Use of Phenolic Extract from Peanut Skin as a Natural Antioxidant in Chia Oil-Based Mayonnaise ^†

Abstract

Currently, the antioxidants (AOs) used in foods are mainly synthetic, often questioned on health grounds. So, the need for innocuous natural AOs has increased in last years. The peanut skin (PS) is an industrial by-product of low added value, but rich in bioactive phenolic compounds. In this study, the antioxidant capacity of a PS extract (PSE) was examined in a chia oil-based mayonnaise stored during six months (25 °C). The mayonnaise was made using chia oil (68% w/w) added with PSE (2 mg/g fat) and without any AO (control). For the storage test, 30 g were placed in 100 mL amber bottles and at 2, 4 and 6 months the oily phase was extracted (chloroform: methanol). Peroxide index (PI), acidity index (AI), K₂₃₂, K₂₇₀, p-Anisidine (pAnV) and TOTOX values were measured. Moreover, the presence of 2,4 heptadienal and 3,5-octadiene-2-one was analyzed by static headspace GC-MS. At the end of the assay, PI, AI, K₂₃₂, K₂₇₀, and pAnV for control and PSE mayonnaises were 74.7 and 13.4 meq O₂/kg oil; 2.4 and 2.0 mg KOH/kg oil, 10 and 3.55, 1.34 and 0.64, 3.7 and 0.98, respectively. The TOTOX value of the control was approximately six times higher than PSE mayonnaise. 2,4 Heptadienal and 3,5-octadiene-2-one were not detected at initial time but in the control treatment at the end reached 3.75 and 2.15 µg/g, respectively. Differently, in PSE mayonnaise, 3,5-octadiene-2-one was undetected and 2,4 Heptadienal was 0.83 µg/g. In conclusion, PSE represents a potential natural AO to preserve the oxidative stability of chia oil-based mayonnaise.

1. Introduction

Mayonnaise, a widely used semi-solid dressing, is an O/W emulsion with a low pH and 65–85% (w/w) of fat. It is formulated by mixing vegetable oil (dispersed phase), water and vinegar (continuous phase), egg yolk (emulsifier), salt and sugar. Mayonnaise is susceptible to spoilage because to autooxidation, but this mechanism is more complex than oxidation in bulk oils due the great oil area exposed to an aqueous phase that turns interfacial oxidation relevant. Moreover, many factors such as oil droplet size, processing conditions, pH, surface charge and area, affect lipid oxidation in emulsion systems [1].

The use of antioxidants (AOs) is very common in the food industry. Synthetic AOs such as butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), tertiary butylhydroquinone (TBHQ) are widely used to delay or prevent lipid oxidation. Some of them, however, are being questioned because both in vitro and in vivo studies have demonstrated possible toxicity. Moreover, the unavoidable AOs release to the environment may cause potential risks to both human and environment health [2]. Some natural polyphenolic compounds have the ability of reducing the rate of lipid oxidation; so, they represent an alternative to synthetic AOs.

The peanut skin (PS), an industrial waste of peanut blanching operations, is an abundant source of phenolic compounds, mostly monomeric and condensed flavonoids. Among the attractive bioactivities of PS phenolic extracts, the antioxidant capacity is probably the most studied [3]. Much of food applications of PS phenolic-based extracts (PSE) have been made on meat products [4], as chemical preservatives that improve oxidative and microbiological spoilage. Additional applications could include their use as AOs in emulsions or others multiphase systems. In this line, Verstraeten et al. [5] found that procyanidin dimers and trimers isolated from PS can protect the integrity of lipid bilayers. According to these, recently, PSE showed good antioxidant efficacy when tested in a highly unsaturated lipid system such as a chia O/W model acid emulsion [6].

As previously stated, the objective of the present study was to evaluate PSE antioxidant efficacy during chia oil-based mayonnaise storage.

2. Materials and Methods

2.1. Chia Oil and Peanut Skin Extract (PSE) Extraction

Chia oil was obtained by cold pressing using a Komet screw press (Komet CA 59G; IBG Monforts, Monchengladbach, Germany). The PS was acquired from peanut Runner-type using a typical industrial blanching process (90 °C, 10 min). Skins were cleaned, milled and sieved to obtain uniform particle sizes (0.5 mm). Phenolic compounds were extracted from PS by means of subcritical fluid extraction following conditions reported previously (220 °C, flow 7 g/min and pressure 7 MPa) [7]. Distilled water and ethanol (40:60, v/v) were used as solvents. After the extraction process, the liquid extract was centrifuged (15 min at 9.000 rpm) and then concentrated under vacuum (40 °C).

Finally, the remaining residue was lyophilized and the obtained dry extract (PSE) was stored (−20 °C) in amber glass bottles under nitrogen. This extract has already been characterized for its antioxidant properties and phenolic composition [6,7].

2.2. Preparation of Chia Oil Based Mayonnaise

Mayonnaise batches of 250 g were prepared using a universal mixer. The ingredients and their proportions were those used by Alizadeth et al. [8] with some modifications. Each batch contained: 170.30 g (68.12% w/w) of chia oil, 35.75 g (14.30% w/w) of water, 2.03 g (0.81% w/w) of vinegar, 22.85 g (9.14% w/w) egg yolk, 2.50 g (1% w/w) lemon juice, 12.50 g (5% w/w) sugar, 3.80 g (1.52% w/w) salt and antimicrobial agents: sodium benzoate and potassium sorbate 0.15 g (0.06% w/w) each one. At first, egg yolk powder and dry materials were mixed manually with water and vinegar. Then, oil was added slowly to the aqueous phase and mixed until a homogeneous emulsion was obtained. For PSE treatment preparation, the PSE was dissolved in 1.5 mL of water:ethanol (40:60 v/v) and subsequently added to the aqueous phase, giving a final PSE concentration of 2 mg/g total fat (oil + egg yolk lipids). This concentration was chosen based on previous studies in an O/W acid emulsion model [7]. Finally, the control treatment was prepared using AOs free chia oil following the same procedure.

2.3. Physical Properties of Chia Oil Based Mayonnaise

The physical properties of the mayonnaise such as color, pH, water activity (a_w) and particle size distribution were measured following the methodologies previously reported [9].

2.3.1. Color Measurements

Mayonnaise samples were measured by CIE L* a* b* scale using a portable colorimeter with a D65 illuminant at a 10° observation (spectrophotometer Konica-Minolta, Tokyo, Japan). Black and white calibrations were necessary before the measurements. Then, an amount of each mayonnaise was poured into the measurement plate and the L* (lightness 0 to 100), a* (red/green coordinate) and b* (yellow/blue coordinate) parameters were recorded. The whiteness index (WI = L* − 3b*) and yellowness index (YI = 142.86 b*/L*) were calculated.

2.3.2. pH and a_w Measurements

pH was measured with a pH meter (Hanna Instruments S.A., Buenos Aires, Argentina) and the a_w by means of a Decagon AquaLab (208 series 3, Decagon Devices Inc., Pullman, WA, USA, EUA water activity meter) equipment at 25 °C.

2.3.3. Particle Size Measurements

Forty mg of mayonnaise sample was mixed with 150 mL of a 0.1% sodium dodecyl sulfate (SDS) solution and stirred until it was completely dispersed. The size distribution of oil droplets was determined by laser diffraction with the Horiba analyzer at 25 °C. The refractive indexes used were 1.47 and 1.33 of oil and water, respectively. The droplet size as the volume-weighted mean diameter (d_4.3) and the span number were reported.

2.3.4. Texture

Texture profile analysis (TPA) was carried out using a TA-TX2i Texture Analyzer (Universal Testing Machine Model 3342, Instron, EUA). The sample were scoped into the 25 mm cylindrical probe. The test was performed with one cycle at a speed of 1.0 mm s⁻¹ to compress the sample to 80% of its original height. The firmness and adhesiveness of the samples are reported.

2.4. Chia Oil Based Mayonnaise Storage Assay

After preparation (Section 2.2), 30 g of the mayonnaise were put into 100 mL brown bottles with film caps that allows some gas exchange. These samples were stored at ambient temperature (25 °C) in a chamber and sampling in triplicate was performed after 2, 4 and 6 months for further analyses. Moreover, for the determination of volatile compounds (VC) an aliquot of mayonnaise (10 g), from both treatment (PSE and control), was placed into a 40 mL headspace vial light cutlery, with silicon septum and sealed cap, and stored for 6 months under the same conditions.

2.4.1. Volatile Compounds by Static Headspace Gas Chromatography

The samples were prepared by adding 3 mL of a sodium chloride saturated solution to break the emulsion [8]. After shaking, 20 µL of internal standard (cyclohexanol 3.8 mg/mL methanol) was added, then the vials were equilibrated (40 min at 50 °C).

Volatile compounds (VC) were sampled from the headspace using a syringe (Hamilton. Model: 81,456 volume: 2.5 mL. Reno, NV, USA) and immediately injected into a PerkinElmer Clarus 600T chromatograph-mass spectrometer (Perkin Elmer, Shelton, CT, USA) operating in electron impact mode at 70 eV and a scan range of 40–300 atomic mass unit (amu). The gas chromatograph was equipped with an Zebron ZB-Wax (30 m × 0.25 mm × 0.25 µm) column (Phenomenex. Torrance, CA, USA). The GC/MS injector and detector were set at 250 °C and 200 °C, respectively. The column temperature was initially held at 35 °C for 5 min, then increased at 5 °C/min to 200 °C, and held for 5 min. GC/MS analytical samples were run in triplicate and VC were identified based on comparisons of retention indices and mass spectra with literature data and by comparison with the NIST-14 Mass Spectral Library (US National Institute of Standards and Technology).

2.4.2. Extraction of Lipids from Chia Oil Based Mayonnaise

The determination of degradation products in the mayonnaise samples was carried out by extracting the lipids using chloroform/methanol (1:1 v/v) as solvent. Two extractions were made from each sample and the extracted lipids were stored at −20 °C under nitrogen until analysis.

2.4.3. Measurement of Degradation Products

Oil samples extracted from each treatment were used to measure, using official methods, acidity (AI) and peroxide indexes (PI), dienes and trienes conjugates (k₂₃₂–k₂₇₀), and concentrations of secondary oxidation products through the p-anisidine value (p-AnV). The TOTOX value (2PI + p-AnV)—which provides an idea of the total oxidative state of a system—was also calculated.

2.5. Statistical Analysis

All experiments were performed in triplicate. The results were depicted in the form of average and standard deviation. Comparisons between treatments were performed by means of ANOVA test using INFOSTAT/Professional 2014 software (FCA-UNC, Argentina). Normality of data was tested using Shapiro–Wilk test. Results giving p values < 0.05 were considered significantly different.

3. Results and Discussion

3.1. Physical Properties of Mayonnaise

The addition of PSE did not affect the pH and a_w of the chia oil-based mayonnaise; their values were 5.73 ± 0.05 and 0.901–0.925, respectively.

Table 1. Color and particle size parameters of chia oil based mayonnaise without antioxidants (Control) and with peanut skin extract (PSE) (2 mg/g) at initial storage time.

Treatment	Color					Particle Size
	L*	a*	b*	WI	YI	d4,3 (µm)	Span
Control	67.01 ± 0.94 b	0.81 ± 0.19 a	31.10 ± 0.77 b	−26.3 ± 1.71 a	66.31 ± 1.99 b	1.29 ± 0.18	0.95 ± 0.03
PSE	39.19 ± 0.77 a	4.55 ± 0.15 b	12.38 ± 0.63 a	2.06 ± 1.40 b	45.12 ± 1.62 a	1.22 ± 0.02	1.13 ± 0.14

Different letters in the same column indicates significative differences (p > 0.05).

summarized the color and particle size results. The incorporation of PSE caused a change in the color of the mayonnaise compared to the control, but it did not affect the mean particle size. The parameter with the highest variation was a*, which indicated an increase in the red tones of the samples when PSE was incorporated into the formulation. Both parameters, WI and YI, are significantly different in both mayonnaise treatments.

Regarding the texture of the mayonnaise, firmness is an important parameter as it can affect sensory characteristics and applicability. However, the addition of PSE did not have an effect on the firmness (1.76 ± 0.03 in both treatments) or adhesiveness (17.27 ± 1.49).

3.2. Degradation Products during Mayonnaise Storage

The PSE used in this study, composed mainly of procyanidin and proanthocyanidin oligomers [3], has been found to display strong antioxidant capacities as measured by different reducing power and radical scavenging methods [6,7]. However, solubility is also important for characterizing the antioxidant activity of phenolic natural extracts. Partition coefficients (log P) of the PSE showed negative values (pH 3.5= −1.19; pH 5.5 = −1.21) [6]. But the absolute values were not as high as might be predictable for an extract with high polyhydric phenols content. Therefore, PSE phenolics could cover a relatively wide range of log P, which in turn could favor their partial dissolution in oil. The information mentioned above could be significant in emulsion systems because the components of PSE have the potential to diffuse through the aqueous phase and reach the interface. This could alter the physical and chemical properties of the interface, potentially affecting the oil stability. Indeed, PSE demonstrated high antioxidant efficacy when tested in an acid model O/W emulsion made with chia oil, Tween 80 and acid buffer, maintained at 40 °C for 15 days [6].

Table 2. Peroxide Index (PI), k₂₃₂, k_270, Acidity Index (AI), p-Anisidine and TOTOX value of lipids extracted from chia oil based mayonnaise without antioxidants (Control) and with peanut skin extract (PSE) (2 mg/g) at different storage (25 °C) time.

Mayonnaise Treatment	Month	PI (meq O2/kg Chia Oil)			K232			K270			AI (mg KOH/ kg Chia Oil)			P-Anisidine Value			TOTOX Value
	0	1.36 Aa	±	0.43	2.28 Aa	±	0.05	0.60 Aa	±	0.05	1.98 Aa	±	0.02	ND			2.72 Aa	±	0.70
Control	2	24.38 Bb	±	2.37	3.90 Bb	±	0.43	0.55 Ba	±	0.01	1.98 Aa	±	0.16	0.19 a	±	0.08	48.97 Bb	±	4.82
	4	50.41 Bc	±	1.02	6.66 Bc	±	0.02	0.83 Bb	±	0.01	1.91 Aa	±	0.23	2.13 b	±	0.02	102.95 Bc	±	2.07
	6	74.69 Bd	±	7.31	10.05 Bd	±	0.70	1.34 Bc	±	0.04	2.37 Bb	±	0.11	3.68 Bc	±	0.47	153.08 Bd	±	14.32
	0	1.36 Aa	±	0.43	2.28 Aa	±	0.05	0.60 Aa	±	0.05	1.98 Aa	±	0.02	ND			2.72 Aa	±	0.70
PSE	2	3.83 Ab	±	0.58	2.47 Ab	±	0.05	0.49 Aa	±	0.01	1.90 Aa	±	0.08	ND			7.67 Ab	±	1.17
	4	9.22 Ac	±	0.38	2.97 Ac	±	0.12	0.54 Aa	±	0.06	1.91 Aa	±	0.12	ND			18.44 Ac	±	0.76
	6	13.36 Ad	±	0.50	3.55 Ad	±	0.01	0.64 Aa	±	0.01	2.01 Aa	±	0.02	0.98 A	±	0.19	27.70 Ad	±	1.19

ND: no detected. Different letters indicate statistically significant differences (p > 0.05). Capital letters indicate differences between treatments in the same month and lower-case letters differences between months within the same treatment.

shows values of oxidative degradation products. In both treatments, at the initial storage time (t = 0), the peroxide index (PI) was 1.36 meq O₂/kg oil; at the final storage time in control it reached a value of 74.7 meq O₂/kg oil. This value was significantly higher than that reached in PSE-added mayonnaise (13.4 meq O₂/kg oil). On the other hand, the acidity (AI) did not show significant variations (p > 0.05) among the different times in each treatment, except for the last month where the differences between the PSE and control treatments were significant (Table 2).

Oils that contain high amounts of polyunsaturated fatty acids (PUFAs), such as chia oil, presents a faster increase in conjugated dienes compared to oils or fats with lower levels of PUFAs. As a result, the increase in K₂₃₂ during storage was noticeable in both treatments (Table 2), but it was much more prompt in the control mayonnaise, reaching a final value of 10.05, while in the PSE treatment, the final value was 3.55. On the other hand, K₂₇₀ remained constant (about 0.6) throughout the storage in mayonnaise with PSE, and in the control from the fourth month they increased slightly until reaching a final value of 1.34.

Decomposition of fatty acid hydroperoxides generates a large variety of secondary oxidation products, which can be measured by pAnV. This index is indicative of aldehydic compounds, mainly 2-alkenals and 2,4-alkadienals. These compounds were not detected in PSE-added mayonnaise until sixth month with a value of 0.98. In the control treatment, the value was 0.19 at the second month, and increased to 3.68 at the end of the storage period (Table 2). The TOTOX index at this time showed a value of 153.08 in control mayonnaise, while in the PSE treatment, it was 27.70, six times less.

Furthermore, lipid oxidation can result in off-flavors, and various volatile compounds (VC) are responsible for this. Changes in selected VC found in the chia oil-based mayonnaise at 0 and 6 months are shown in

Table 3. Concentration of majority volatile compound (VC) from chia oil based mayonnaise without antioxidants (Control) and with peanut skin extract (PSE) (2 mg/g) at initial (t0) and final time (6 month) of storage (25 °C) assay.

	Concentration (µg VC/g Mayonnaise)
Volatile Compounds	t0	Control			PSE
(VC)		6 month/25 °C
2.4 Heptadienal	ND	3.75 b	±	0.44	0.83 a	±	0.21
3.5-Octadiene-2-one	ND	2.15	±	0.84	ND

ND: no detected. Different letters in the same row indicates significative differences (p > 0.05).

. Autoxidized linolenic acid produce decatrienal methyl octanoate from the 9-hydroperoxide; 2,4-heptadienal from the 12-hydroperoxide; 3-hexenal and 2-pentenal from the 13-hydroperoxide; propanol and ethane from the 16-hydroperoxide [10]. In this study, only 2,4-heptadienal and 3,5-octadiene-2-one were quantified due to the method detection limits. These compounds have been previously reported as the predominant VC in a chia oil accelerated oxidation test [10]. In the control mayonnaise, the final concentration of 2,4-heptadienal was 3.75 µg/g, while in the PSE treatment, the value was approximately five times lower (0.83 µg/g). In contrast, 3,5-octadiene-2-one was not detected in the mayonnaise with PSE, while in the control treatment the value was 2.15 µg/g.

4. Conclusions

Despite the unfavorable conditions for food preservation (water activity 0.9, pH 5.73, air-oxygen, and 25 °C), the chia oil-based mayonnaise with 2 mg/g of a natural antioxidant extract from peanut skin (PSE) was highly stable for six months. Therefore, the results of this storage test demonstrate that PSE phenolic compounds have a high potential to reduce primary and secondary oxidation products in this emulsion food system.

Although the effective concentration of PSE could be much higher than those usually used for synthetic antioxidants (0.1–0.2 mg/g), it should be noted that worldwide food regulations recognize natural antioxidants as GRAS additives; so, there are no regulations that limit their use.