Fortification of Cookies with Peanut Skins - American Chemical Society

Oct 28, 2014 - added at 1.3, 1.8, and 2.5% to cookies to increase their polyphenol content. ... cookie formulation fortified with peanut skins as a so...
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Fortification of Cookies with Peanut Skins: Effects on the Composition, Polyphenols, Antioxidant Properties, and Sensory Quality Adriano Costa de Camargo,†,§ Carolina Maldonado Martins Vidal,§ Solange Guidolin Canniatti-Brazaca,§ and Fereidoon Shahidi*,† †

Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 Department of Agri-Food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Av. Pádua Dias 11, P.O. Box 9, CEP 13418-900 Piracicaba, SP, Brazil

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S Supporting Information *

ABSTRACT: Food fortification may be carried out to improve the health status of consumers. In this study, peanut skins were added at 1.3, 1.8, and 2.5% to cookies to increase their polyphenol content. Insoluble fiber was increased by up to 52%. In addition, total phenolic content and the corresponding antioxidant capacities also increased as evidenced by increases of epicatechin and procyanidin dimers A and B. In addition, trimers and tetramers of procyanidins were identified only in peanut skin-fortified cookies. Addition of 2.5% peanut skins rendered an increase of up to 30% in the total polyphenols as evaluated by high-performance liquid chromatography−diode array detection−electrospray ionization multistage mass spectrometry (HPLCDAD-ESI-MSn). Sensory evaluation results demonstrated that peanut skin-fortified cookies were well accepted, which suggests that the present formulation may lend itself for commercial exploitation. KEYWORDS: byproduct, fiber, moisture, polyphenol, proanthocyanidin

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INTRODUCTION Much attention has been paid to polyphenols, their sources, health effects, and potential use as food ingredients and/or supplements. There are also many literature reports on potential antimicrobial, anti-inflammatory, anticarcinogenic, and antioxidant properties of polyphenols.1−4 Phenolics in peanuts belong to several classes, namely, phenolic acids, stilbenes, monomeric flavonoids, and proanthocyanidins;5,6 however, their highest concentration is found in peanut skins, which are byproducts from the blanching process of peanuts. The major phenolic compounds from peanuts and peanut products are protocatechuic, caffeic, and p-coumaric acids as well as (+)-catechin, (−)-epicatechin, and proanthocyanidins.7,8 The use of peanut skins has so far been limited to animal feed. Due to their low cost and high potential health benefits, an effective utilization would be beneficial to peanut producers, the food industry, and consumers. Although studies about phenolic composition and antioxidant properties of peanuts skins began in the 1990s, only a few papers have appeared on their use as food ingredients. Their high proanthocyanidin content is beneficial to health; however, it may cause bitterness and astringent mouthfeel. Thus, the correct concentration to achieve a significant increase in antioxidant properties of fortified foods using ingredients with high content of proanthocyanidins should also provide a minimum or undetectable negative sensory effect. Although the use of peanut skins to produce beverages and to increase the antioxidant properties of peanut butter has been reported,9−11 to the best of our knowledge, there are no data in the literature on the use of peanut skins as a food ingredient to fortify bakery products. © XXXX American Chemical Society

Cookies are traditional nonperishable snacks that could easily be fortified with polyphenols from selected resources as well as byproducts. Because bakery goods have a high concentration of carbohydrates in their formulation, several studies have indicated partial replacement of refined wheat flour with different ingredients such as whole barley flour and wheat bran, as well fiber from apple, lemon, and wheat.12,13 Thus, peanut skins may serve as a potential source of fiber and also phenolic compounds to promote health and reduce disease risk. Some studies have reported the use of different sources of phenolics to increase the dietary intake of antioxidants. In general, these studies have successfully demonstrated enhancement of total phenolic contents as well as their scavenging ability against radicals such as 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS) as evaluated by methods such as ferric radical absorbance potential (FRAP) and oxygen radical absorbance capacity (ORAC). Furthermore, identification and quantification of the bioactive compounds regarded as health beneficial would be helpful. However, a quick scrutiny of the literature demonstrates that some food development studies do not receive support from sensory evaluation. Thus, the present study was focused on the development of a cookie formulation fortified with peanut skins as a source of phenolics such as monomeric flavonoids and proanthocyanidins. The product was evaluated for its composition, total phenolic Received: August 2, 2014 Revised: October 2, 2014 Accepted: October 28, 2014

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upper layer was collected and extraction was repeated twice. The combined supernatant was evaporated to remove the organic solvent in vacuo at 40 °C (Buchi, Flawil, Switzerland), and the residue containing soluble phenolics (free plus soluble esters) was stored at −20 °C until used for further analysis, within 1 month. Total Phenolic Contents (TPC). TPC were evaluated using the Folin−Ciocalteu method15 with minor modifications.16 Phenolic extracts were diluted to 5 mg/mL, and the solution so obtained (0.5 mL) was mixed with distilled water (4.0 mL) and Folin−Ciocalteu reagent (2.5 mL). After 3 min, 1 mL of a saturated solution (30%, w/v) of Na2CO3 was added, and the mixture was kept in the dark at room temperature (23 °C) for 2 h. The absorbance was read at 760 nm using an Agilent UV−visible spectrophotometer (Agilent 8453, Agilent Technologies, Palo Alto, CA, USA). The results were expressed as milligram catechin equivalents (CE) per gram dry weight of cookies. DPPH Radical Scavenging Activity. The DPPH assay was carried out using a slightly modified version of the method previously explained.17,18 The phenolic extracts were diluted in methanol (20 mg/mL), and 1 mL of the solution so obtained was mixed with 250 μL of a methanolic solution of DPPH (0.5 mM). The mixture was kept in the dark at room temperature (23 °C) for 10 min, after which it was injected onto the capillary tubing that guides the sample through the sample cavity of a Bruker e-scan EPR spectrophotometer (Bruker E-Scan, Bruker Biospin Co., Billercia, MA, USA). The spectrum was recorded with the following parameters: 5.02 × 102 receiver gain, 1.93 G modulation amplitude, 2.62 s sweep time, 8 scans, 100.000 G sweep width, 3495.258 G center field, 5.12 ms time constant, 9.793220 GHz microwave frequency, and 86.00 kHz modulation frequency. The DPPH scavenging activity of the extracts was calculated using the equation

content, antioxidant activity, phenolic profile (HPLC-DAD-ESIMSn), and sensory acceptability.

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MATERIALS AND METHODS

Dry-blanched peanut skin (cv. Runner IAC 886) samples were kindly donated by CAP−Agroindustrial, Dumont, São Paulo, Brazil. White sugar, vanilla, wheat flour, baking powder, margarine, eggs, salt, brown sugar, oatmeal, sodium bicarbonate, and bittersweet chocolate were procured from a local market in São Paulo, Brazil. Folin−Ciocalteu’s reagent, DPPH, ABTS, mono- and dibasic potassium phosphates, hydrogen peroxide, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ferrous sulfate, protocatechuic acid, p-coumaric acid, (+)-catechin, and (−)-epicatechin were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Sodium carbonate, sodium hydroxide, potassium persulfate, hexane, methanol, acetonitrile, formic acid, and sodium hydroxide were purchased from Fisher Scientific (Ottawa, ON, Canada). Cookie Making. The cookie formulation was developed by our research group. Control and fortified formulations are shown in Table 1.

Table 1. Formulation of Control and Fortified Cookies ingredient white sugar vanilla wheat flour baking powder margarine eggs salt brown sugar oatmeal sodium bicarbonate bittersweet chocolate peanut skins

wet basis (%) 8.96 0.15 22.5 0.15 8.96 6.41 0.15 7.72 12.8 0.15 32.0

8.84 0.15 22.2 0.15 8.84 6.33 0.15 7.62 12.7 0.15 31.6 1.30

8.80 0.15 22.1 0.15 8.80 6.29 0.15 7.58 12.6 0.15 31.5 1.80

8.73 0.15 21.9 0.15 8.73 6.25 0.15 7.53 12.5 0.15 31.2 2.50

DPPH scavenging activity (%) = [(EPR control − EPR sample)/(EPR control)] × 100 where EPRcontrol is the signal intensity of DPPH radical + methanol and EPRsample is the signal intensity of DPPH radical + phenolic extract or catechin. The results were expressed as catechin equivalents per gram dry weight of cookies. ABTS Radical Cation Scavenging Activity. The ABTS assay19 was performed using a modified version of the method described by de Camargo et al.16 The ABTS radical (7.00 mM) was generated in 100 mM phosphate buffer saline solution (PBS) (pH 7.4, 0.15 M sodium chloride) via potassium persulfate oxidation (2.45 mM). Prior to analysis, the stock solution of ABTS was diluted with PBS to reach an absorbance value of 0.70 (734 nm). Phenolic extracts were diluted in PBS (30 mg/mL), and 20 μL of the extract so obtained was added to 2 mL of the ABTS radical cation solution. The absorbance was read at 734 nm after 6 min using an Agilent UV−visible spectrophotometer (Agilent 8453). ABTS radical cation scavenging activity was calculated using the equation

The process was carried out in two steps. In a first bowl, margarine, brown sugar, and white sugar were homogenized in a mixer (Mondial, Barueri, SP, Brazil), which was followed by the addition of eggs and vanilla, a further mixing step for 5 min, and the addition of wheat flour. In a second bowl, oatmeal, salt, baking powder, and sodium bicarbonate were homogenized. Both mixtures were then blended with chocolate chips and fractionated in four parts to which different amounts of peanut skins (excluding control) were mixed. Each cookie weighed 32 g. Cookies were baked for 15 min at 280 °C using an Esmaltec oven (Esmaltec, Fortaleza, CE, Brazil). The samples were stored at room temperature (25 °C) until checked for their microbiological safety (within 2 days) followed by sensory analyses on the third day. The remaining samples (chemical analyses) were immediately frozen (−20 °C) for further analysis, within 1 month. Proximate Composition. The ash, lipid, protein, and fiber contents of the sample were determined using AOAC14 methods. Total carbohydrates were calculated by difference, and results are reported as percentage of the sample on a dry weight basis. Extraction of Phenolic Compounds. Cookies were ground using a coffee bean grinder (model CBG5 series, Black & Decker Canada Inc., Brockville, ON, Canada), and the powder so obtained was passed through a mesh 16 (sieve opening 1 mm, Tyler test sieve, Mentor, OH, USA) sieve. The samples were defatted three times using hexane (solid/ solvent, 1:5, w/v) in a Waring blender (model 33BL73, Waring Products Division Dynamics Co. of America, New Hartford, CT, USA). Defatted samples were immediately used for the extraction of phenolic compounds. Defatted cookies were extracted with 70% acetone in a gyratory water bath shaker (model G76, New Brunswick Scientific Co. Inc., New Brunswick, NJ, USA) at 30 °C for 20 min, at a solid to solvent ratio of 2.5% (w/v). After centrifugation at 4000g (IEC Centra MP4, International Equipament Co., Needham Heights, MA, USA), the

ABTS radical scavenging activity (%) = [(Abscontrol − Abssample )/(Abscontrol )] × 100 where Abscontrol is the absorbance of the ABTS radical + PBS and Abssample is the absorbance of ABTS radical cation + phenolic extract or trolox. The results were expressed as micromoles of trolox equivalent (TE) per gram dry weight of cookies. Hydrogen Peroxide Scavenging Activity. The hydrogen peroxide scavenging activity of phenolic extracts was evaluated as previously explained.20 Phenolic extracts (10 mg/mL) and 0.4 mM hydrogen peroxide solution were prepared in 0.1 M phosphate buffer (pH 7.4). The extracts (0.4 mL) were mixed with hydrogen peroxide solution (0.6 mL), and the final volume was made to 2.0 mL with the same buffer. The samples were kept in a gyrotory water bath shaker (model G76, New Brunswick Scientific Co. Inc.) for 40 min, and the absorbance was read at 230 nm in an Agilent UV−visible spectrophotometer (Agilent 8453). Blanks devoid of hydrogen peroxide (in phosphate buffer) were prepared for background corrections. The hydrogen peroxide scavenging activity was calculated using the equation B

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Table 2. Proximate Composition Cookies with Different Levels of Peanut Skinsa component

control

1.3%

1.8%

2.5%

peanut skins

ash lipid protein insoluble fiber soluble fiber carbohydrate

1.78 ± 0.01b 19.1 ± 1.28a 4.10 ± 0.05b 6.40 ± 0.91c 2.17 ± 0.30b 66.3

2.08 ± 0.09b 21.3 ± 1.11a 4.21 ± 0.01b 7.54 ± 0.04bc 1.99 ± 0.03b 63.3

1.89 ± 0.35b 20.7 ± 1.26a 4.19 ± 0.11b 7.81 ± 0.88bc 2.43 ± 0.10b 63.2

1.92 ± 0.06b 21.6 ± 0.76a 4.30 ± 0.04b 9.70 ± 0.96b 2.00 ± 0.33b 60.0

2.89 ± 0.38a 11.2 ± 0.31b 4.66 ± 0.04a 76.6 ± 1.48a 4.11 ± 0.25a 0.31

a Data represent mean values for each sample ± standard deviation (n = 3). Means with the same letters within a row are not statistically (p > 0.05) different. Carbohydrates were calculated by difference. Results are expressed on a dry weight basis of cookies.

were filtered before injection using a 0.45 μm PTFE membrane syringe filter (Thermo Scientific, Rockwood, TN, USA). HPLC-ESI-MSn analysis was carried out using the same conditions as described above in an Agilent 1100 series capillary liquid chromatography−mass selective detector (LC-MSD) ion trap system in electrospray ionization (ESI) in the negative mode. The data were acquired and analyzed with Agilent LC-MSD software (Agilent). The scan range was from m/z 50 to 1200, using smart parameter setting, drying nitrogen gas at 350 °C, flow 12 L/min, and nebulizer gas pressure of 70 psi. Protocatechuic acid, (+)-catechin, and (−)-epicatechin were identified by comparing their retention times and ion fragmentation with coded and authentic standard and the same conditions as the samples. Dimers, trimers, and tetramers of proanthocyanidins were tentatively identified using tandem mass spectrometry (MSn), UV spectral, and literature data.8 Sensory Evaluation. The present study was approved by the ethics committee of “Luiz de Queiroz” College of Agriculture, University of São Paulo, Brazil. Sensory analyses were performed using the acceptability test. Sixty volunteers, students and employees, from “Luiz de Queiroz” College of Agriculture (ESALQ/USP), aged between 18 and 60 years, participated in the acceptability test. The panel was composed by 72% women, with no history of allergy to peanuts or other ingredients of the formulation. Samples were placed in white ceramic plates coded with three-digit random numbers and delivered to the panel. The members received a set of four samples (control and cookies with added 1.3, 1.8, and 2.5% peanut skins) and water (25 °C). Participants were requested to rinse their mouths between each sample and evaluate color, odor, taste, texture, and overall impression. Scores were recorded on a paper ballot using a five-point hedonic scale (5 = extremely like, 4 = like, 3 = neither like nor dislike, 2 = dislike, and 1 = extremely dislike). Acceptable scores were ≥3 points. Statistical Analysis. Unless stated otherwise, the experimental design was randomized with three replications, and the results were analyzed using Tukey’s test (p < 0.05) and SAS software.

hydrogen peroxide scavenging activity (%) = [(Abscontrol − Abssample )/(Abscontrol )] × 100 where Abscontrol is the absorbance of H2O2 + phosphate buffer and Abssample is the absorbance of H2O2 + phenolic extract or catechin. The results were expressed as catechin equivalents per gram dry weight of cookies. Reducing Power. The reducing power assay was performed according to a method described by Alasalvar et al.21 The extracts (6 mg/mL) were prepared in phosphate buffer (pH 6.6, 0.2 mM). Extracts (1.0 mL) were then mixed with phosphate buffer (2.5 mL) and 1% (w/ v) potassium ferricyanide solution (2.5 mL), followed by their incubation at 50 °C for 20 min in a gyrotory water bath shaker (model G76, New Brunswick Scientific Co. Inc.), and subsequently a 10% (w/v) solution of trichloroacetic acid (2.5 mL) was added to it. The mixture was centrifuged at 1750g for 10 min, and the supernatant (2.5 mL) was added to distilled water (2.5 mL) and 0.1% (w/v) ferric chloride solution (0.5 mL). The absorbance was read at 700 nm, as before. The results were expressed as micromoles of trolox equivalents per gram dry weight of cookies. Hydroxyl Radical Scavenging Activity. The ability of phenolic compounds in scavenging hydroxyl radicals generated by Fenton reaction was evaluated by electron paramagnetic resonance (EPR) spectroscopy using a slightly modified version of a method previously reported.20 Extracts were prepared in 0.1 M phosphate buffer (pH 7.4). A 0.2 mL portion of the solution so obtained was mixed with 0.2 mL of H2O2 (10 mM), 0.4 mL of 17.6 mM DMPO, and 0.2 mL of FeSO4 (10 mM). After 3 min, the EPR spectrum was recorded using a Bruker e-scan EPR spectrophotometer (Bruker E-Scan, Bruker Biospin Co.) The spectrum was recorded with the same parameters as for DPPH. The hydroxyl radical scavenging activity of the extracts was calculated using the equation

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hydroxyl radical scavenging activity (%)

RESULTS AND DISCUSSION Proximate Composition. A cookie formulation (Table 1) was developed by a gastronomy professional from our research group for different addition levels of peanut skins. Such concentrations were decided on the basis of the saturation point, at which an increase in peanut skin concentration would result in a loss of pasting properties. Chocolate flavor was chosen to avoid or mask potential bitterness and astringent mouthfeel due to the presence of peanut skins, which was reported by the sensory panel in a first formulation developed (data not shown). The proximate composition of the cookies and peanut skins, reported on a dry weight basis, is summarized in Table 2. The moisture content is shown in Figure 1. Industrial and homemade cookies have several differences, and one of them is the moisture content. Homemade cookies tend to be softer, mainly because of the moisture content and freshness. In the present study, the addition of peanut skins increased the moisture retention of cookies in a concentration-dependent manner upon addition of up to 1.8% peanut skins, from which no difference was found,

= [(EPR control − EPR sample)/(EPR control)] × 100 where EPRcontrol is the signal intensity of hydroxyl radical + phosphate buffer and EPRsample is the signal intensity of hydroxyl radical + phenolic extract or catechin. The results were expressed as micromoles of catechin equivalents per gram dry weight of cookies. HPLC-DAD-ESI- MSn Analysis. The identification of the major soluble phenolics (free plus soluble esters) was performed using an Agilent 1100 system (Agilent) equipped with a G1311A quaternary pump, a G1379A degasser, a G1329A ALS automatic sampler, a G1130B ALS Therm, a G1316 Colcom column compartment, a G1315B diode array detector, and a system controller linked to Chem Station Data handling system (Agilent). Separations were performed with a SUPELCOSIL LC-18 column (4.6 × 250 mm × 5 μm, Merck, Darmstadt, Germany). The binary mobile phase consisted of 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The flow rate of mobile phase was 0.5 mL/min, and the elution gradient used was as follows: 0 min, 100% A; 5 min, 90% A; 35 min, 85% A; 45 min, 60% A; held at 60% A from 45 to 50 min; afterward, mobile phase A was increased to 100% at 55 min, followed by column equilibration from 55 to 65 min. The compounds were detected at 280 nm, and the samples C

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however, they have a different qualitative and quantitative polyphenol composition when compared to peanut skins. The addition of 2.5% peanut skins to cookies increased the TPC of the product by almost 50%. Although no recommended daily intake has been established for polyphenols, the extensive literature available lends support for the consumption of dietary sources of polyphenols. DPPH Radical Scavenging Activity Using Electron Paramagnetic Resonance (EPR). The radical scavenging activity results are given in Table 3 and Figure 2A, showing a Figure 1. Moisture content of cookies fortified with peanut skins. Data represent the mean ± standard deviation of each sample (n = 3). Means with different letters indicate significant differences (p < 0.05).

suggesting that the saturation point for moisture retention was achieved. The addition of 2.5% peanut skins resulted in a 50% increase of insoluble fiber content. These data are supported by the high content of insoluble fiber in peanut skins. Dietary fiber intake has been recognized for the reduction of cardiovascular disease risk and colorectal cancer,22,23 management of diabetes, and body weight gain.24,25 The insoluble fiber increase is in good agreement with a previous study reporting the use of peanut skins to fortify peanut butter.9 A negative correlation existed between the addition of peanut skins and the carbohydrate content (r = −0.9663); a 10% reduction in total carbohydrates was noted. According to a randomized intervention study,26 a low-carbohydrate Mediterranean diet improved cardiovascular disease risk factors and diabetes control in overweight patients with type 2 diabetes mellitus. In the present study, the addition of peanut skins to cookies increased the content of insoluble fiber and moisture retention and decreased the total carbohydrate content. Total Phenolic Content. Phenolic compounds are secondary metabolites found in nuts, vegetables, cereals, legumes, and certain vegetable oils as well as in alcoholic and nonalcoholic beverages.17,27−31 The TPC of peanut skins used in the present study as well as their contribution to the TPC of whole peanuts has already been reported.16,32 Although their contribution as a weight percentage of peanuts was very low, around 2.6%,33 the same does not apply for their contribution to the TPC in whole peanuts as their removal during the blanching process induced a reduction of 45% in TPC.32 Most peanuts for industrial purposes, such as production of candies, chocolates, and stuffing, are subjected to a blanching process that removes their skins, and these become a byproduct. In the current study, cookies were fortified with peanut skins, and their contribution to the TPC is summarized in Table 3. Food ingredients such as wheat flour, oatmeal, and chocolate, which have been used in the present study, also have polyphenols;34−36

Figure 2. Electron paramagnetic resonance signals of phenolics of control cookies and cookies fortified with 2.5% peanut skins as evaluated by DPPH (A) and DMPO-OH (B) assays.

decrease of the EPR signal. Although TPC displayed an increase of around 50%, DPPH radical scavenging activity increased by up to 250% upon addition of peanut skins. The increase of both TPC and DPPH scavenging activity demonstrates the potential enhancement of beneficial health effect of the product due to increase in the content of bioactives present. Furthermore, the data suggest that the heating process did not have any detrimental effect on either TPC or scavenging activity. In fact, the phenolic content and antioxidant activity of food products may be increased upon heating.18

Table 3. Total Phenolic Content (TPC), Antioxidant Activity, and Reducing Power (RP) of Cookies Fortified with Peanut Skinsa addition level (%)

TPC (mg CE/g)

DPPH (μmol CE/g)

ABTS (μmol TE/g)

H2O2 (μmol CE/g)

RP (μmol TE/g)

HRSA (μmol CE/g)

control, 0 1.3 1.8 2.5

61.2 ± 3.1d 68.5 ± 1.2c 78.5 ± 2.8b 91.4 ± 2.0a

1028 ± 119d 1715 ± 13.2c 2437 ± 190b 3607 ± 169a

1119 ± 81.0c 1769 ± 40.3b 1986 ± 229b 2579 ± 176a

372.0 ± 14.3d 520.6 ± 19.8c 692.3 ± 12.3b 997.5 ± 22.4a

57.14 ± 5.34d 97.18 ± 7.59c 113.3 ± 5.23b 134.9 ± 3.25a

563 ± 46c 657 ± 6.7b 706 ± 2.9ab 766 ± 6.8a

Data represent mean values for each sample ± standard deviation (n = 3). Means followed by the same letters within a column are not significantly (p > 0.05) different. CE, catechin equivalents; TE, trolox equivalents. HRSA, hydroxyl radical scavenging activity. Results are expressed on a dry weight basis of cookies.

a

D

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Table 4. Content of Soluble Polyphenols (Micrograms per Gram Dry Weight) of Cookies Fortified with Peanut Skinsa,b compound

addition level

phenolic compounds protocatechuic acidc (+)-catechinc (−)-epicatechinc procyanidin dimer Ad procyanidin dimer Bd procyanidin trimer Ad procyanidin tetramer Ad

control, 0% 56.94 ± 2.03a 219.2 ± 3.78a 340.3 ± 19.4b 118.5 ± 2.05b 2708 ± 64.4bc nd nd

1.3% 55.96 ± 0.83a 219.2 ± 8.90a 355.6 ± 11.1ab 131.8 ± 2.23a 2688 ± 35.0c 108.3 ± 1.33c 42.48 ± 0.44c

1.8% 56.61 ± 0.25a 220.1 ± 16.8a 385.1 ± 4.78ab 129.6 ± 2.56a 2826 ± 33.2ab 318.9 ± 6.36b 55.97 ± 4.94b

2.5% 58.14 ± 1.69a 241.7 ± 13.3a 426.0 ± 58.4a 132.7 ± 2.43a 2859 ± 68.0a 601.4 ± 22.7a 173.0 ± 4.98a

total

3443

3601

3992

4492

Data represent mean values for each sample ± standard deviation (n = 3). Means followed by the same letters within a row are not significantly (p > 0.05) different. bThe contents (μg/g) of soluble phenolics in the original peanut skins were 118.9 ± 1.09 (protocatechuic acid); 1211 ± 49.0 ((+)-catechin); 450.7 ± 15.5 ((−)-epicatechin); 3623 ± 60.1 (procyanidin dimer A); 1477 ± 27.5 (procyanidin dimer B); 6494 ± 296 (procyanidin trimer A); 827.6 ± 51.7 (procyanidin tetramer A). cIdentified with authentic standards. dValues are given as catechin equivalents. a

ABTS Radical Cation Scavenging Activity. The antioxidant evaluation carried out using the ABTS method demonstrated the same trend as that from the DPPH assay (Table 3). The total antioxidant activity, which comprises hydrophilic and lipophilic antioxidants, was increased from 1119 to 2579 μmol TE/g. As demonstrated by the proximate composition, peanut skins still have a lipid content of 11%. Tocopherol, which is found in the lipid fraction of peanuts,37 renders antioxidant properties. However, in the present study, the samples were defatted prior to the analysis for phenolics and antioxidant activity; thus, tocopherol is not affecting the assay results. According to Francisco and Resurreccion,10 infusions prepared with Runner type peanut skins had the highest antioxidant activity as evaluated by the ABTS assay when compared to other preparations using Virginia or Spanish cultivars. It is interesting to note that peanut skins have been reported to possess a higher antioxidant activity than green tea.38 Hydrogen Peroxide Scavenging Activity. Hydrogen peroxide in the presence of ferrous ion generates hydroxyl radicals via Fenton’s reaction, which makes the hydrogen peroxide scavenging assay a biologically relevant one. Hydroxyl radicals are highly reactive, leading to changes in DNA39 and inactivating enzymes.40 Furthermore, hydrogen peroxide induces cell damage.41 Data from the present study (Table 3) demonstrated that control cookies already had scavenging activity against hydrogen peroxide, which, as mentioned before, is due to the presence of antioxidant compounds from its ingredients such as wheat flour, oatmeal, and chocolate. Furthermore, the baking process generates Maillard reaction products; these are produced in the presence of reducing sugars and amino acids. Such compounds also have antioxidant properties and may influence the evaluation. However, there was a clear concentration-dependent increase in the ability of phenolic groups in scavenging hydrogen peroxide, which was significant upon addition of peanut skins at 1.3, 1.8, and 2.5%. Reducing Power. The reducing power measures the ability of phenolic compounds to reduce ferric to ferrous ion. As already mentioned, ferrous ions participate in Fenton’s reaction, as well as in the oxidation of proteins and lipids. Phenolic compounds demonstrated the ability to scavenge hydrogen peroxide to different extents. Thus, evaluating the ability of phenolic compounds in preventing oxidative damages through other mechanisms provides useful information to anticipate additional protective effects of the bioactive compounds.

The reducing power of cookies fortified with 2.5% peanut skins rose by 136% (Table 3). The reducing power has been correlated with the degree of hydroxylation and conjugation in the phenolic compounds.42 Peanut skins have a high content of proanthocyanidins, which possess several hydroxyl groups; this may explain the increase in reducing power despite the low addition level of peanut skins. The addition of 2.5% peanut skins in peanut butter9 induced an increase of 747% in reducing power as evaluated by ferric reducing antioxidant power assay (FRAP); however, a direct comparison with the present study is not possible due to the different starting material (peanut butter) and method used by these authors. Whereas the FRAP assay is evaluated in acidic medium (pH 3.6), the method applied in the present study was carried out at pH 6.6. In fact, the antioxidant/ reducing power of phenolic compounds has been demonstrated to be dependent on the pH of the medium.43 Hydroxyl Radical Scavenging Activity. Reactive oxygen species (ROS) are involved in several pathologies and the aging process; among them, hydroxyl radicals are of special concern and, although short-lived, they play an important role in the development of chronic diseases. They can be produced by Fenton’s reaction in the presence of ferrous ions, via decomposition of hydroperoxides, which are intermediate in lipid oxidation process, and UV light. In the present study, the Fenton’s reaction was carried out to produce hydroxyl radicals, and DMPO was used in the reaction medium as a spin trap to enable the study of scavenging activity of phenolics as evaluated by EPR. The addition of peanut skins increased the hydroxyl scavenging activity of cookies (Table 3), which can also be seen by the decrease in the intensity of the EPR signals (Figure 2B). As monomeric flavonoids and proanthocyanidins are the major phenolic compounds in peanut skins, the results were expressed as catechin equivalents. No difference was found between 1.8 and 2.5% addition levels; however, in other experiments, a significant difference was observed, which suggests that the addition of 2.5% would provide more potential health benefits to consumers. The control cookies and those fortified with 2.5% peanut skins had scavenging activities in the range of 563 and 766 μmol CE/g, respectively. Peanut butter fortified with peanut skins also exhibited higher ORAC values. According to Ma et al.,9 peanut butter fortified with dry-blanched peanut skins was more effective in neutralizing peroxyl radicals than that fortified with roasted peanut skins. Another study11 reported similar results E

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Table 5. Sensory Acceptability of Cookies Fortified with Peanut Skinsa addition level (%)

color

odor

taste

texture

overall impression

control, 0 1.3 1.8 2.5

3.7 ± 1.0a 4.1 ± 0.8a 3.9 ± 0.7a 4.2 ± 0.8a

3.9 ± 0.9a 4.0 ± 0.9a 4.0 ± 0.9a 3.9 ± 1.0a

4.1 ± 1.0a 4.2 ± 1.0a 4.1 ± 0.8a 3.9 ± 1.1a

3.9 ± 1.1a 3.9 ± 1.0a 4.0 ± 0.9a 3.6 ± 1.0a

4.0 ± 1.1a 4.1 ± 0.8a 4.2 ± 1.0a 3.9 ± 1.0a

Data represent the mean scores for each sample ± standard deviation (n = 60); means with the same letters within a row are not significantly (p > 0.05) different from each other; 5 = extremely liked, 4 = liked, 3 = neither liked nor disliked, 2 = disliked, and 1 = extremely disliked). Acceptable scores were ≥3 points. a

Figure 3. Complementary information collected during sensory evaluation of cookies fortified with peanut skins: (A) how often do you consume cookies? (B) what influences your cookie purchase decision? (C) would you increase your cookie consumption in the case of having additional health benefits?; (D) which kind of cookie do you consume more often?

using the ORAC assay for peanut butter and peanut paste with added peanut skins. The present study demonstrated increases in the antioxidant activity of cookies in the range of 36−251%, depending on the assay, and all results were statistically significant (p < 0.05), which suggests that cookies with added peanut skins have potential health benefits, yet to be fully substantiated and exploited. HPLC-DAD-ESI-MSn Analysis. The data presented so far have evidenced improvement in the potential health benefits of cookies fortified with peanut skins. Peanut skins have several phenolic compounds, namely, phenolic acids, stilbenes, monomeric flavonoids, and dimeric to oligomeric proanthocyanidins. However, in the present study the major phenolic compounds were evaluated as biomarkers to explain the potential functionality of cookies with added peanut skins (Table 4). Here, we also focused only on the soluble phenolic as they accounted for >90% of total phenolic compounds in peanut skins (data not shown). Protocatechuic acid was positively identified by comparison of its retention time and fragmentation pattern with those of authentic standard. The MS and MS2 spectra of protocatechuic acid gave ions at m/z 153 and 109, respectively. The deprotonated molecular ion [M − H]− of both (+)-catechin and (−)-epicatechin exhibited an m/z signal at 289 and MS2 spectra at m/z 245, and their identities were confirmed according to their different retention times by comparison with those of authentic standards. In accordance with the literature,17,28

protocatechuic acid, (+)-catechin, and (−)-epicatechin showed loss of CO2, giving [M − H − 44]− as their characteristic ion in MS2. The major procyanidins, which consist exclusively of (epi)catechin units, were tentatively identified according to the literature.8,44 Procyanidin dimer A showed a deprotonated molecular ion at m/z 575 and at m/z 449, 423, 407, 289, and 285 in MS2. Procyanidin dimer B exhibited an m/z at 577 and in MS2 at m/z 451, 425, 407, 289, and 287. The MS fragmentation of procyanidin trimer A gave deprotonated ion at m/z 863 and at m/z 737, 711, 693, 575, 559, and 449 in MS2. Procyanidin tetramer A showed an m/z signal at 1149 in MS and at m/z 861, 737, and 575 in MS2. The mass spectra of the most representative compound tentatively identified as procyanidin dimer B is provided as Supporting Information. Prodelphinidins, which have (epi)gallocatechin in their structures, were also present in peanut skins; however, their concentration was very low and had no effect on their concentration in cookies fortified with peanut skins. Protocatechuic acid and (+)-catechin did not show any significant increase (p > 0.05). Protocatechuic acid and (+)-catechin were present at less than 1 and 2% in the free nonconjugated phenolic fraction of peanut skins in the present study (data not shown), which may explain the lack of increase for such compounds in cookies fortified with peanut skins. On the other hand, the content of (−)-epicatechin increased upon F

dx.doi.org/10.1021/jf503625p | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

found in the control samples, were also increased. Sensory evaluation demonstrated no significant difference among the control and the remaining samples; thus, cookies fortified with peanut skins offer promise for possible commercialization.

addition of peanut skins, and a small, but significant, increase was found for procyanidin dimers A and B. The formulation used in the present study included a high proportion of chocolate, which also has a high content of (−)-epicatechin and procyanidins;36 this may explain why the threshold for the increase of (−)-epicatechin and procyanidins B was achieved only with 2.5 and 1.8%, respectively, in comparison with the control. However, procyanidin dimer A, which is the major one in peanut skins, also increased with the lowest addition (1.3%). Furthermore, procyanidin trimers and tetramers were not found in the control sample, but were gradually increased with increasing addition levels of peanut skins. The addition of peanut skins to cookies rendered an increase of up to 30% in major phenolics as evaluated by HPLC-DADESI-MSn. Data from the present study demonstrated that proanthocyanidins accounted for the highest increase in phenolics of cookies fortified with peanut skins. The degree of polymerization of proanthocyanidins has an effect on their absorption. According to Ou and Gu,45 proanthocyanidins with a degree of polymerization of