 2015; Zorić et al., 2017).

Conclusions

This study defines simple and low-cost conditions that guarantee the preservation of the PSE composition and the continuity of its antioxidant properties during prolonged storage. Consistent with the stated hypothesis, results showed that the composition and selected antioxidant capacities of the PSE were preserved when stored in darkness and refrigerated (4 °C) conditions. Under these conditions, most of the identified compounds did not show significant losses in concentration at the final storage time (12 months). When tested in a highly unsaturated lipid system (bulk chia oil), this extract also showed the same antioxidant efficacy as the original extract (t0). Meanwhile, reducing power and radical scavenging activities of PSE stored in darkness were virtually retained; irrespective of the temperature condition, the parameters analysed remained almost constant over all the storage time. Overall, all these findings highlight the importance of the dark condition as a key factor for the PSE's composition and antioxidant activity preservation. Considering the potential use in food, the dry solid powder quality of the extract achieved should also be emphasised. This attribute is important as it facilitates handling and dosing.

Acknowledgments

This study was financed with grants from SeCyT-UNC and ANPCyT. The authors are indebted to Dr. Romina Di Paola Naranjo for her assistance with the HPLC-ESI-MS/MS analysis.

Author contributions

Yanina Rossi: Investigation (equal); methodology (supporting); writing – review and editing (equal). Alexis Velez: Formal analysis (supporting); methodology (supporting); resources (supporting); writing – review and editing (equal). Mariana Montenegro: Conceptualization (supporting); resources (supporting); writing – review and editing (equal). Marcela L Martinez: Formal analysis (supporting); investigation (supporting); methodology (equal); resources (supporting); writing – review and editing (equal). Pablo Daniel Ribotta: Formal analysis (supporting); funding acquisition (supporting); project administration (supporting); resources (supporting); writing – review and editing (equal). Damián Modesto Maestri: Conceptualization (equal); funding acquisition (lead); methodology (equal); project administration (supporting); resources (lead); supervision (lead); visualization (supporting); writing – review and editing (equal).

Ethical guidelines

Ethics approval was not required for this research.

Conflict of interest

Authors have no conflict of interest to declare.

Peer review

The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.15964.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

© 2022 Institute of Food Science and Technology

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