Influence of different types of acids and pH in the recovery of bioactive compounds in Jabuticaba peel (Plinia cauliflora)

Accepted Manuscript Influence of different types of acids and pH in the recovery of bioactive compounds in Jabuticaba peel (Plinia cauliflora)

Helena D.F.Q. Barros, Andressa M. Baseggio, Célio F.F. Angolini, Gláucia M. Pastore, Cinthia B.B. Cazarin, Mario R. Marostica-Junior PII: DOI: Reference:

S0963-9969(19)30010-9 https://doi.org/10.1016/j.foodres.2019.01.010 FRIN 8200

To appear in:

Food Research International

Received date: Revised date: Accepted date:

15 April 2018 18 December 2018 7 January 2019

Please cite this article as: Helena D.F.Q. Barros, Andressa M. Baseggio, Célio F.F. Angolini, Gláucia M. Pastore, Cinthia B.B. Cazarin, Mario R. Marostica-Junior , Influence of different types of acids and pH in the recovery of bioactive compounds in Jabuticaba peel (Plinia cauliflora). Frin (2018), https://doi.org/10.1016/j.foodres.2019.01.010

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ACCEPTED MANUSCRIPT Influence of different types of acids and pH in the recovery of bioactive compounds in Jabuticaba peel (Plinia cauliflora) BARROS, HELENA D.F.Q.a; BASEGGIO, ANDRESSA M.a; ANGOLINI, CÉLIO F.F.b; PASTORE, GLÁUCIA M.b; CAZARIN, CINTHIA B.B.a; MAROSTICAJUNIOR, MARIO Ra*. a

Department of Food and Nutrition, University of Campinas – UNICAMP, 13083-862 Campinas, SP, Brazil b

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Department of Food Science, University of Campinas – UNICAMP, 13083-862 Campinas, SP, Brazil

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* Corresponding author. Tel.: +55 19 ++55 19 35214059; fax: ++55 19 35214060

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E-mail address: [email protected]

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ABSTRACT

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Jabuticaba peel presents a high content of bioactive compounds such as phenolic acids, flavonoids, and anthocyanins, normally considered as a food residue. Nowadays, there is a great interesting in the recovery of bioactive compounds from food residue due to health benefits of the ingredients produced, environmental issues and economic aspects. For the success of phenolic compounds extraction, the solvent and pH influence recovery of these compounds. However, studies that evaluate the use of different weak acids bioactive compounds recovery are scarce. Thus, the aim of the present work was to evaluate the effect of formic, acetic, and phosphoric acids addition in the extraction solvent, to adjust the pH to 1.0, 2.0 and 3.0, in bioactive compounds recovery and antioxidant capacity of jabuticaba peel. The extracts were analyzed as antioxidant capacity (ORAC, FRAP), total phenols content monomeric anthocyanin’s and a qualitative analysis of phenolics by Liquid Chromatography with mass spectrometry (LC-MS). The kind of acid used in the extraction process affected mainly in the extraction of anthocyanins. The acid that presented a better recovery of anthocyanin (3.4 mg/g raw material) and a better antioxidant capacity (ORAC) (841 µmol TE/g raw material) was formic acid in pH 1.0. Keywords: phenolic compounds; extraction; acidified solvent; pH; jabuticaba peel; recovery; antioxidant activity; bioactive compounds.

ACCEPTED MANUSCRIPT 1. INTRODUCTION High amounts of bioactive compounds can be found in generally nonedible fractions of fruits and vegetables, as seeds or peel. These compounds have a high antioxidant capacity, what can protect cells from oxidative stress (Liu, 2003) or still, influence directly in gene expression and consequently,

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causing changes in several cell mechanisms (Pandima Devi et al., 2017). Due

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to this, the use of these residues has received attention, to generate new by-

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products with capacity for use in food or cosmetics formulations, for example (R. G. C. Barros, Andrade, Denadai, Nunes, & Narain, 2017; Moure et al., Mainly, several berries residues present a high content of phenolic

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2001).

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compounds, as pigments (anthocyanins) and phenolic acids (Tian et al., 2017). The jabuticaba, also known as “Brazilian berry” is a small dark-colored

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fruit native to Brazil belongs to the Myrtaceae family. The fruit pulp has a sweet

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taste, due to the high sugar content. The peel, which is commonly not consumed, concentrates large amounts of anthocyanins, as cyanidin and delphinidin (Baseggio et al., 2018), and phenolic acids, as ellagic and gallic

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acids (da Silva et al., 2017; Leite-Legatti et al., 2012; Plaza et al., 2016). In this

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context, studies demonstrated the direct relationship between jabuticaba peel consumption and health improvement (Baseggio et al., 2018; Batista et al., 2014; Batista et al., 2017; Dragano et al., 2013; Lamas et al., 2018; Lenquiste, Batista, Marineli, Dragano, & Maróstica, 2012). The extraction procedure is the first and most important step to recovery the phenolic compounds from the plant matrix (Cong Cong, Bing, Yi Qiong, Jian Sheng, & Tong, 2017). Ultrasound is an important technology that achieves the

ACCEPTED MANUSCRIPT objective of sustainable green chemistry and extraction due to the reduced use of the solvent, reduction of unit operations, reduction of extraction time and energy employed (Chemat et al., 2017). The principle of ultrasound-assisted extraction (UAE) consists mainly in the sample and solvent molecular movement due to acoustic cavitation. The agitation of solvent promoted by

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ultrasound increases the contact between the solvent and the compounds of

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interest, improving the extraction efficiency (Chen et al., 2007).

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Extraction efficiency can be directly affected by the sonication time, temperature, and by the type of solvent applied (polarity) (Cong Cong, Bing, Yi

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Qiong, Jian Sheng, & Tong, 2017). In this context, solutions contained water and ethanol are frequently used in different polyphenol extraction techniques

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(Lao & Giusti, 2018). In mixtures, each solvent will exert a specific role in the extraction, ethanol enhances the solubility of phenolic compounds, while water

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aids the desorption of the solute from the sample (Mustafa & Turner, 2011). In

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addition, even that temperature and extraction time affects the extraction efficiency, studies already demonstrated that elevated temperatures and

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prolonged extraction time affect negatively phenolic compounds, leading to a decrease in extraction yield mainly due to the degradation of these compounds

2005).

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(Blackhall, Berry, Davies, & Walls, 2018; Lapornik, Prošek, & Golc Wondra,

The addition of acid to the extraction solvent can increases the phenolic compounds extraction, in special anthocyanin’s, for two mechanisms: 1) the acidic environment leads to cell membrane denaturation increasing the interaction between the solvent and the compound and 2) the free hydrogen ions leads to stabilization of flavylium cation form of the anthocyanin (Blackhall

ACCEPTED MANUSCRIPT et al., 2018). Usually strong acids are applied to acidify the extraction solvent, such as hydrochloric and sulfuric acids. However, anthocyanin’s are degraded in solvents containing mineral acids and should be extracted with solvents acidified with organic acids such as acetic and formic acid (Salamon, Mariychuk, & Grulova, 2015).

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Several studies evaluated the acidification effects in the extraction

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process using a single acid and/or one pH condition (Ferarsa et al., 2018;

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Garcia-Mendoza et al., 2017; Lao & Giusti, 2018; Schulz et al., 2015). However, until the moment, no study was performed to evaluate if the type of acid applied

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to acidify solvent of extraction exerts positive effects in the recovery of anthocyanin’s and phenolic compounds, and consequently in the antioxidant

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capacity of the extract from jabuticaba powder peel (JPP). Thus, this work aimed to evaluate the effect of acid type (formic acid, acetic acid, and

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phosphoric acid) in extraction solvent, concomitantly to changes in pH, in the

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recovery of bioactive compounds and antioxidant capacity of jabuticaba peel.

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2. MATERIAL AND METHODS 2.1 SAMPLE PREPARATION

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Jabuticaba fruits were obtained from Casa Branca (São Paulo state – Brazil, 21º 46' 26" S; 47º 05' 11" W) in 2016. The fruits were manually washed with water, the peel was separated and then frozen at – 20 °C. The frozen peel was freeze-dried (LP 1010, Liobrás, São Carlos/São Paulo/Brazil), grounded into a powder, sieved (12 mesh) and stored in dark airtight flasks at - 20°C until analyses. 2.2 PROXIMATE COMPOSITION

ACCEPTED MANUSCRIPT The proximate composition of freeze-dried jabuticaba powder peel (JPP) was determined by analyses of moisture, total protein and ash according to (AOAC, 1997) and total lipids (Bligh & Dyer, 1959). Carbohydrates were calculated by difference.

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2.3 EXTRACTION PROCEDURE

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2.3.1 Selection of extraction parameters

Extraction solvent was selected based on studies (Garcia-Mendoza et

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al., 2017; Machado, Pasquel-Reátegui, Barbero, & Martínez, 2015; Rabelo,

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Machado, Martínez, & Hubinger, 2016) which demonstrated that hydroalcoholic mixture (50 v/v) is more efficient than pure solvents in the extraction of

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amphiphilic or moderate polar molecules, such as polyphenols. Mass transfer is a time-dependent process, and excessive extraction time can degraded and

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oxidized the phenolic compounds (Xu, Burton, Kim, & Sismour, 2015). Based

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on this, extraction time was selected according to Dias et al., (2017); Lao & Giusti, (2018); Mohd Jusoh et al., (2018) that obtained satisfactory recovery of

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phenolic acids with one hour of extraction. To select the acids employed on the extraction process, it was

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considered that the extraction of phenolic compounds, especially anthocyanin’s, must be performed with solvents that avoid any chemical modification, once hydrochloric acid may hydrolyze acylated anthocyanins (Salamon et al., 2015). For this reason, to prevent or at least minimize the breakdown of acylated anthocyanins, we have selected organic acids commonly used in foods and considered as GRAS (Generally recognized as safe), such as formic acid, acetic acid and orthophosphoric acid (Quitmann, Fan, & Czermak, 2014).

ACCEPTED MANUSCRIPT The other parameter evaluated was the influence of pH in the recovery of phenolic compounds. Studies performed with acidified solvents does not present a standard range of pH applied, it can vary from pH 2.0 – 2.5 (F. Barros, Dykes, Awika, & Rooney, 2013; Garcia-Mendoza et al., 2017; Pereira, Tarone, Cazarin, Barbero, & Martínez, 2019) until application of different

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percentages of acid (from 0.1 to 15 %) (Đurović et al., 2018; Mokrani & Madani,

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2016; Rao et al., 2018; Ryu & Koh, 2018; Sánchez Maldonado, Mudge, Gänzle,

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& Schieber, 2014) in the solvent extraction, with no mention on the final pH value of the solvent. Based on this piece of information, , three values of pH in

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the final mixture (sample + extraction solvent) were selected 1.0, 2.0 and 3.0 with the intention of evaluating which one is more effective in phenolic

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compounds recovery. The total volume of acid added varied according to pH and acid type: for pH 1.0, the minor acid volume was 1mL for orthophosphoric

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acid while 11mL was the highest volume added, for acetic acid; for pH 2.0, the

for

pH

3,

the

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acid range variated from <1mL to formic acid until 7mL, for acetic acid. Finally, acid

volume

added

oscillated

since

<1mL

for

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formic/orthophosphoric acid until 4mL for acetic acid.

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2.3.2 Ultrasound Assisted Extraction (UAE) JPP was subjected to extraction using a hydroalcoholic mixture (50 v/v). To evaluate the effect of acidified media to increase the recovery of phenolic compounds, three different acids were applied formic acid 88 % (LabSynth, Diadema, São Paulo, Brazil), acetic acid 99.7 % (LabSynth, Diadema, São Paulo, Brazil) and orthophosphoric acid 85 % (Merck, Darmstadt, Germany) in three pH ranges: 1.0, 2.0 and 3.0. Extract without acid addition was used as control (pH: 4).

ACCEPTED MANUSCRIPT The extraction procedure was performed in an ultrasonic bath (USC1800A, Unique, Indaiatuba, São Paulo, Brazil) with frequency and powder fixed at 40 kHz and 150 W, respectively. In all experiments, 0.5 g of freezedried jabuticaba powder peel was mixed with 25 mL of a hydroalcoholic mixture (50 v/v) in beakers protected from light and sonicated for 60 minutes at ambient

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pressure and 30 °C. Acids were added to the hydroalcoholic mixture containing

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JPP until reaches the pH range intended. After the process, all the extracts

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were filtered using Whatman No. 4 filter paper. All the extractions were performed in duplicate. The representative scheme of the extraction procedure

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is presented in Figure 1.

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2.4 EXTRACTS ANALYSES

2.4.1 Colorimetric and fluorometric analyses

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For extracts evaluation analyses were performed using BioTek Synergy

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HT Microplate Reader (Winooski, USA) coupled to the data software program Gen5™ 2.0. Antioxidant activity was determined by ORAC (Ou, Hampsch-

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Woodill, & Prior, 2001) and FRAP (Benzie & Strain, 1996) assay. Phenolic content was determinate according to Singleton, Orthofer, and Lamuela-

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Raventós (1999) with some adaptations to microplate assay. The absorbance was read at 725 nm. Monomeric anthocyanins were determinate in absorbance readings at 520 and 700 nm (Francis, 1989). 2.4.2 Liquid Chromatographic – Mass spectrometry (LC-MS) analysis of peel extracts Extracts

were

analyzed

by

Liquid

Chromatography

with

mass

spectrometry. Samples were diluted to a 10 – 25.6 mg mL-1 extract solution and

ACCEPTED MANUSCRIPT 1 µL were injected. A Poroshell 120 SB-Aq 2.7 μm column (2.1 x 100 mm, Agilent) was used to performe the LC-MS/MS analysis in an UHPLC (Hewlett Packard, Agilent Technologies 1290 series) coupled to a Q-ToF iFunnel 6550 mass spectrometer using electrospray ionization (ESI) source. Mobile phase A was milli-Q water with 0.1 % of formic acid (FA); and mobile phase B was

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acetonitrile (ACN) with 0.1 % of FA. The flow rate of 0.45 mL min-1 was used

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following the linear gradient: 0 - 1 min, 5% B; 1–10 min, 5 % B to 18 % B; 10-13

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min 18 % B to 70 % B; 13-15 min, 70 % B to 100 % B; 15-17 min, 100 % B and 3 min of post time at 5 % B to column re-equilibration. The mass spectrometer

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voltages and temperatures was set as: VCap 3000 V; fragmentor voltage at 150 V; OCT 1RF Vpp at 750 V; Gas Temperature at 290 °C; Sheath Gas

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Temperature at 350 °C; Drying Gas at 12 L min-1 and the fragmentation were performed using normalized collision energy (NCE) of 30. Mass spectra were

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acquired in profile and negative ion mode and the acquisition range was 100 -

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1200 m/z. Data was treated using Agilent MassHunter Qualitative Analysis B0.7 software.

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2.5 STATISTICAL ANALYSES

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Analyses of the influence of the parameters on total phenolic content, monomeric anthocyanin and antioxidant activity were performed by analysis of variance (ANOVA) using the Minitab 16® software (Minitab Inc., State College, PA, USA) with a 95 % confidence level (p-value ≤0.05). The parameters were evaluated with a randomized full factorial design (3 × 3) with pH (1, 2 and 3), and kind of acid solvent (formic acid, acetic acid and phosphoric acid), with 18 experimental runs. The acidified extracts were compared with a control sample (without acid) to evaluate if the acid employed on the extraction process

ACCEPTED MANUSCRIPT interferes on recovery of bioactive compounds by one-way ANOVA test for variances analysis were performed, followed by Tukey test to compare averages, with significance of 5 %. Software GraphPad Prism 5.0 was used. Unsupervised segregation was assessed with principal components analysis (PCA) using mean-centred scaled data and it was performed through

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MetaboAnalyst 4.0 (www.metaboanalyst.ca). The matrix data for PCA was

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created using software profinder. PCA data were visualized by plotting the PCA

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scores in which each point represents one sample. The biplot was visualized by plotting the PCA and the variables contribution gives, therefore, an indication of

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3. RESULTS AND DISCUSSION

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which compounds that most strongly influence the patterns in the score plot.

3.1 CHARACTERIZATION OF JABUTICABA PEEL

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The proximate composition of freeze-dried jabuticaba peel is presented

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in Table 1. Carbohydrate is the main constituent (85 % m/m), and other constituents were found in smaller proportions, proteins (7.32 %), ashes (1.97

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%) and lipids (1.77 %). The protein and lipid results are in according to the literature data related by Lenquiste et al. (2015), 7.31 % and 1.72 %,

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respectively.

3.2 EXTRACTS ANALYSIS

3.2.1 Effects of process parameters on antioxidant capacity, phenolic content and monomeric anthocyanin’s Table 2 presents the analysis results for antioxidant activity, total phenolic compounds and monomeric anthocyanin’s content for each extract. The results of the analysis of variance (ANOVA, α = 0.05) indicated that kind of

ACCEPTED MANUSCRIPT solvent (acid employed) was significant to ORAC and to total phenolic assays (p < 0.000). The pH applied for the extracts was significant to FRAP and total phenolic content (p < 0.004) and the interaction between solvent (acid employed) and pH was significant to all the parameters (p < 0.000) except to anthocyanin’s (p > 0.2).

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Figure 2 shows the process parameters effects on the antioxidant

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capacity and bioactive content in the extracts. The total bioactive compounds

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and antioxidant capacity observed in the extracts varied according to the kind of acid employed in the extraction process as well as with the extract pH.

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When Figure 2 (a) and (b) is evaluated, it is possible to observe an

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opposite behavior among the antioxidant methods applied. For the ORAC method, higher antioxidant activity was measured for formic acid followed by

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acetic acid and finally for orthophosphoric acid. Whereas for the FRAP method,

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the acidified extract with orthophosphoric acid presented higher antioxidant activity, and the extracts prepared with acetic and formic acid have lower

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antioxidant activity.

This difference in behavior between both antioxidant methods can be

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explained for two factors: 1) the measurement of antioxidant activity by FRAP method consists on the ability of analyzed samples antioxidants in reduces the ferric 2,4,6-tripyridyl-s-triazine complex [Fe(III)-(TPTZ)2]3+ to the intensely blue colored ferrous complex [Fe(II)-(TPTZ)2]2+. This reaction is performed in 30 minutes (Benzie & Strain, 1996). Pulido, Bravo, and Saura-Calixto (2000) observed that few phenolic compounds, such as quercetin do not reduce the Fe(III) at a rate fast enough to allow it is measurement within the observation

ACCEPTED MANUSCRIPT time of 30 minutes requiring longer reaction times (≥30min) for total quantification. The second factor is the reduction of Fe(III)/ Fe(II) occurs much faster in the presence of orthophosphoric acid (Cher & Davidson, 1955). The two factors explained above justifies the reason why the extract acidified with orthophosphoric acid presets higher antioxidant activity for FRAP assay.

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The determination of antioxidant capacity in vitro of a complex matrix, as

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an extract is difficult, once may be influenced by the chemical complexity of the

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antioxidant compounds presents. Despite this, the ORAC method is the most indicated one to mimic the antioxidant activity of phenol in biological systems

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since it is adopted biologically relevant free radicals and integrates both time

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and degree of activity of antioxidants (Dai & Mumper, 2010). 3.2.2 Interaction between pH and acid employed on antioxidant activity and bioactive compounds content on the extracts

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The interaction between pH and type of acid is available in Figure 3.

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According to Figure 3, the amount of total phenolic content in pH 3.0 have increased for all types of acid employed, except for formic acid, in which is

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observed a reduction in the total phenolic content.

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The most significant recovery of phenolic compounds was obtained with solvent acidification with acetic acid until pH 3.0, which was accomplished with good FRAP and ORAC values, demonstrating the influence of phenolic concentration in antioxidant capacity. The same comportment was observed by Garcia-Mendoza

et

al.

(2017),

where

the

high

phenolic

compounds

concentration, obtained with acidified solvent, was related to the highest values for ORAC assay.

A study performed by Tian et al. (2017) showed that

acidification of aqueous ethanol with 1% acetic acid presented high efficiency in

ACCEPTED MANUSCRIPT extracting phenolic compounds from berries, recommending the use of this solvent mixture for food application. It is widely accepted that the amount of phenolic compounds in foods is related to a significant antioxidant activity (Xu et al., 2015). The solubility of the phenolic compounds is dependent on the chemical nature of the plant sample,

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also by the polarity of the solvent employed in the extraction process. The

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chemical structure of phenolic compounds in plant material can range from

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simple compounds (such as phenolic acids and anthocyanin’s) to polymerized compounds (tannins) in different amounts. Therefore, depending on the solvent

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used during extraction different phenolic compounds will be solubilized and

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consequently extracted from the plant matrix (Dai & Mumper, 2010) Acid conditions may enhance the solubility of phenolic compounds due to

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the rupture of the cell wall that increases the extraction yield, and,

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consequently, the rate of bioactive compounds diffusion into the solvent (Ribeiro et al., 2018). The best recover of monomeric anthocyanins were found in extraction performed in pH 1.0, using formic acid for acidification, what was

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accomplished with a high ORAC value. The great relation obtained between

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ORAC assay and anthocyanins concentration could indicate the extract application as a food ingredient, both as a natural colorant and an antioxidant (Abdel-Aal, Hucl, & Rabalski, 2018). In the same way, the best condition for monomeric anthocyanins recovery in jabuticaba peel obtained by Rodrigues, Fernandes, de Brito, Sousa, and Narain (2015) was in HCl acidified ethanol:water (46%), pH 1.0.

ACCEPTED MANUSCRIPT Concerning the anthocyanin’s the pH possess a strong influence since their structures can undergo transformation when the pH of the solution varies. At pH 1.0 anthocyanin’s are predominantly in the flavylium cation form, whereas the proportion of this form significantly decreases at pH 3.0. Therefore, in acidified solvent extractions, special care should be taken to avoid

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anthocyanin’s degradation (Castañeda-Ovando, Pacheco-Hernández, Páez-

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Hernández, Rodríguez, & Galán-Vidal, 2009)

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Despite the use of acetic acid until pH 2.0 (sample 5) have extracted a low content of total phenolic, a great ORAC value was observed in this

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condition. This fact could be attributed to the high myricetin-O-rhamnoside abundance found in the extract (Table 4), once this flavonol presents a high (Tabart, Kevers, Pincemail,

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Defraigne, & Dommes, 2009).

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capacity to scavenging the peroxyl radical

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The commonly added acids for phenols extraction are hydrochloric acid, citric acid, formic acid, and trifluoroacetic acid. In the present work, solvents that are less toxic were selected to ensure its application for in food products, as

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formic acid, orthophosphoric, and acetic acid. Our results demonstrate that the

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acid choice was as crucial as the pH of the medium since the lowest concentration of anthocyanins and ORAC values was also found at pH 1.0, although with orthophosphoric acid for acidification. 3.3 PROFILE OF PHENOLIC COMPOUNDS Table 3 shows the tentative of identification of all ESI(-)-MS compounds by their exact mass and fragmentation pattern, while Table 4 shows the abundance of the phenolic compounds identified in each extract produced. The

ACCEPTED MANUSCRIPT identified compounds were phenolic acids, as ellagic acid and it is related compounds, flavonoids, like quercetin and its isomers, myricetin and anthocyanin’s. The intense purple color verified in jabuticaba peel has been attracted the attention for the anthocyanins responsible for this characteristic, either due

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to technological applications such natural pigments in foods or due to the

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possible health benefits attributed to this compounds. In this context, several

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studies involving jabuticaba peel identified and quantified the delphinidin-3glucoside and the cyanidin-3-glucoside in this matrix, (Baseggio et al., 2018;

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Inada et al., 2015; Plaza et al., 2016), what is according with the observed in this study. Is known that cyanidin and delphinidin are the anthocyanidins, the

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aglycone form of anthocyanins, most distributed in plants, with abundance

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approximately of 50% and 12%, respectively (Castañeda-Ovando et al., 2009)

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The best condition for recovery of delphinidin-3-glucoside was found in lowest pH (1.0), independently of acid employed. The form in which extremes pH values, reaches with acid employment in solvents, can improve the

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extraction efficacy is due to the stabilization of flavylium cation anthocyanin

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form, the denaturation of cell membranes, generated by pH decrease, what possibilities the more interaction between solvent:solute (Blackhall et al., 2018). Similar behavior was observed for cyanidin-3-glucoside, but in this case, the no acidified solvent was also able to recover a large amount of this anthocyanin. The use of weak acids (as acetic and formic acids) could improve the extraction of different and more anthocyanins, once due to these conditions the acylated and 3,5-diglc anthocyanins are preserved against hydrolysis (CastañedaOvando et al., 2009).

ACCEPTED MANUSCRIPT We also found the higher abundance of the flavanol myricetin-Orhamnoside (m/z 301.0365) in extracts with pH 2.0, with all acids employed. This fact could be explained due to the physicochemical compounds characteristics: the myricetin is most stable at pH 2.0, while the substance degradation is related with pH increase (Yao et al., 2014). In another hand, the

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extracts prepared using orthophosphoric acid do not extract the flavanol

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quercetin-C-rhamnoside for any pH applied (Table 4), showing that the use of

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orthophosphoric acid as the acidifying agent to quercetin extraction is less efficient than formic and acetic acid.

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All the applied conditions can recovery ellagic acid and derivatives in great amounts, being the extraction with orthophosphoric acid until pH 3.0 the

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condition with the largest pure compound abundance. The recovery of ellagic acid is improved in acidified solvents (Rodrigues et al., 2015), while the ellagic

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acid content seems to reduce with the pH increase, being that how much basic

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is the solvent, higher is degradation product formation (Panichayupakaranant, Itsuriya, & Sirikatitham, 2010).

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A principal component analysis (PCA) was performed with intention of

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correlating the extraction condition with the phenolic compounds profile of the jabuticaba’s peel extracts. The PCA presents the profile of the different extracts in relation to the pH variation and kind of acid used in the extraction process (Figure 4). The dimensions presented in the PCA graph explain 83.7 % of the original variance data. Lao, Giusti (2018) evaluated the extraction of phenolic compounds in purple corn using different concentrations of ethanol/water (EtOH/H2O) acidified

ACCEPTED MANUSCRIPT with 0.01 % HCl, for the best extraction condition, 50 % EtOH/H 2O, they had an amount of 14.3 mg of monomeric anthocyanin/g of sample. The content of anthocyanins in the present study varied from 0.1 to 3.4 mg/g depending on the acid employed and pH condition. The difference in the amount of anthocyanins obtained in the present

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study compared to the aforementioned can be explained may be due to the fact

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that Lao and Giusti (2018) made an exhaustive extraction (until obtaining a

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faintly colored filtrate). In addition, they use HCl as an acidifying agent, the study developed by Oancea, Stoia, and Coman (2012) noticed that using

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hydrochloric acid instead of acetic acid improves the anthocyanin extraction

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yield. 4. CONCLUSIONS

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The kind of acid used in the extraction process affected mainly in the

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extraction of anthocyanins. The acid that presented a better recovery of anthocyanin and a better antioxidant capacity (ORAC) was formic acid in pH

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1.0. It is well known that the application of weak organic acids during the extraction process can acylate the anthocyanins, protecting them of the

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nucleophilic attack by the water and sulfite in the media, contributing to the high anthocyanin stability. For further studies, we suggest a stability analysis of the extracts to confirm which acid employed on the extraction process (formic, acetic and phosphoric) is more effective in phenolic compounds stability. ACKNOWLEDGEMENTS HDFQB

thanks

São

Paulo

Research

Foundation

(FAPESP)

(2017/04231-8) for the Ph.D. scholarship. C.F.F.A. acknowledges FAPESP for

ACCEPTED MANUSCRIPT Post-doc

scholarship

(2017/10753-7).

MRMJ

acknowledges

CNPq

301108/2016-1 for support. FORMATTING OF FUNDING SOURCES The

Faepex

Unicamp

(2734/17),

FAPESP

(2017/04231-8

and

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2015/50333-1), supported this work. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil

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(CAPES) - Finance Code 001.

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REFERENCES

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Blackhall, M. L., Berry, R., Davies, N. W., & Walls, J. T. (2018). Optimized extraction of anthocyanins from Reid Fruits’ Prunus avium ‘Lapins’ cherries. Food Chemistry, 256, 280-285. doi: https://doi.org/10.1016/j.foodchem.2018.02.137 Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911917. https://doi.org/10.1139/o59-099 Castañeda-Ovando, A., Pacheco-Hernández, M. d. L., Páez-Hernández, M. E., Rodríguez, J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food Chemistry, 113(4), 859-871. doi: https://doi.org/10.1016/j.foodchem.2008.09.001 Chemat, F., Rombaut, N., Sicaire, A. G., Meullemiestre, A., Fabiano-Tixier, A. S., & Abert-Vian, M. (2017). Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrason Sonochem, 34, 540-560. doi: 10.1016/j.ultsonch.2016.06.035 Chen, F., Sun, Y., Zhao, G., Liao, X., Hu, X., Wu, J., & Wang, Z. (2007). Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using highperformance liquid chromatography–mass spectrometry. Ultrasonics Sonochemistry, 14(6), 767-778. doi: https://doi.org/10.1016/j.ultsonch.2006.12.011 Cher, M., & Davidson, N. (1955). The Kinetics of the Oxygenation of Ferrous Iron in Phosphoric Acid Solution. Journal of the American Chemical Society, 77(3), 793-798. doi: 10.1021/ja01608a086 da Silva, J. K., Batista, Â. G., Cazarin, C. B. B., Dionísio, A. P., de Brito, E. S., Marques, A. T. B., & Maróstica Junior, M. R. (2017). Functional tea from a Brazilian berry: Overview of the bioactives compounds. LWT - Food Science and Technology, 76, 292-298. doi: http://dx.doi.org/10.1016/j.lwt.2016.06.016 Dai, J., & Mumper, R. J. (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules, 15(10), 7313-7352. doi: 10.3390/molecules15107313 Dias, A. L. B., Arroio Sergio, C. S., Santos, P., Barbero, G. F., Rezende, C. A., & Martínez, J. (2017). Ultrasound-assisted extraction of bioactive compounds from dedo de moça pepper (Capsicum baccatum L.): Effects on the vegetable matrix and mathematical modeling. Journal of Food Engineering, 198, 36-44. doi: https://doi.org/10.1016/j.jfoodeng.2016.11.020 Dragano, N. R. V., Marques, A. y. C., Cintra, D. E. C., Solon, C., Morari, J., Leite-Legatti, A. V., . . . Maróstica-Júnior, M. R. (2013). Freeze-dried jaboticaba peel powder improves insulin sensitivity in high-fat-fed mice. British Journal of Nutrition, 110(3), 447-455. doi: 10.1017/S0007114512005090 Đurović, S., Nikolić, B., Luković, N., Jovanović, J., Stefanović, A., Šekuljica, N., . . . Knežević-Jugović, Z. (2018). The impact of high-power ultrasound and microwave on the phenolic acid profile and antioxidant activity of the extract from yellow soybean seeds. Industrial Crops and Products, 122, 223-231. doi: https://doi.org/10.1016/j.indcrop.2018.05.078

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Ferarsa, S., Zhang, W., Moulai-Mostefa, N., Ding, L., Jaffrin, M. Y., & Grimi, N. (2018). Recovery of anthocyanins and other phenolic compounds from purple eggplant peels and pulps using ultrasonic-assisted extraction. Food and Bioproducts Processing, 109, 19-28. doi: 10.1016/j.fbp.2018.02.006 Francis, F. J. (1989). Food colorants: anthocyanins. Crit Rev Food Sci Nutr, 28(4), 273-314. doi: 10.1080/10408398909527503 Garcia-Mendoza, M. d. P., Espinosa-Pardo, F. A., Baseggio, A. M., Barbero, G. F., Maróstica Junior, M. R., Rostagno, M. A., & Martínez, J. (2017). Extraction of phenolic compounds and anthocyanins from juçara (Euterpe edulis Mart.) residues using pressurized liquids and supercritical fluids. The Journal of Supercritical Fluids, 119, 9-16. doi: https://doi.org/10.1016/j.supflu.2016.08.014 Inada, K. O. P., Oliveira, A. A., Revorêdo, T. B., Martins, A. B. N., Lacerda, E. C. Q., Freire, A. S., . . . Monteiro, M. C. (2015). Screening of the chemical composition and occurring antioxidants in jabuticaba (Myrciaria jaboticaba) and jussara (Euterpe edulis) fruits and their fractions. J. Functional Foods, 17, 422-433. doi: http://dx.doi.org/10.1016/j.jff.2015.06.002 Lamas, C. A., Lenquiste, S. A., Baseggio, A. M., Cuquetto-Leite, L., Kido, L. A., Aguiar, A. C., . . . Cagnon, V. H. A. (2018). Jaboticaba extract prevents prediabetes and liver steatosis in high-fat-fed aging mice. Journal of Functional Foods, 47, 434-446. doi: https://doi.org/10.1016/j.jff.2018.06.005 Lao, F., & Giusti, M. M. (2018). Extraction of purple corn (Zea mays L.) cob pigments and phenolic compounds using food-friendly solvents. Journal of Cereal Science, 80, 87-93. doi: https://doi.org/10.1016/j.jcs.2018.01.001 Lapornik, B., Prošek, M., & Golc Wondra, A. (2005). Comparison of extracts prepared from plant by-products using different solvents and extraction time. Journal of Food Engineering, 71(2), 214-222. doi: https://doi.org/10.1016/j.jfoodeng.2004.10.036 Leite-Legatti, A. V., Batista, Â. G., Dragano, N. R. V., Marques, A. C., Malta, L. G., Riccio, M. F., . . . Maróstica, M. R. (2012). Jaboticaba peel: Antioxidant compounds, antiproliferative and antimutagenic activities. Food Research International, 49(1), 596-603. doi: http://dx.doi.org/10.1016/j.foodres.2012.07.044 Lenquiste, S. A., Batista, Â. G., Marineli, R. d. S., Dragano, N. R. V., & Maróstica, M. R. (2012). Freeze-dried jaboticaba peel added to high-fat diet increases HDL-cholesterol and improves insulin resistance in obese rats. Food Research International, 49(1), 153-160. doi: http://dx.doi.org/10.1016/j.foodres.2012.07.052 Lenquiste, S. A., Marineli, R. d. S., Moraes, É. A., Dionísio, A. P., Brito, E. S. d., & Maróstica Junior, M. R. (2015). Jaboticaba peel and jaboticaba peel aqueous extract shows in vitro and in vivo antioxidant properties in obesity model. Food Research International, 77, Part 2, 162-170. doi: https://doi.org/10.1016/j.foodres.2015.07.023 Machado, A. P. D. F., Pasquel-Reátegui, J. L., Barbero, G. F., & Martínez, J. (2015). Pressurized liquid extraction of bioactive compounds from blackberry (Rubus fruticosus L.) residues: a comparison with

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conventional methods. Food Research International, 77, 675-683. doi: https://doi.org/10.1016/j.foodres.2014.12.042 Mohd Jusoh, A.A., I., Khairuddin N, D.N, A. Z., Hashim Z, N.A., M., . . . I.I;, M. (2018). Microwave Power and Extraction Time on Microwave-Assisted Extraction of Hibiscus rosa-sinensis. Chemical Engineering Transactions, 93, 541-546. doi: 10.3303/CET1863091 Mokrani, A., & Madani, K. (2016). Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit. Separation and Purification Technology, 162, 68-76. doi: https://doi.org/10.1016/j.seppur.2016.01.043 Moure, A., Cruz, J. M., Franco, ., om nguez, J. M., Sineiro, J., om nguez, H., . . . Parajó, J. C. (2001). Natural antioxidants from residual sources. Food Chemistry, 72(2), 145-171. doi: https://doi.org/10.1016/S03088146(00)00223-5 Oancea, S., Stoia, M., & Coman, D. (2012). Effects of Extraction Conditions on Bioactive Anthocyanin Content of Vaccinium Corymbosum in the Perspective of Food Applications. Procedia Engineering, 42, 489-495. doi: https://doi.org/10.1016/j.proeng.2012.07.440 Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. Journal of Agricultural and Food Chemistry, 49(10), 4619-4626. doi: 10.1021/jf010586o Panichayupakaranant, P., Itsuriya, A., & Sirikatitham, A. (2010). Preparation method and stability of ellagic acid-rich pomegranate fruit peel extract. Pharm Biol, 48(2), 201-205. doi: 10.3109/13880200903078503 Pereira, D. T. V., Tarone, A. G., Cazarin, C. B. B., Barbero, G. F., & Martínez, J. (2019). Pressurized liquid extraction of bioactive compounds from grape marc. Journal of Food Engineering, 240, 105-113. doi: https://doi.org/10.1016/j.jfoodeng.2018.07.019 Plaza, M., Batista, Â. G., Cazarin, C. B. B., Sandahl, M., Turner, C., Östman, E., & Maróstica Júnior, M. R. (2016). Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study. Food Chemistry, 211, 185-197. doi: http://dx.doi.org/10.1016/j.foodchem.2016.04.142 Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant Activity of Dietary Polyphenols As Determined by a Modified Ferric Reducing/Antioxidant Power Assay. Journal of Agricultural and Food Chemistry, 48(8), 33963402. doi: 10.1021/jf9913458 Quitmann, H., Fan, R., & Czermak, P. (2014). Acidic Organic Compounds in Beverage, Food, and Feed Production. In H. Zorn & P. Czermak (Eds.), Biotechnology of Food and Feed Additives (pp. 91-141). Berlin, Heidelberg: Springer Berlin Heidelberg. doi: 10.1007/10_2013_262 Rabelo, R. S., Machado, M. T. C., Martínez, J., & Hubinger, M. D. (2016). Ultrasound assisted extraction and nanofiltration of phenolic compounds from artichoke solid wastes. Journal of Food Engineering, 178, 170-180. doi: https://doi.org/10.1016/j.jfoodeng.2016.01.018 Rao, S., Santhakumar, A. B., Chinkwo, K. A., Wu, G., Johnson, S. K., & Blanchard, C. L. (2018). Characterization of phenolic compounds and antioxidant activity in sorghum grains. Journal of Cereal Science, 84, 103-111. doi: https://doi.org/10.1016/j.jcs.2018.07.013

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Ribeiro, L. O., Pereira, R. N. C., Tonon, R. V., Cabral, L. M. C., Santiago, M. C. P. A., Vicente, A. A., . . . Freitas, S. P. (2018). Antioxidant Compounds Recovery from Juçara Residue by Thermal Assisted Extraction. [journal article]. Plant Foods for Human Nutrition, 73(1), 68-73. doi: 10.1007/s11130-017-0651-0 Rodrigues, S., Fernandes, F. A. N., de Brito, E. S., Sousa, A. D., & Narain, N. (2015). Ultrasound extraction of phenolics and anthocyanins from jabuticaba peel. Industrial Crops and Products, 69, 400-407. doi: https://doi.org/10.1016/j.indcrop.2015.02.059 Ryu, D., & Koh, E. (2018). Application of response surface methodology to acidified water extraction of black soybeans for improving anthocyanin content, total phenols content and antioxidant activity. Food Chem, 261, 260-266. doi: 10.1016/j.foodchem.2018.04.061 Salamon, I., Mariychuk, R., & Grulova, D. (2015). Optimal Extraction of Pure Anthocyanins from fruits of Sambucus nigra. https://doi.org/10.17660/ActaHortic.2015.1061.6 Sánchez Maldonado, A. F., Mudge, E., Gänzle, M. G., & Schieber, A. (2014). Extraction and fractionation of phenolic acids and glycoalkaloids from potato peels using acidified water/ethanol-based solvents. Food Research International, 65, 27-34. doi: https://doi.org/10.1016/j.foodres.2014.06.018 Schulz, M., Borges, G. d. S. C., Gonzaga, L. V., Seraglio, S. K. T., Olivo, I. S., Azevedo, M. S., . . . Fett, R. (2015). Chemical composition, bioactive compounds and antioxidant capacity of juçara fruit (Euterpe edulis Martius) during ripening. Food Research International, 77, 125-131. doi: https://doi.org/10.1016/j.foodres.2015.08.006 Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent Methods in Enzymology (Vol. 299, pp. 152-178): Academic Press. https://doi.org/10.1016/S00766879(99)99017-1 Tabart, J., Kevers, C., Pincemail, J., Defraigne, J.-O., & Dommes, J. (2009). Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chemistry, 113(4), 1226-1233. doi: https://doi.org/10.1016/j.foodchem.2008.08.013 Tian, Y., Liimatainen, J., Alanne, A.-L., Lindstedt, A., Liu, P., Sinkkonen, J., . . . Yang, B. (2017). Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants. Food Chemistry, 220, 266-281. doi: https://doi.org/10.1016/j.foodchem.2016.09.145 Xu, Y., Burton, S., Kim, C., & Sismour, E. (2015). Phenolic compounds, antioxidant, and antibacterial properties of pomace extracts from four Virginia-grown grape varieties. Food science & nutrition, 4(1), 125-133. doi: 10.1002/fsn3.264 Yao, Y., Lin, G., Xie, Y., Ma, P., Li, G., Meng, Q., & Wu, T. (2014). Preformulation studies of myricetin: a natural antioxidant flavonoid. Pharmazie, 69(1), 19-26. doi: 10.1691/ph.2014.3076

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Appendices Residual Plots for ORAC Normal Probability Plot

Versus Fits

99

100

Residual

50 10 1

50 0 -50 -100

-100

-50

0 Residual

50

100

PT

Percent

90

200

400 600 Fitted Value

50

2 1

-50 -100

-50

0 Residual

50

100

2

4

6 8 10 12 14 Observation Order

NU

-100

16

18

99

Residual

10 -50

0 Residual

50

PT E

-100

D

50

40 0

-40 -80 200

100

300

Histogram

-80

AC

0

CE

2

-40

0 Residual

400 Fitted Value

500

600

Versus Order 80 40

Residual

3

1

Versus Fits

80

90

1

MA

Residual Plots for FRAP Normal Probability Plot

Percent

0

0

SC

3

Residual

100

RI

Versus Order

4

Frequency

Frequency

Histogram

800

0 -40 -80

40

80

2

4

6 8 10 12 14 Observation Order

16

18

ACCEPTED MANUSCRIPT Residual Plots for Phenols Normal Probability Plot

Versus Fits

99

2

Residual

50 10 1

1 0 -1 -2

-4

-2

0 Residual

2

4

20

1,5

1

1,0 0,5

PT

2

0 -1 -2

-2

-1

0 Residual

1

2

2

4

6 8 10 12 14 Observation Order

16

18

SC

0,0

40

Versus Order

2,0

Residual

Frequency

Histogram

30 Fitted Value

RI

Percent

90

Normal Probability Plot 99

Residual

0,0 Residual

0,2

Histogram

6 4

-0,3

-0,2

CE

2

-0,1 0,0 0,1 Residual

0,2

0,00

0,3

-0,15 -0,30

0,4

PT E

8

0,15

MA

-0,2

D

1 -0,4

0,0

0,5

1,0 1,5 Fitted Value

2,0

Versus Order 0,30 0,15

Residual

Percent

50 10

Frequency

Versus Fits

0,30

90

0

NU

Residual Plots for Antocyanins

0,00 -0,15 -0,30

2

4

6 8 10 12 14 Observation Order

16

18

AC

Figure A1. Residual Plots for ORAC, FRAP, total phenolic content and monomeric anthocyanin’s assays

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Figure 1. Representative scheme of extraction procedure

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Figure 2. Effects of the process parameters on the extraction of bioactive compounds and the antioxidant capacity

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Figure 3. Effects of the interaction between different process parameters on the extraction of bioactive compounds and on the antioxidant activity

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Figure 4. Principal component analysis (PCA) plot representing a) spatial representation of the samples; b) phenolic compounds identified as supplementary variables (biplot). For the same extraction condition see Table 2.

ACCEPTED MANUSCRIPT Table 1. Proximate composition of freeze-dried Jabuticaba peel powder Composition (g/100g)

Water

4.46 ± 0.28

Ashes

1.97 ± 0,.02

Total lipids

1.77 ± 0.07

Proteins

7.32 ± 0.03

Total carbohydrate

84.48

PT

Constituents

pH

CT A/B

4

246E ± 0.3

488A ± 43

3 A/B

1

841A ± 21

342A ± 66

1 A/B

2

2 A/B

3

4 A/B

NU

(µmol TE/g RM)

(µmol TE/g RM)

Phenolic content (mg GAE/ g RM)

31BCDEF ± 2 ABCDE 32 ± 4

Monomeric anthocyanin (mg/ g RM)

2.8AB ± 0.2 3.4A ± 0.1

492A ± 45

35ABC ± 1

2.1BC ± 0.2

413CDE ± 14

371A ± 24

28DEFG ± 2

1.8CDE ± 0.4

1

248 B ± 108

19H ± 3

0.8FG ± 0.3

D

540BCD ± 127

362CDE ± 92

5 A/B

2

722AB ± 18

373A ± 90

16H ± 1

1.9CD ± 0.1

6 A/B

3

664ABC ± 61

474A ± 73

39A ± 3

1.2DEF ± 0.3

7 A/B

1

153E ± 76

238B ± 20

CE

Formic acid

ORAC

MA

Control

8 A/B

2

257CDE ± 35

542A ± 58

9 A/B

3

395CDE ± 16

564A ± 24

Acetic acid

AC

Orthophosphoric acid

FRAP

Sample Identification

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Acid

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Table 2. Process parameters and results of antioxidant activity and bioactive compounds for the extracts

23.8FGH ± 0.3 35.5ABCD ± 0.5 37AB ± 1

RM: Raw Material TE: Trolox Equivalent GAE: Gallic Acid Equivalent Uppercase different letters on the same column indicate significant difference (p ≤ 0.05).

0.1G ± 0.02 1.3CDEF ± 0.2 1.6CDEF ± 0.02

ACCEPTED MANUSCRIPT 30

Peak Number

Phenolic compound

m/z

MM/RT

1

Bis-HHDPglucose

783.0687

784.076/1.77

783.0690/30

2

Bis-HHDPglucose

783.0689

784.075/2.97

783.0690/30

3

Quercetin-Crhamnoside

447.0937

448.1/3.78

4

Delphinidin-3glucoside

463.0888

464.095/4.88

463.090

5

Cyanidin-3glucoside

447.0944

SC

Table 3. Chromatographic and mass spectrometry characteristics of phenolic compounds from jabuticaba peel, obtained by LC-MS/MS

447.0948/284.033

6

Bis-HHDPglucose

783.0683

784.075/5.92

783.066

7

Quercetin isomer

301.0365

8

Unknown

509.1297

510.137/7.15

9

Unknown

509.1291

510.137/7.74

10

Ellagic acid pentoside

433.0421

434.048/8.33

433.0388/30

11

Myricetin-Orhamnoside

463.089

464.095/9.1

463.0875/316.0234/30

12

Ellagic acid pentoside

433.0418

434.048/9.44

433.0408/30

Myricetin-Orhamnoside

463.0885

464.096/9.49

463.0893/301.034

14

Quercetin isomer

301.0365

302.043/9.49

15

Ellagic acid

300.9998

302.006/9.75

301.0002/283.9969/245.008

16

Ellagic acid pentoside

433.0413

434.047/9.83

433.0374/30

17

Quercetin-Orhamnoside

447.0941

448.1/11.08

447.0925/300.0283/27

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448.1/5.74

MA

D

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CE

AC

13

M

447.0934/28

302.043/6.24

ACCEPTED MANUSCRIPT 31

18

Galloyl-bis-HHDPglucose

935.0779

936.087/11.42

19

Quercetin isomer

301.0365

302.043/11.64

300.9993/165.0200/13

20

Quercetin

301.0365

302.043/12.03

301.0359/178.9887/15

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m/z: mass-to-charge ratio MM: Molecular mass RT: retention time (min) MS/MS: mass spectrometry/mass spectrometry

SC

Table 4. Abundance (%) of the phenolic compounds identified for each extract

MM/RT

CT

Bis-HHDP-glucose

784.076/1.77

NU

Compounds

Bis-HHDP-glucose

784.075/2.97

Quercetin-C-rhamnoside

448.1/3.78

-7

5x10

8x10

-6

1

2

-6

1x10

8x10

-6

0.76

1x10

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3

Samples Identification 4

-5

1x10

1x10

-5

0.08

5

-5

5x10

1x10

-5

0.12

6

-6

8x10

-6

4x10

9x10

-6

1x10

-5

1x10

0.21

0.05

0.07

0.0

464.095/4.88

2.5

4.0

2.7

2.4

4.0

3.0

2.2

Cyanidin-3-glucoside

448.1/5.74

70

63

39

48

51

38

25

784.075/5.92

0.63

0.51

0.71

0.64

0.36

0.53

0.6

Quercetin isomer

302.043/6.24

0.32

1.71

6.37

5.02

3.29

4.96

7.4

Unknown

510.137/7.15

0.85

0

2.38

4.68

0.51

1.91

7.9

Unknown

510.137/7.74

0

0

0

0.15

0

0.07

0.4

Ellagic acid pentoside

434.048/8.33

0.63

0.60

0.65

0.67

0.58

0.64

0.7

Myricetin-O-rhamnoside

464.095/9.1

1.55

1.32

1.43

1.45

1.10

1.05

1.3

CE

Delphinidin-3-glucoside

AC

Bis-HHDP-glucose

ACCEPTED MANUSCRIPT

434.048/9.44

3.08

2.79

2.59

3.04

2.41

2.58

2.8

Myricetin-O-rhamnoside

464.096/9.49

1.60

6.99

16.7

9.18

13.7

18.3

15.

Quercetin isomer

302.043/9.49

0

3.88

9.77

4.87

8.36

11.4

9.4

Ellagic acid

302.006/9.75

6.87

6.69

6.22

6.79

5.70

6.12

7.1

Ellagic acid pentoside

434.047/9.83

0.49

0.44

0.50

0.46

0.50

0.6

Quercetin-O-rhamnoside

448.1/11.08

4.6

4.4

5.0

3.5

3.6

4.7

Galloyl-bis-HHDP-glucose

936.087/11.42

1.3

1.48

1.39

1.72

0.97

1.32

1.6

Quercetin isomer

302.043/11.64

0.69

2.01

3.06

1.37

2.43

5.1

Quercetin

302.043/12.03

0.38

0.70

0.80

0.30

0.61

2.6

AC

CE

SC

NU 2.11

MA

D

PT E

MM: Molecular Mass RT: Retention Time CT: Control Sample 1: Formic Acid pH 2.0 2: Formic Acid pH 3.0 3: Formic Acid pH 1.0 4: Acetic Acid pH 1.0 5: Acid Acetic pH 2.0 6: Acid Acetic pH 3.0 7: Ortho-phosporic Acid pH 1.0 8: Ortho-phosporic Acid pH 2.0 9: Ortho-phosporic Acid pH 3.0

3.9

0.01

PT

Ellagic acid pentoside

RI

32

0.43

ACCEPTED MANUSCRIPT 33

Highlights

The kind of acid employed on the extraction had influence on anthocyanin yield. Formic acid in pH 1 enhanced the anthocyanin content and antioxidant capacity. Phosphoric acid in pH 1 reduced the extraction of anthocyanins.

PT

Acetic acid in pH 3 increased the content of phenolic compounds.

AC

CE

PT E

D

MA

NU

SC

RI

Cyanindin-3-glucoside was the mainly compound identified in all extratcs.