Effect of the extraction process on the phenolic compounds profile and the antioxidant and antimicrobial activity of extracts of pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell

Industrial Crops and Products 52 (2014) 552–561

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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Review

Effect of the extraction process on the phenolic compounds profile and the antioxidant and antimicrobial activity of extracts of pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell Ana Cristina Pinheiro do Prado a , Helen Silvestre da Silva a , Sheila Mello da Silveira b , Pedro Luiz Manique Barreto a , Cleide Rosana Werneck Vieira a , Marcelo Maraschin c , Sandra Regina Salvador Ferreira d , Jane Mara Block a,∗ a Department of Food Science and Technology, Federal University of Santa Catarina (UFSC), Av. Admar Gonzaga, 1346, Itacorubi, 88034-001 Florianopolis, SC, Brazil b Santa Catarina Federal Institute of Education, Science and Technology (IFC), SC 283, Km 08, Vila Fragosos, 89700-000 Concordia, SC, Brazil c Department of Phytotechny, Federal University of Santa Catarina (UFSC), Av. Admar Gonzaga, 1346, Itacorubi, 88034-001 Florianopolis, SC, Brazil d Department of Chemical and Food Engineering, Federal University of Santa Catarina (UFSC), R. João Pio Duarte Silva, 523-645, 88037-000 Florianopolis, SC, Brazil

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Article history: Received 16 June 2013 Received in revised form 18 November 2013 Accepted 20 November 2013 Keywords: Pecan nut shell Phenolic profile Antioxidant activity Antimicrobial activity

a b s t r a c t In this study, the effect of the extraction processes (infusion, infusion followed by spray drying, ethanol extraction and supercritical extraction) on the total content and profile of phenolic compounds, antioxidant and antimicrobial activities of extracts of pecan nut shell were studied. The extract obtained through infusion followed by atomization in a spray dryer showed significantly higher (p < 0.05) contents of total phenolic compounds (590.78 mg GAE/g) and condensed tannins (412.10 mg CE/g), and significantly greater antioxidant activity (ABTS and DPPH assays) as compared with extracts obtained by infusion only, ethanol extraction and supercritical extraction. Five major phenolic compounds (gallic, chlorogenic and p-hydroxybenzoic acids, epigallocatechin and epicatechin gallate) were identified and quantified by HPLC in the obtained extracts. The minimum inhibitory concentration and minimum bactericidal concentration against Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus and Bacillus cereus were significantly lower (p < 0.05) for the extract obtained through infusion followed by atomization in a spray dryer when compared to the other extracts. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Chemical reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Obtaining the powder and extracts of the pecan shell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Total phenolics, condensed tannins and phenolic profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Determination of total phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Determination of condensed tannin content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Determination of phenolic profile by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Antioxidant and antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. Antioxidant activity in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +55 48 3721 5367; fax: +55 48 3721 9943. E-mail addresses: [email protected] (A.C.P. do Prado), helen [email protected] (H.S. da Silva), [email protected] (S.M. da Silveira), [email protected] (P.L.M. Barreto), [email protected] (C.R.W. Vieira), [email protected] (M. Maraschin), [email protected] (S.R.S. Ferreira), [email protected] (J.M. Block). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.11.031

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3.

4.

2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Total phenolics, condensed tannins and antioxidant activity in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Phenolic profile determined by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Every year the food industry produces a significant volume of waste and serious issues are associated with its disposal. In recent years, several studies have been conducted, aimed at developing new alternatives for the use of these byproducts, which have high chemical and nutritional potential (Orzua et al., 2009; Graminha et al., 2008; Rodríguez-Couto, 2008). In southern Brazil, in the processing of pecan nuts approximately 40–50% of the total industrial output is in the form of the shell, which is sold in pieces to prepare tea (Divinut, 2011). The pecan nut shell is rich in phenolic compounds, such as phenolic acids, flavonoid acids and proanthocyanidins, which have been extensively studied due to their antioxidant properties (Malick et al., 2009; Villarreal-Lozoya et al., 2007; Dimitrios, 2006; Senter et al., 1980). In vivo studies have reported that phenolic compounds in tea from the pecan nut shell can minimize the liver damage in rats caused by oxidative stress after chronic ethanol intake. Moreover, pecan nut tea was found to prevent anxiety caused by cigarette abstinence, acting as a natural anxiolytic (Müller et al., 2013; Reckziegel et al., 2011). Moreover, phenolic compounds have been reported to function as antibacterial agents against microorganisms (Serrano et al., 2009; Rauha et al., 2000). Phenolic acids such as caffeic, gallic, p-coumaric, protocatechuic and ferulic acid are capable of inhibiting the growth of various bacteria (Bacillus cereus, Escherichia coli spp., Salmonella spp.). Furthermore, some studies have showed that flavonoid compounds (such as catechin and quercetin) can exhibit bacteriostatic or bactericidal activity against Bacillus spp., E. coli, Shigella spp., Salmonella spp., Staphylococcus aureus and Vibrio spp. (Vaquero and Nadra, 2008; Naz et al., 2007; Puupponen-Pimiä et al., 2001; Rauha et al., 2000; Herald and Davidson, 1983). Different extraction techniques used to obtain plant extracts rich in natural antioxidants have been widely investigated. Traditional methods, such as Soxhlet extraction, are very time consuming, require relatively large quantities of solvents and may cause the loss of volatile and thermolabile compounds due to the higher temperatures used. Extraction with organic solvents presents major restrictions such as the presence of solvent residues in the extracts, which are often toxic, and the obtainment of undesirable compounds in the product. Thus, the use of alternative extraction technologies such as supercritical fluid (a process carried out at moderate temperatures usually with non-toxic carbon dioxide and in which the use of co-solvents is possible in order to improve the selective extraction yield of certain compounds) may represent an alternative to conventional extraction processes for the extraction of bioactive compounds of interest to the food and pharmaceutical industries (Mendiola et al., 2007; Dinc¸er et al., 2005; Zancan et al., 2002; Pokorny and Korczak, 2001). The effectiveness of the use of extracts rich in polyphenols is dependent, among other factors, on the preservation of the stability, bioactivity and bioavailability of the active ingredients. Moreover, the unpleasant taste associated with some phenolic compounds also limits their application. Using encapsulated polyphenols obtained by spray drying, instead of free compounds,

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may help to solve these problems (Fang and Bhandari, 2010). Microparticles of various bioactive compounds such as fruit fiber, probiotics and plant extracts with antioxidant properties have been obtained using this technology (Homayouni et al., 2008; Chiou and Langrish, 2007). However, further studies are required to assess the influence of the use of the spray drying technique on the phenolic content of the extracts, especially those that are susceptible to thermal degradation, since high temperatures are often used with this procedure. The determination of the phenolic profile of plant extracts can be performed by high performance liquid chromatography (HPLC). In complex matrices such as nut shells, which are rich in lignocellulosic material, the phenolic profile analysis may not be simple and sometimes it requires additional methods of sample preparation. Villarreal-Lozoya et al. (2007) and De La Rosa et al. (2011) employed methods of basic/acid hydrolysis (8 N NaOH/6 M HCl) and acid/basic hydrolysis (2 M HCl/10 M NaOH), respectively, and both treatments proved to be ineffective for the detection of flavonoids and condensed tannins, as these conditions proved too harsh for the preparation of the sample. Conditions of analysis which do not affect the sample such as the use of specific enzymes (␤-glucosidase and tannase) for the cleavage of specific chemical bonds, and lipophilic column (LH-20 Sephadex® resin) have also been used in the determination of the phenolic profile of plant extracts (Macedo et al., 2011; La Torre et al., 2004; Malick et al., 2009). The objective of this study was to investigate the effect of the extraction process on the total content of phenolic compounds, antioxidant activity (using ABTS and DPPH methods) and antimicrobial activity (by determination of the minimum inhibitory concentration and minimum bactericidal concentration values) of the extracts of pecan nut shell. 2. Material and methods 2.1. Samples Pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell of the variety Barton was provided by EMATER (a Brazilian government agency that conducts research on the genetic improvement of pecan nuts). Shells from nuts harvested in the 2010 were used. 2.2. Chemical reagents Folin–Ciocalteau phenol reagent, tannase (E.C.3.1.1.20 of Aspergillus ficcum), gallic acid, ABTS [2,2 -azino-bis-(3ethylbenzothiazoline-6-sulfonic acid)], DPPH (2,2-diphenyl1-picrylhydrazyl), Trolox, vanillin and (−)-gallocatechin and ferulic acid were obtained from Sigma–Aldrich (Germany); (+)catechin hydrated and (−)-epicatechin gallate were obtained from Sigma–Aldrich (China); chlorogenic acid and (−)-epigallocatechin were obtained from Sigma–Aldrich (India); (−)-epicatechin, 4–5 dicaffeoylquinic acid, p-hydroxy-benzoic acid, protocatechuic were obtained from Sigma–Aldrich (USA); caffeic acid was obtained from Sigma–Aldrich (Switzerland); Lipophilic Sephadex resin (Sephadex® LH-20) was obtained from Sigma–Aldrich (Sweden);

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Müller Hilton Agar, Potato Dextrose Agar, Tryptose Soya Agar and Tryptose Soya Broth were obtained from the Acumedia brand – Sovereign (Brazil); bacterial and fungal strains were obtained from Fiocruz (Brazil). All other chemical reagents and solvents used in the experiment were of analytical grade (P.A.) obtained from Vetec Química Fina and Sigma–Aldrich. 2.3. Sample preparation 2.3.1. Obtaining the powder and extracts of the pecan shell The shells were dried at 40 ◦ C in an oven with forced air circulation (model 400/D 200 ◦ C, Nova Ética® ) to a reduced moisture content and then milled in a Mill analytical laboratory (model A-11 IKA Works® ). The powder was sieved to 60-mesh size and stored in amber bottles with nitrogen atmosphere at 24 ◦ C for later analysis (Prado et al., 2009). Infusions were prepared according to Prado et al. (2009). The powder samples were placed in distilled water (20 g/L on a dry basis) at 98◦ for 10 min. In addition the alcoholic extracts were obtained according to procedure performed by Prado et al. (2010) with minor modifications. The powder samples were placed in absolute ethanol (20 g/L on a dry basis) under constant stirring for 1 h in the dark and at room temperature. The extracts were filtered using Whatman filter paper (no 541, 125 mm) and the volume completed to 100 mL with distilled water and absolute ethanol in order to obtain the infusion and the alcoholic extract, respectively. The extracts were stored in amber bottles with nitrogen atmosphere at −24 ◦ C for later analysis. The extractions with supercritical CO2 were performed using 15 g of pecan nut shell, at 50 ◦ C, first at 100 bars and then at 200 bars, with pure CO2 , using a flow rate of 0.7 kg/h and 10% of absolute ethanol as co-solvent. The infusions prepared as previously described were dried by spray drying process using a BÜCHI Mini Spray Dryer (model B290). Spray drying conditions were as follows: air temperature inlet and outlet 150 ◦ C and 50 ◦ C, respectively, with aspirator set at 100% and pump at 25% (Sahin Nadeem et al., 2011). The sample powder was collected and stored for later analysis at −24 ◦ C. The determination of the dry extract of all extracts was performed by gravimetric analysis, according to AOAC (2005). 2.4. Total phenolics, condensed tannins and phenolic profile 2.4.1. Determination of total phenolic compounds The total phenolic content was estimated using Folin–Ciocalteau colorimetric method with some modifications (Prado et al., 2009; Budini et al., 1980). Quantification was performed using a standard curve generated with dilutions (50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 mg/L) of a gallic acid stock solution (0.00005 g/mL or 50 ppm of gallic acid). After incubation for 2 h in the dark at room temperature, the absorbance of the resulting blue solution was measured at 764 nm with a spectrophotometer (Spectrophotometer model SP 2000 UV, trademark Bel Photonics® ). Results were expressed as milligrams of Gallic Acid Equivalents per gram (mg GAE/g) of dry weight. 2.4.2. Determination of condensed tannin content The determination of condensed tannins was carried out according to the method described by Price et al. (1978) and adapted by Villarreal-Lozoya et al. (2007). The absorbance was compared to a standard curve obtained with dilutions (0.1, 10, 37.5, 75, 150, 300, 600 and 1200 mg/L) of a catechin stock solution (0.0012 g/mL or 50 ppm of catechin). The results were expressed in milligrams of Cathechin Equivalent per gram (mg CE/g) of dry weight.

2.4.3. Determination of phenolic profile by HPLC 2.4.3.1. Sample preparation. The phenolic profile was determined showing the concentrations of total phenolics and condensed tannins most significant in the extracts (infusion, infusion dried by spray dryer and ethanol extract). Before HPLC analysis the samples were prepared according to three different methodologies as follows: basic/acid extraction was performed according to AbdelAal et al. (2001) and Gelinas and McKinnon (2006); enzymatic hydrolysis was performed according to Macedo et al. (2011) using the enzyme Tannase (E.C.3.1.1.20 of A. ficcum); and separation by lipophilic Sephadex resin (Sephadex® LH-20) was performed according to Malick et al. (2009) with the following modifications: the resin Sephadex® LH-20 was conditioned (0.750 g in 3 mL of extraction solvent) in column (9 mm × 100 mm) over a 24-h period; then the column was washed with 10 mL of extraction solvent (0.5 mL min−1 ) in vacuum. An aliquot of 300 ␮L of the filtered extract (0.45 ␮m) was eluted with 10 mL of 80% ethanol (0.5 mL min−1 ) in vacuum and five fractions of 2 mL were collected for analysis. 2.4.3.2. HPLC analysis. 10 ␮L of the samples were injected onto the HPLC (Shimadzu LC-10, Tokyo, Japan) system equipped with a reversed phase column (Shim-pack C18, 4.6 mm i.d. × 250 mm, Shimadzu), at 40 ◦ C, and UV–visible detector (Shimadzu SPD 10 A,  = 280 nm). An isocratic mobile phase consisting of a mixture of water:acetic acid:n-butanol (350:1:10, v/v/v) was used at a flow rate of 0.8 mL min−1 . For quantitative analysis, analytical curves were obtained by plotting the peak area versus different concentrations (1–100 ␮g/mL) for each standard compound. The injections were performed in triplicate, using the mean of three injections for quantification of the compounds in the sample. The identification of compounds of interest was confirmed by chromatographic analysis of reference compounds (gallic acid, chlorogenic acid, p-coumaric acid, caffeic acid, epicatechin, epicatechin gallate, epigallocatechin, 4–5 dicaffeoylquinic, p-hydroxybenzoic acid, protocatechuic acid) comparing the relative retention times of these compounds. 2.5. Antioxidant and antimicrobial activity 2.5.1. Antioxidant activity in vitro 2.5.1.1. ABTS method. The ABTS method [2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] assay was carried out according to Re et al. (1999). The concentration of the ABTS radical was 7 mM. Trolox (15 ␮M) was used as the standard. The results were expressed in ␮mol TEAC g−1 of dry weight (␮mol Trolox equivalent antioxidant capacity/g of defatted sample). 2.5.1.2. DPPH method. The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was carried out according to Brand-Williams et al. (1995) modified by Prado et al. (2009) and Mensor et al. (2001). An aliquot of 2.9 mL of the DPPH (0.1 mM = 0.03943 g DPPH dissolved in 10 mL of 80% ethanol) was used. Trolox was used as standard (150 mg/L). The results were expressed as the TEAC g−1 mg of dry weight (mg Trolox equivalent antioxidant capacity/g of defatted sample). 2.5.2. Antimicrobial activity All extracts, except those obtained with supercritical technology as well as standards of phenolic compounds were tested for antibacterial and antifungal activity. Extracts from supercritical extraction were not tested due to showing results much lower for total phenolics, condensed tannins and antioxidant activity in vitro, compared the others extracts. Extracts used were concentrated in a rotary evaporator (40 ◦ C) until the concentration of 200 mg/mL. B. cereus (ATCC 11778), E. coli (ATCC 25922), L. monocytogenes (ATCC 19117), L. monocytogenes serotype II (ATCC 19112), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC

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14028), S. aureus (ATCC 29213), S. aureus (ATCC 6538), Vibrio parahaemolyticus (ATCC 17802), Aspergillus niger (IOC 0206), Aspergillus flavus (FC 1087), Penicillium roquefortii (IOC 2242), Rhizopus sp. (IOC 1109) and Fusarium sp. (IOC 1200) were used to evaluate the antibacterial and antifungal activity of the extracts. For reactivation, all bacterial strains were grown in Tryptose Soya Broth (TSB) incubated at 35–37 ◦ C for 24 h, and for fungal strains a spore suspension was prepared. 2.5.2.1. Inhibition assays on solid medium. The inhibition assays on solid medium (agar diffusion method) were performed according to Pinto et al. (2003) on 10 mm × 90 mm Petri plates. The bacterial inoculums were uniformly spread using sterile cotton swab on a Petri dish Muller Hinton (MH) agar. The fungal inoculums were applied with a Drigalski spatula on a Potato Dextose Agar (PDA). After the complete absorption of the inoculums cavities of 6 mm diameter were made, 50 ␮L of each extract were inoculated. The plates were pre-incubated for 2 h at room temperature to the diffusion of the extracts and then the bacterial inoculums were incubated at 37 ◦ C (24–48 h) and the fungal inoculums were incubated at 25–26 ◦ C (for five days). After the incubation period, the diameter of the zone with no growing of the tested strain (inhibition zone) was measured according to the Farmacopéia Brasileira (1988). 2.5.2.2. Determination of minimum inhibitory concentration (MIC). Extracts were evaluated for the minimal inhibitory concentration (MIC) by a micro dilution method based on the document CLSI (2009). The following strains of bacteria were tested: L. monocytogenes (ATCC 19117), L. monocytogenes serotype II (ATCC 19112), S. aureus (ATCC 29213), S. aureus (ATCC 25923), S. aureus (ATCC 6538), B. cereus (ATCC 11778) and V. parahaemolyticus (ATCC 17802). The cultures in Tryptose Soya Agar (TSA) were transplanted to blood agar and incubated at 35 ◦ C for 12–18 h in order to check the colony morphology and purity of the culture. From these plates, 3–5 isolated colonies were transferred to Tryptose Soya Broth (TSB) and incubated at 35 ◦ C for 2–6 h, to obtain an actively growing culture. The inoculum was prepared from an active culture of each bacterial strain, diluted in 0.9% saline solution to a concentration of approximately 108 CFU/ml, comparable to McFarland standard solution of 0.5 absorbance units recorded by spectrophotometer at 625 nm. The suspension was diluted to approximately 108 CFU/ml in saline solution, and this suspension was used to inoculate the wells of micro dilution plates. Culture mediums and diluents used for culture of V. parahaemolyticus were added of 3% NaCl. The extracts were diluted in dimethylsulfoxide (DMSO) at a concentration of 100 mg/mL and a series of successive dilutions was prepared in the range of 10–0.075 mg/mL in Mueller-Hinton Broth. 100 ␮L of each diluted solution and 5 ␮L of bacterial suspension were added to the wells of microdilution plates. A sterility control (no added inoculum) and a growth control (Mueller-Hinton Broth added from DMSO and inoculum) were made in each plate. The plates were incubated at 36 ◦ C for 18 h and the microbial growth visually detected and confirmed by adding 20 ␮L of aqueous solution of 2,3,5-triphenyltetrazolium chloride 0.5% (m/v) with further incubation of 1 h at the same temperature. All assays were performed in triplicate and results expressed in mg/mL. The MIC was defined as the lowest concentration of the extract that completely inhibited the microbial growth (Smânia et al., 1995). 2.5.2.3. Determination of minimum bactericidal concentration (MBC). The minimum bactericidal concentration (MBC) was determined according to Celiktas et al. (2007). From each well of the microplate used for determination of MIC where no visible microbial growth occurred, 10 ␮L aliquots were transferred to plates with Tryptose

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Soya Broth (TSA), and TSA supplemented with 3% NaCl for V. parahaemolyticus. The plates were incubated at 36 ◦ C for 24 h and the growth of colonies was observed. All assays were performed in triplicate and the results expressed in mg/mL. The MBC was defined as the lowest concentration of each extract that completely prevented microbial growth on plates without the presence of other antimicrobial agents. 2.6. Statistical analysis The statistical analysis of data was performed using SAS “for Windows” version 6.11 and Statistica® version 7.0 software. Results were submitted to analysis of variance (ANOVA), Tukey test (p < 0.05) and Principal Component Analysis (PCA). Moreover, three-dimensional graphics using response surface methodology were generated, in Statistica® version 7.0, to relate the results obtained by the HPLC, the antioxidant activity and total phenolic content for each of the fractions analyzed. All analyses were carried out in triplicate. 3. Results and discussion 3.1. Total phenolics, condensed tannins and antioxidant activity in vitro Table 1 shows the results obtained for the dry matter content, total phenolics, condensed tannins and antioxidant activity in vitro of different pecan nut shell extracts. In extracts obtained by infusion the concentrations of total phenolics and antioxidant activity measured by ABTS and DPPH systems (181.49 ± 6.97 mg GAE g−1 , 1809.01 ± 27.18 ␮mol TEAC g−1 and 612.24 ± 26.73 mg TEAC g−1 , respectively) were significantly higher (p < 0.05) when compared to alcoholic extracts (167.85 ± 3.89 mg GAE g−1 , 1562.51 ± 33.15 ␮mol TEAC g−1 and 524.77 ± 40.72 mg TEAC g−1 , respectively). However, the content of tannins was significantly higher in alcoholic extracts (412.1 ± 9.46 mg CE g−1 ), compared to the levels observed in the extract obtained by infusion (36.94 mg CE g−1 ). The differences observed between the samples for the concentration of condensed tannins can be explained by the fact that the water-soluble fraction of these compounds consists principally of hydrolyzable tannins, sugars, some pigments and proanthocyanidins with a low degree of polymerization, and these compounds represent a less significant fraction of condensed tannins extracted in the infusion. The compounds soluble in organic solvents include esterified lignocellulosic molecules, which have a more complex molecular structure with a higher degree of polymerization (Trugilho et al., 2003). For the extracts obtained through supercritical fluid extraction using ethanol as a co-solvent the parameters evaluated were much lower compared to conventional methods of extraction. The use of a pressure of 200 bar significantly increased all parameters compared to the results obtained at 100 bar. The dry extract, total phenolics and condensed tannins contents were 10, 25 and 60 times higher, respectively, when the experimental pressure condition was changed from 100 to 200 bar. Consequently, a significant increase in the antioxidant activity in vitro was observed for the extracts obtained at 200 bar, with values being 20 and 40 times higher for the ABTS and DPPH methods, respectively. This behavior can be explained by changes in the density of the solvent with increasing pressure at constant temperature, which may improve the extraction of phenolic compounds (Brunner, 2005). For the extract obtained by infusion followed by atomization, a significant increase in the solids, phenolic compounds and

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Table 1 Dry matter content, total phenolics, condensed tannins and antioxidant activity in vitro of different extracts of the pecan nut shell. Determination

DE (g/100 g−1 ) TF (mg GAE g−1 ) CT (mg CE g−1 ) ACABTS (␮mol TEAC g−1 ) ACDPPH (mg TEAC g−1 )

Conventional extraction method

Supercritical extraction method

Infusion

100 bar

32.12A 181.49A 36.94B 1809.01A 612.24A

Alcohol ± ± ± ± ±

0.43 6.97 3.20 27.18 26.73

32.09A 167.85B 412.1A 1562.51B 524.77B

± ± ± ± ±

0.77 3.89 9.46 33.15 40.72

0.83B 0.34B 0.48B 4.95B 1.91B

± ± ± ± ±

Infusion + spray dryer

200 bar 0.02 0.01 0.01 0.04 0.03

8.40A 9.30A 29.00A 100.00A 79.20A

± ± ± ± ±

0.90 0.10 2.00 5.00 0.80

46.36* 590.78* 48.70* 4124.83* 1210.97*

± ± ± ± ±

0.50 4.41 1.50 57.09 25.24

DE = dry extract – g 100−1 (assay gravimetric); TF = total phenolic – mg GAE g−1 (equivalents in gallic acid) in dry weight (Folin–Ciocalteau assay); CT = condensed tannins – mg CE g−1 (catechin equivalents) of dry weight (Vanillin test); AC = antioxidant capacity – ␮mol TEAC g−1 (equivalent antioxidant activity in Trolox) in dry weight (ABTS), AC = antioxidant capacity – mg TEAC g−1 (equivalent in Trolox) in dry weight (DPPH–TEAC). The same letters in the same row do not differ significantly (Tukey test, p < 0.05) for the extracts within the same extraction method employed. * Significant difference (Tukey test, p < 0.05) between the sample subjected to the process of Spray Drying and other extraction methods, mean ± standard deviation (n = 3).

condensed tannins content was observed as well as enhanced antioxidant activity which was attributed to the increased concentration of solids after the drying process. Prado et al. (2009) studied the infusion of pecan nut shells from a mixture of commercial varieties (Barton, Shoshone, Shawnee, Choctaw and Cape Fear) and reported lower values for the dry extract (23.0 g 100 g−1 ), total phenolic compounds (138 mg GAE g−1 ) and condensed tannins (43 mg CE g−1 ) contents as well as the antioxidant activity (1404 ␮mol TEAC g−1 and 385 mg TEAC g−1 for ABTS and DPPH systems, respectively) compared with the results obtained in the study reported herein. These differences could be related to the different pecan nut varieties and years of collection of the samples studied (Prado et al., 2013). Prado et al. (2010) using sequential extractions, reported values for the dry extract, total phenolics and antioxidant activity using the DPPH system (23 g 100 g−1 , 118 mg GAE g−1 and 453 mg TEAC g−1 , respectively) in alcoholic extracts of pecan nut shell which are lower than those found in the present study. The same authors, however, reported significantly higher levels of condensed tannins (736 mg CE g−1 ) and antioxidant activity assessed through the ABTS-analysis (2600 ␮mol TEAC g−1 ). The process of sequential extraction, which leads to depletion of the sample, may facilitate the extraction of some compounds at the expense of others, and this may explain the differences in the results. Pérez-Jiménez and Saura-Calixto (2006) reported significant differences in the results for antioxidant activity using the same method for samples solubilized with different solvents. The ABTS method, in which more polar solvents are used, leads to a greater variation in the results obtained than the DPPH method. Furthermore, the authors observed that food constituents, such as amino acids and uronic acids, may cause interference in the measurement suggesting that the antioxidant activity results can only be compared when the samples are analyzed by the same methodology and extracted with the same solvent. 3.2. Phenolic profile determined by HPLC According to the results obtained when testing different methods of sample preparation for HPLC injection to determine the phenolic compounds profile of different extracts (infusion, infusion followed by spray drying and ethanol extract), basic/acidic and enzymatic hydrolysis techniques using tannase (EC3.1.1.20 A. ficcum) were inefficient in the separation of phenolic compounds. However, with the use of Sephadex ® LH-20 resin, the components which interfered in the chromatograms were removed, making it possible to identify three phenolic acids (gallic acid, chlorogenic acid and p-hydroxybenzoic) and two compounds from the class of flavonoids (flavan-3-ol subclass: epigallocatechin and epicatechingallate) in the different extracts evaluated. Fig. 1 shows the three-dimensional graphs for the response surface of the different fractions obtained from the separation process

using Sephadex® LH-20 resin. For the infusion extract, the first fraction (IF1) gave the best response for the total phenolic content, antioxidant activity (DPPH) and concentrations of gallic acid, chlorogenic acid, p-hydroxybenzoic acid and epigallocatechin. In extracts obtained by infusion followed by atomization in a spray dryer, the best responses for the same parameters were observed in the second fraction (ISD2), except for chlorogenic acid, which was equivalent to that found in the first fraction. For the fractions collected from the ethanol extract, a variation in the best responses is observed. The results for the first fraction (ET1) were superior to those of the other fractions in relation to gallic acid, chlorogenic acid and epigallocatechin. On the other hand, the second fraction (ET2) resulted in the best responses for antioxidant activity (DPPH) and epicatechingallate. In order to better understand the relationship between phenolic compounds and the antioxidant properties of the different extracts obtained, PCA (principal component analysis) was applied to the results obtained for total phenolics, antioxidant activity (DPPH) and the phenolic compounds in the five fractions obtained using the Sephadex® LH-20 resin. Fig. 2A shows that seven compounds were obtained in the correlation matrix of the variables. The first two dimensions together account for 73.29% of the variability of the data, and the principal components 1 and 2 (CP1 and CP2 ) were responsible for 44.78% and 28.51% of the total data variance, respectively (Fig. 2B). CP1 showed a strong positive correlation with the levels of chlorogenic acid (r = 0.9774) and epigallocatechin (r = 0.9214), this being less pronounced for the variables p-hydroxybenzoic acid (r = 0.7673) and antioxidant activity obtained through the DPPH system (r = 0.6358) with low significance for the levels of gallic acid (r = 0.4621) and epicatechingallate (r = 0.2477). In the case of CP2 , there was a negative correlation with the concentration of total phenolics (r = −0.8016), this decreasing for the variables of antioxidant activity evaluated by the DPPH method (r = −0.6583), epicatechin gallate (r = −0.6438) and gallic acid (r = −0.2283), and the correlation was positive for the variables of chlorogenic acid (r = 0.1382), epigallocatechin (r = 0.3455) and p-hydroxybenzoic acid (r = 0.5605), as shown in Fig. 3. This indicates that the higher the content of phenolic compounds, particularly gallic acid and epigallocatechin gallate, in the extracts, the higher the antioxidant activity will be. These results are in agreement with studies performed by Prudêncio (2011) on aqueous extracts of yerba mate (Ilex paraguariensis St. Hil.) concentrated by nanofiltration. Concentrations of gallic acid, chlorogenic acid, phydroxybenzoic acid, epigallocatechin and epicatechin gallate in the different extracts were observed in decreasing order in the extracts as follows: ET1 > ISD2 > IF1; IF1 > ET1 > ISD2; IF1 > ISD2 = ET1; IF1 > ISD2 > ET1 and ISD2 > ET1 > IF1, respectively. For the extract obtained by infusion followed by atomization in spray dryer, a significant increase was observed in the gallic acid and epitecatechin gallate contents. On the other hand, there was

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Fig. 1. Response surface for antioxidant activity (DPPH), total phenolic compounds (TF) and phenolic compounds (A: gallic acid, B: chlorogenic acid, C: p-hydroxybenzoic acid, D: epigallocatechin, E: epicatechin gallate) from the 5 sample fractions obtained through the resin Sephadex® LH-20 for the studied extracts.

a notable decrease in the chlorogenic acid, p-hydroxybenzoic acid and epigallocatechin contents. These results of a higher total phenolics content and antioxidant activity in the second fraction obtained by means of spray drying when compared to the other fractions of all three extracts evaluated, indicate the presence of phenolic compounds in fraction ISD2 different than those identified by HPLC. Moreover, the high degree of interdependence between the variables total phenolics (TP), DPPH (antioxidant activity) and epicatechin gallate (EG) (Fig. 2B) is a possible indicator that this flavonoid, followed by gallic acid (GA), belongs to the compounds with the greatest contribution to the antioxidant activity exerted by this extract. The PCA analysis showed a distinct distribution for the phenolic compounds in each of the five fractions obtained in the Sephadex®

LH-20 column for the three different extracts. There was a particularly strong influence of the separation process on the fractions IF1 (extract obtained by infusion) and ISD2 (extract obtained by infusion followed by atomization in a spray dryer), which showed significantly higher values for most of the parameters studied when compared to the other fractions for the same extracts analyzed. In fraction ET1 of the ethanol extract, values close to the average for most variables were observed for the five fractions obtained using Sephadex® LH-20. Table 2 shows the structural formula and the concentrations of major phenolic compounds determined in extracts of pecan nut shell. According to the results shown in Table 2, the extracts obtained by atomization in a spray dryer (ISD2) showed twice the

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Fig. 2. Eigenvalues obtained from principal component analysis (A) and the graphical representation of PCA performed for the antioxidant properties (total phenolics and antioxidant activity) of the different extracts (infusion, infusion followed by spray dryer and ethanol extract), and the phenolic compounds identified in the five fractions obtained from the resin Sephadex® LH-20 (B).

concentration of gallic acid compared to the extract obtained by infusion (IF1) and the epicatechin gallate concentration increased to levels detectable by the HPLC methodology. Moreover, for this extract a reduction in the level of p-hydroxybenzoic acid was observed, along with significant decreases in the concentrations of chlorogenic acid (by around 50%) and epigallocatechin (by around 75%) when compared to the extract obtained by infusion. The decrease in these phenolic compounds may have occurred as a function of the high temperatures used in the spray drying technology since some of these compounds are thermolabile (Moure et al., 2001). The concentration of gallic acid determined in the ethanol extract (ET1) was approximately seven times greater than that

detected in the infusion extract (IF1), and showed significantly reduced concentrations of chlorogenic acid, epigallocatechin and epicatechin gallate when compared to the extract obtained by infusion (IF1). Different factors such as the high concentration of gallic acid, the organic nature of the solvent and the high concentration of condensed tannins in this extract indicate the possible presence of monomers and dimers of complex tannins, such as catechins and gallocatechins, and molecules with a higher degree of polymerization. For these molecules the methods applied for the sample preparation and/or separation used are shown not to be efficient. Due to the high photo-sensitivity of these compounds, they may also have been degraded during the preparation process (Serrano et al., 2009; Trugilho et al., 2003).

Fig. 3. Antibiograms for Listeria monocytogenes (A), Staphylococcus aureus (B), Vibrio parahaemolyticus (C) and Bacillus cereus (D), measured in extracts of infusion (I), infusion followed by atomization in spray dryer (SD) and ethanol extract.

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Table 2 Major phenolic compounds determined by HPLC in extracts of the pecan shell from the fractions obtained using the separation column Sephadex® LH-20. Structural formula

Infusion (IF1)

Infusion + spray dryer (ISD2)

Ethanol extract (ET1)

Gallic acid (␮g/mL)

124.26B ± 14

238.83B ± 23

828.68A ± 32

Chlorogenic acid (␮g/mL)

233.36A ± 44

93.14A ± 7.33

137.91A ± 6.44

p-hydroxybenzoic acid (␮g/mL)

148.86 ± 8.06

n.d

n.d.

Epigallocatechin (␮g/mL)

5184.34A ± 100

1325.95B ± 116.54

120.21C ± 29.72

Epicatechin gallate (␮g/mL)

n.d.

0.97A ± 0.02

0.34B ± 0.01

n.d. = levels not detected; same letters in the same row do not differ significantly (Tukey test, p < 0.05), mean ± standard deviation (n = 3).

The extracts with the highest levels of phenolic compounds also showed the highest antioxidant activity which is consistent with studies by Soobrattee et al. (2005) who reported the antioxidant capacity (as Trolox equivalents – TEAC) for phenolic compounds decreasing in the following order: epicatechin gallate > epigallocatechin > gallic acid > chlorogenic acid > phydroxybenzoic acid, with the results being significantly higher for the group of oligomeric procyanidins (B1 and B2 ). Among the phenolic compounds present in the pecan nut kernel, some authors have reported the presence of larger quantities of hydroxybenzoic, ellagic and gallic acids, and flavonoids such as catechin and epicatechin. Smaller quantities of compounds such as p-hydroxybenzoic, protocatechuic and vanillic and p-coumaric acids and trace amounts of syringic acid have also been detected (De La Rosa et al., 2011; Malick et al., 2009; Villarreal-Lozoya et al., 2007; Senter et al., 1980). However, due to analytical difficulties, few studies on the pecan nut shell phenolic composition have been published. De La Rosa et al. (2011), using HPLC coupled to a mass spectrometer, detected only the presence of gallic and ellagic acids. 3.3. Antimicrobial activity Table 3 and Fig. 3 show the inhibition zones and the classification of inhibitory capacity for the different extracts and major phenolic compounds detected. According to the results, the extracts were

effective against L. monocytogenes, S. aureus, V. parahaemolyticus and B. cereus. Antimicrobial activity was not observed for the other bacterial and fungal strains tested such as E. coli (ATCC 25922), A. niger (IOC 0206), A. flavus (FC 1087), P. roquefortii (IOC 2242), Rhizopus sp. (IOC 1109) and Fusarium sp. (IOC 1200). The infusion and the alcoholic extract were able to inhibit growth of S. aureus (ATCC 25923), and for the strain V. parahaemolyticus (ATCC 17802) inhibition zones were observed only when the alcoholic extract was used. All assays showed more pronounced inhibition halos for L. monocytogenes (ATCC 19117). When antibiograms of isolated phenolic substances were evaluated, the antimicrobial activity was observed only for epicatechin gallate (L. monocytogenes, V. parahaemolyticus and B. cereus) and epigallocatechin (S. aureus). This results are in agreement with a study carried out by Puupponen-Pimiä et al. (2001) which tested the antimicrobial activity of 17 pure phenolic compounds representing flavonoids and phenolic acids, and eight berry extracts which were measured against selected Gram-positive and Gramnegative bacteria. This study reported that the strongest inhibition was observed for the extract compared to the pure phenolics compounds. The results of the assays conducted in this research suggested that the inhibitory effects of extracts may not be due to simple phenolics but to complex phenolic compounds, many of which are still unidentified. Moreover, the combination of different phenolic compounds, might be responsible for the enhanced antimicrobial effects. Thus, such results indicate that the extracts

Table 3 Antimicrobial activity of extracts obtained by different extraction processes and major phenolic compounds present in the pecan nut shell against Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus and Bacillus cereus.

Infusion Infusion + spray dryer Ethanol extract Gallic acid Chlorogenic acid Epigallocatechin Epicatechin gallate

Listeria monocytogenes ATCC 19117

Staphylococcus aureus ATCC 25923

Vibrio parahaemolyticus ATCC 17802

Bacillus cereus ATCC 11718

++ ++ ++ − − − +

+ − + − − + −

− − + − − − +

+ + + − − − +

Average diameter of inhibition zones (mm) already subtracted the value of the well bore: + = 7–11 mm, ++ = 12–16 mm, − = no inhibition zone.

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Table 4 Evaluation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) from the extracts (mg/mL) of pecan nut shell against Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus and Bacillus cereus. Infusion (mg/mL) MIC* L. monocytogenes ATCC 19117 L. monocytogenes IIATCC 19112 S. aureus ATCC 25923 S. aureus ATCC 29213 S. aureus ATCC 6538 V. parahaemolyticus ATCC 17802 B. cereus ATCC 11778

A

2.5 1.25A 0.15A 0.46A 0.46A 0.15B 0.11A

MBC** ± ± ± ± ± ± ±

0.01 0.02 0.01 0.13 0.01 0.01 0.01

a

2.5 1.25a 0.63a 1.25a 1.25a 0.15b 0.23a

± ± ± ± ± ± ±

0.03 0.04 0.01 0.10 0.02 0.02 0.01

Infusion + spray dryer (mg/mL)

Ethanol extract (mg/mL)

MIC

MIC

MBC B

0.93 0.62B 0.15A 0.23A 0.23A 0.31A 0.075B

± ± ± ± ± ± ±

0.01 0.01 0.01 0.09 0.03 0.01 0.01

b

0.93 0.93a 0.63a 0.31b 0.31b 0.31a 0.31a

± ± ± ± ± ± ±

0.01 0.01 0.03 0.05 0.04 0.01 0.01

A

1.87 1.25A 0.15A 0.62A 0.62A 0.31A 0.15A

MBC ± ± ± ± ± ± ±

0.01 0.02 0.01 0.16 0.05 0.02 0.01

1.87a 1.25a 0.47a 1.25a 1.25a 0.31a 0.15a

± ± ± ± ± ± ±

0.03 0.01 0.09 0.07 0.04 0.01 0.01

*

MIC: minimum inhibitory concentration. MBC: minimal bactericidal concentration. Equal capital letters in the same row do not differ significantly for MIC; equal lowercase letters in the same row do not differ significantly for MBC; mean ± standard deviation (n = 3); (Tukey test, p < 0.05). **

possess higher antimicrobial activity when compared to the phenolic compounds in isolated form, especially in relation to inhibiting the growth of L. monocytogenes (ATCC 19117). It has been reported that tannins affect bacterial growth through different mechanisms, such as inhibiting the action of extracellular microbial enzymes, complexing the substrates required for growth and metal ions and by direct action on microbial metabolism by oxidative phosphorylation (Serrano et al., 2009; Scalbert, 1991). However, according to Scalbert (1991), some microorganisms, such as A. niger, Penicillium sp. and E. coli, can grow in the presence of tannins and their monomers and dimers, using these compounds as a carbon source. Some species of Penicillium have the ability to use phenols of simpler molecular structure, such as gallic acid and catechins, as a metabolic substrate. This is because some tannases are able to maintain a high metabolic activity even in the presence of tannins, being active mainly in residues of galloyl esters of ellagitannins. This explains the behavior of the fungal strains tested in this study, showing growth capacity in high concentrations of the pecan nut shell extracts. Table 4 shows the results obtained for the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the extracts (in mg/mL) of pecan nut shell in relation to L. monocytogenes, S. aureus, V. parahaemolyticus and B. cereus. All extracts tested showed bactericidal activity and although the inhibition zones had larger diameters for L. monocytogenes, the lowest MIC and MBC values were observed with S. aureus, V. parahaemolyticus and B. cereus. In tests against L. monocytogenes (ATCC 19112), the extracts were more effective at lower concentrations when compared to the results against the strain ATCC 19177. The same behavior was observed in tests against S. aureus where the ATCC 25923 strain showed concentrations for MIC and MBC lower than for ATCC 29213 and ATCC 6538 strains. When assessing the MIC and MBC, it was observed that the extract obtained by infusion followed by spray drying was more effective at lower concentrations against all strains of L. monocytogenes tested, compared to the extracts obtained by infusion and by ethanol extraction. The extract obtained by infusion followed by spraying drying also presented a lower MBC in relation to S. aureus. No significant differences were observed for the MIC values obtained for the different extracts tested in relation to this microorganism for the statistical test (Tukey). The extract obtained by infusion showed the best MIC and MBC in relation to V. parahaemolyticus. The extract obtained by infusion followed by atomization in a spray dryer was effective at lower concentrations according to the MIC and did not show statistically significant differences (p < 0.05) in the case of the MBC. According to Puupponen-Pimiä et al. (2001), the results showed that different bacterial species exhibit different sensitivities toward phenolics. Moreover, different strains of the same bacterial species

showed different response to one specific phenolic compound tested. Ho et al. (2010) evaluated the antimicrobial potential of plant extracts obtained from Orthosiphon stamineus with methanol at concentrations of 25, 50 and 75%. The authors reported a weak antimicrobial activity against the microorganisms S. aureus and B. cereus, and a moderate activity against the growth of V. parahaemolyticus, for which MIC and MBC values ranging from 1.56 to 3.13 mg/mL were observed, depending on the methanol concentration of the extract. Hayrapetyan et al. (2012) evaluated the antimicrobial activity of extracts from pomegranate shell (methanol:ethanol:water 2:2:1) and reported that the growth of L. monocytogenes (ATCC 35152 and 23074) was successfully inhibited in chilled beef (MBC 24.5 mg/mL). However, the same effect was not observed when the phenolic compounds (gallic and ellagic acids) were tested individually. 4. Conclusions Among the three different extraction methods evaluated, infusion and alcohol extraction provided higher levels of total phenolics, condensed tannins and antioxidant activity (DPPH and ABTS) whereas the spray dryer process applied to the infusion extract significantly concentrated its solids content increasing the amount of dry matter, total phenolics and condensed tannins, and, as a consequence, the antioxidant activity. Using the Sephadex® LH-20 resin made it possible to identify gallic acid, chlorogenic acid and p-hydroxybenzoic acid (epigallocatechin and epicatechin gallate) in the pecan nut shell using HPLC analysis. The extracts studied were effective in inhibiting the growth of L. monocytogenes, S. aureus, V. parahaemolyticus and B. cereus, indicating that such extracts present antimicrobial and bactericidal activities against micro-organisms related with foods. Acknowledgements The authors are grateful to Divinut Ind. de Nozes Ltda (Cachoeira do Sul – RS) for providing the raw material and to Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) for providing the scholarship for Ana Cristina Pinheiro do Prado. The authors would like to thank the following students: Fernanda Ramlov from the Laboratory of Plant Biochemistry and Morphogenesis (UFSC); Simone Mazzutti, Patrícia Beneli and Kátia Andrade from the Laboratory of Thermodynamics and Supercritical Extraction (UFSC); and Priscila de Brito Policarpi and Priscilla dos Santos de Oliveira from the Laboratory of Fats and Oils (UFSC) for their assistance with chemical analysis. Also the authors would like to thank Gustavo Althoff (PhD in Translation Studies at Universidade Federal de Santa

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