Effects of gluconic and alcoholic fermentation on anthocyanin composition and antioxidant activity of beverages made from strawberry

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in LWT – Food Science and Technology on June 2016, available at: https://doi.org/10.1016/j.lwt.2016.01.070 .” 1 Effects of gluconic and alcoholic fermentation on anthocyanin composition and 1 antioxidant activity of beverages made from strawberry 2 Ruth Hornedo-Ortega,a Stéphanie Krisa,b M.Carmen García-Parrillaa* and Tristan 3 Richardb 4 a Area of Nutrition and Food Science, Faculty of Pharmacy, University of Seville, C/P. 5 García González No 2., Sevilla 41012, Spain. 6 b University of Bordeaux, ISVV Bordeaux-Aquitaine, 71 Avenue Edouard Bourleaux, 7 33883 Villenave d’Ornon Cedex, France. 8 e-mails: 9 Ruth Hornedo-Ortega : rhorned[email protected] 10 Stéphanie Krisa: [email protected] 11 M. Carmen García-Parrilla: [email protected] 12 Tristan Richard: [email protected] 13 14 * Correspondence author: M. Carmen García-Parrilla, Area of Nutrition and Food 15 Science, Faculty of Pharmacy, University of Seville, C/P. García González No 2., Sevilla 16 41012, Spain. E-mail: [email protected]. 17 18 19 20 21 2 22 Abstract 23 Strawberry is a very perishable fruit, well-known for being a source of bioactive 24 compounds. The elaboration of the beverages by alcoholic and gluconic fermentation 25 process has been explored as a worthy strategy for preventing food losses as well as 26 preserving bioactive compounds with antioxidant properties. 27 To this end, this paper aims to characterize the anthocyanin composition of the resulting 28 beverages and to evaluate their antioxidant properties with in vitro assays (ORAC, 29 DPPH). Additionally, the protective effect against amyloid-β (Aβ) peptide toxicity in 30 terms of Reactive Oxygen Species (ROS) production and PC12 cells viability was 31 determined. 32 Eleven anthocyanin compounds were identified and quantified by UHPLC-DAD-MS. 33 Pelargonidin 3-glucoside and its derivatives were the major compounds. Gluconic 34 fermentation preserved anthocyanin composition being an advantage of this innovative 35 process. Accordingly the values of antioxidant activity were higher for gluconic than 36 alcoholic fermented beverages. Indeed, both of them increased cell viability (16 to 57% 37 p<0.05) and attenuate the oxidative stress triggered by Aβ (13 to 38 % p<0.05). 38 39 Keywords: process, drink, polyphenols, ROS, amyloid-β 40 41 Pelargonidin 3-glucoside (PubChem CID: 443448); Pelargonidin 3-rutinoside (PubChem CID: 42 44256626); Pelargonidin 3- (6”-malonylglucoside) (PubChem CID: 45256635); Cyanidin 343 glucoside (PubChem CID: 92131208); Delphinidin 3-glucoside (PubChem CID: 443650). 44 3 45 Abbreviations: 46 Aβ: amyloid-β 47 AAPH: 2,2´-diazo-bis-amidinepropane-dihydrochloride 48 DCFDA: 2',7'-dichlorofluorescin diacetate 49 DPPH: 2,2-diphenyl-1-picrylhydrazyl 50 IC50: Half Maximal Inhibitory Concentration 51 MTT: Thiazolyl Blue Tetrazolium Bromide 52 ORAC: Oxygen Radical Absorbance Aapacity 53 ROS: Reactive Oxygen Species 54 UHPLC: Ultra High-Performance Liquid Chromatography 55 56 57 58 59 60 61 62 1. Introduction 63 Strawberry is an important fruit crop worldwide, especially for fresh consumption. An 64 alternative for avoiding economic loss due to its perishable nature is the elaboration of 65 4 derivatives products such as jams, yoghourts, products for biscuits or cakes and 66 beverages made from strawberry pureés. 67 Recently, different studies summarized the evidence for the health benefits of 68 strawberry and other berry fruits (Basu, Nguyen, Betts, & Lyons, 2014; Giampieri et al., 69 2015). Indeed, strawberry is a good source of bioactive compounds. Furthermore, the 70 antioxidant properties of strawberry have been attributed to its polyphenol and vitamin 71 content, being ascorbic acid, ellagitanins and anthocyanins the greatest contributors to 72 its antioxidant capacity (Aaby, Ekeberg, & Skrede, 2007; Manganaris, Goulas, Vicente, & 73 Terry, 2014). Recently, strawberries were included among the 100 richest sources of 74 dietary polyphenols and also listed in rankings of 89 foods and beverages that provide 75 more than 1 mg of polyphenols per serving (Pérez-Jiménez, Neveu, Vos, & Scalbert, 76 2010). Anthocyanins are responsible for the red color of berry fruits, such as blueberries, 77 blackberries and strawberries. It is well known that pelargonidin 3-glucoside is the major 78 anthocyanin in strawberry (150–650 mg kg-1 of fresh weight) (García-Viguera, Zafrilla, & 79 Tomás-Barberán, 1998; Lopes-da-Silva, Escribano-Bailón, Pérez Alonso, Rivas-Gonzalo, 80 & Santos-Buelga, 2007). Indeed, there are crucial factors that influence significantly the 81 stability of anthocyanin compounds such as process, time, and storage temperature 82 (Clifford, 2000). Several efforts have been done to diminish the effect of process in the 83 composition of products made from strawberry using different production systems and 84 the employment of modified atmosphere in the storage (Fan et al., 2012; Oliveira et al., 85 2015). 86 Furthermore, the production of strawberry drinks is an innovative trend. In particular, 87 the fermentation by Gluconobacter japonicus which transforms the glucose content of 88 5 the fruit into gluconic acid to keep the fructose as sweetener (Cañete-Rodríguez et al., 89 2015). Additionally, alcoholic fermentation by Saccharomyces cerevisiae, is used to 90 elaborate strawberry beverages (Hidalgo, Torija, Mas, & Mateo, 2013) The impact of 91 gluconic fermentation has been evaluated in terms of amino acids and biogenic amines 92 (Ordóñez et al., 2015). Besides, non-anthocyanin composition of these beverages 93 (gluconic and alcoholic) has been described before showing their potential as a source 94 of bioactive compounds (Álvarez-Fernández, Hornedo-Ortega, Cerezo, Troncoso, & 95 García-Parrilla, 2014; Álvarez-Fernández, Cerezo, Cañete-Rodriguez, Troncoso, & García96 Parrilla, 2015). 97 Among the healthy properties of strawberries, neuroprotective effects due to the 98 anthocyanin content have been reported (Giampieri et al., 2015) Hence, recent studies 99 show the effectiveness of anthocyanins against Aβ toxicity. Indeed, Badshah, Kim, & Kim 100 (2015) demonstrated that an anthocyanin extract (cyanidin 3-glucoside, delphinidin 3101 glucoside and petunidin 3-glucoside) of black soybean decreased the neuronal death in 102 HT22 cells. Additionally, cyanidin 3-glucoside can inhibit Aβ25–35 spontaneous 103 aggregation into oligomers and their neurotoxicity in human neuronal SH-SY5Y cells 104 (Tarozzi et al., 2010). To the best of our knowledge, the protective effect against Aβ 105 peptide has not been explored neither with strawberry or its derivatives nor for 106 pelargonidin and derivatives compounds. Therefore, our work intends to explore the 107 hypothetical activity these compound and beverages may present. 108 The aims of this paper are to (i) characterize the anthocyanin composition of fermented 109 beverages elaborated from strawberry, (ii) to estimate the effect of alcoholic and 110 gluconic fermentation on anthocyanin compounds, (ii) to evaluate their bioactive 111 potential. 112 6 2. Material and methods. 113 2.1. Samples 114 Hudisa Desarrollo Industrial S.A. (Lepe, Spain) provided strawberry purée. The process 115 of elaboration of the strawberry mash is summarized as follows: the fruit is received, 116 selected, cleaned and the stems and leaves are eliminated. After the strawberry has 117 been mashed, an inactivation enzymatic (2 min, 55 ºC–65 ºC) is performed, followed by 118 a pasteurization process (3 min, >90 ºC). Finally, the temperature is reduced to 5 ºC. The 119 mash is sieved to remove the seeds. 120 In this study, two harvests were analyzed (2012 and 2013). These purées were frozen (- 121 20ºC) until fermentation was carried out. Alcoholic and gluconic fermentations were 122 conducted in the Department of Inorganic Chemistry and Chemical engineering of the 123 University of Córdoba (Córdoba, Spain). Fermentation conditions were previously 124 described (Álvarez-Fernández et al., 2014, 2015). Alcoholic fermentation was carried out 125 with a Saccharomyces cerevisiae (CET 13057 isolated from native strawberry yeast) used 126 as a starter for the submerged fermentation process. The fermentation process was as 127 follows: 3.6 L of strawberry purée were placed into the bioreactor and the conditions 128 set (pH 3.32, 29 ºC, 26.20 rad.s-1); the medium was saturated with oxygen only at the 129 beginning of the fermentation process, before adding the inoculum (10% (w/v) glucose, 130 0.1% (w/v) MgSO4, 0.2% (w/v) KH2PO4, 0.3% (w/v) (NH4)2SO4, 0.4% (w/v) yeast extract 131 and 0.36% (w/v) bacteriological peptone). The end of the fermentation process was 132 established when the glucose had been totally consumed and final pH was 3.30. 133 For gluconic fermentation, 3 L of strawberry purée substrate were placed into the 134 bioreactor and the conditions set (pH 3.24, 29 ºC, 20% O2 and 1250 g); after 10-20 min, 135 7 125 ml of inoculum of Gluconobacter japonicus strain E1 were added (5% (w/v) glucose, 136 1% (w/v) bacteria extract and 2% (w/v) bacteriological peptone) and mixed for 20-30 137 min, then the initial sample was taken. The end of the fermentation process was 138 established when the glucose had been totally consumed and final pH was 2.74. 139 Eight alcoholic fermentation (code A) experiments were performed: four with purées 140 from the 2012 harvest (code 12) and four with those from the 2013 harvest (code 13). 141 Additionally, six gluconic fermentation experiments (code G) were performed: four with 142 purées from the 2012 harvest and two from the 2013 harvest. Samples were taken at 143 the initial point of the fermentation experiment (I), the final point (F) and after 144 pasteurization of the fermented product (FP). Pasteurization was carried out at 70-80 ºC 145 for 15 minutes. All samples were frozen until analysis. Table 1 displays the codes of the 146 samples used in this study. 147 2.2. Chemicals and reagents 148 Amberlite XAD7HP, Dimethyl sulfoxide (DMSO), Dulbecco´s modified Eagle´s medium 149 (DMEM)-Glutamax, Trypsine-EDTA, Thiazolyl Blue Tetrazolium Bromide (MTT), 150 Phosphate Buffered Saline (PBS), L-glutamine, fetal horse serum and fetal bovine serum, 151 streptomycin, 2',7'-dichlorofluorescin diacetate (DCFH-DA), 2,2-diphenyl-1152 picrylhydrazyl (DPPH), 2,2'-diazo-bis-amidinepropane-dihydrochloride (AAPH), and 153 Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic) were purchased from 154 Sigma (Steinheim, Germany). Fluorescein sodium was obtained through Fluka 155 (Steinheim, Germany). 156 VWR Chemicals (Llinars del Vallés, Barcelona) supplied methanol and acetic acid. Formic 157 acid and acetonitrile were obtained by Fisher Chemical. Pelargonidin 3-glucoside was 158 8 obtained from Chloride (Cromadex Inc., USA). Cells PC12-Adh were supplied by ATCC® 159 CRL-1721.1TM (Manassas, USA) and Amyloid β-protein 25–35 (Aβ25-35) by Synvec 160 (Bordeaux, France). 161 2.3. Sample preparation 162 An Amberlite XAD7HP column (30 x 1.5 cm) was conditioned with 200 mL of methanol 163 and then 200 mL of water. A total of 20 g of sample (gluconic and alcoholic fermented 164 samples) were diluted with water (1:1, w/v). The column was loaded with the diluted 165 sample and cleaned with water to eliminate sugars, acids and polar compounds. 166 Subsequently, the anthocyanin fraction was eluted with a mixture of methanol: acetic 167 acid (19:1); flow rate 1 drop s-1. This fraction was collected and concentrated with a 168 rotary evaporator under vacuum (Büchi Rotavapor, R-200/205), frozen (-80ºC) and 169 finally freeze-dried. A total of approximately 20 mg of this extract was obtained from 20 170 g of sample. The respective extracts were used for the subsequent analysis. 171 2.4. Analysis of anthocyanin compounds 172 Analyses were carried out using a UHPLC system model 1290 Infinity (Agilent, Palo Alto, 173 CA), equipped with a binary pump (Agilent Technologies, 1290 VL G4220B), an 174 autosampler (Agilent Technologies, 1290 sampler, G4226A) and a DAD detector (Agilent 175 Technologies, G1316C). The UHPLC system was coupled to a Bruker mass spectrometer 176 (model Esquire 3000+) with electrospray ionization (ESI), Atmospheric Pressure 177 Chemical Ionization (APCI) and an Ion Trap analyzer. Samples were filtered through a 178 Millipore PTFE 0.45 µm filter before injection. 179 15 alcoholic and gluconic fermentation on the activity of the extract. In particular, those 312 extracts with higher concentration in cyanidin 3-glucoside, pelargonidin 3-glucoside, 313 pelargonidin 3-rutinoside and pelargonidin 3-(6’’-malonylglucoside) according to the 314 values displayed in Tables 3 and 4 were used for further experiments. 315 First, the cytotoxic potential of the extracts at different concentrations ranging from 10 316 to 200 µg mL-1 on PC12 cells was measured. We observed a toxic effect for PC12 cells at 317 200 µg mL-1 in all tested extracts (data not given). Figure 2 displays cell viability 318 expressed as a percentage relative to the untreated control cells. After exposure to Aβ25319 35 alone, viability decreased by more than 60%, compared with the control. However, 320 when cells were treated with Aβ25-35 in the presence of the strawberry extracts, cell 321 viability increased significantly (p<0.05) (16 to 57%), in a dose-dependent manner. 322 Specifically, 100 µg mL-1 of gluconic extract reversed amyloid-induced toxicity and 323 increased cell viability up to near the control level (Figure 2). Other authors have 324 reported that acai berry extracts (50 µg mL-1) did not provide any significant degree of 325 protection against Aβ25-35-mediated loss of cell viability PC12; however, they had an 326 effect when tested with Aβ1-42 at 5 µM, with 25 and 29% increased cell viability at 5 and 327 50 µg mL-1, respectively (Wond, Musgrave, Harvey, & Smid, 2013). Additionally, 328 blueberry leaf extracts (50-100 µg mL-1) caused a small cell viability increase of 6-15% 329 on Aβ25-35-induced cytotoxicity in PC12 cells (Jeong et al., 2013). Harvey, Musgrave, 330 Ohlsson, Franson, & Smid (2011) found that grape-skin extract tested at different 331 concentrations of Aβ1-42 provided protection against amyloid toxicity (37%) in PC12 cells. 332 As far as we know, the strawberry extract studied in this paper shows one of the highest 333 protective effects against Aβ-induced damage. 334 16 The effect of the extracts on cell viability could be explained as being due to the presence 335 of pelargonidin 3-glucoside, which is the major compound in our extracts. For this 336 reason, we evaluated the cytoprotective ability of pelargonidin 3-glucoside standard on 337 PC12 cells. Cell viability was decreased after cells were exposed to 300 µM (data not 338 shown). However, a protective effect was determined at concentrations of 50, 100 and 339 200 µM (21.6, 43.2 and 86.4 µg mL-1, respectively), in a dose-dependent manner (Figure 340 2B). In gluconic fermented extract (G13F) at the highest concentration tested (100 µg 341 mL-1), pelargonidin 3-glucoside concentration is 12.9 µg mL-1. Since the pelargonidin 3342 glucoside at concentration 21.6 µg mL-1 (= 50 µM) increased cell viability by just 10%, 343 the protective effects of gluconic fermented extract are not only due to the pelargonidin 344 3-glucoside. Other anthocyanins described here and polyphenols previously identified 345 in strawberry may contribute to the protective activity in our extracts (Cerezo et al., 346 2010). Indeed, other authors have reported that cyanidin 3-glucoside and ellagic acid 347 also contribute to the neuroprotective effect against Aβ25-35 and Aβ1-42, respectively, in 348 SH-SY5Y cells (Tarozzi et al., 2010; Feng et al., 2009). 349 It has been postulated that ROS are important players in degenerative diseases (Emerit, 350 Edeas & Bricaire, 2004). ROS generate protein and lipid oxidations, DNA damage and 351 induce the death of neuronal cells (Wang et al., 2014). The neurotoxic effect of Aβ is 352 associated with the production of ROS. Recent studies showed that cyanidin and 353 malvidin 3-glucoside have protective effects against Aβ-induced neurotoxicity through 354 inhibiting ROS formation (Thummayot et al., 2014; Shih, Wu, Yeh, & Yen, 2011) 355 In this study, we evaluated the effect of G13F and A13F extracts on ROS production by 356 measuring intracellular ROS levels with the DCFH-DA assay. As shown in Figure 3, Aβ25357 35 treatment for 6 h induced a 1.8to 2.4-fold increase in ROS production. Co-treatment 358 17 with different concentrations of strawberry extracts significantly attenuated 359 intracellular ROS accumulation. Strawberry extracts decreased the production of ROS in 360 a dose-dependent manner that can attenuate Aβ-induced oxidative stress proving this 361 specific antioxidant activity. 362 4. Conclusion 363 Alcoholic fermentation of strawberry pureés decreased the anthocyanin content while 364 gluconic fermentation preserved these compounds, which is an advantage of this last 365 process. All these extracts were protective against Aβ-peptide neurotoxicity in PC12 366 cells, with gluconic fermented strawberry extracts being the most effective, as 367 determined by cell viability and intracellular ROS production. Taken together, our 368 experiments suggest that the fermented derivatives of strawberry are a good source of 369 antioxidant bioactives. 370 ACKNOWLEDGMENTS 371 The authors are very grateful to the Spanish Government for its financial assistance 372 (Project MICINN AGL2010-22152-01) and to the University of Seville and the Institute of 373 Vine and Wine Sciences (ISVV, University of Bordeaux) for their financial assistance in 374 the international mobility of Ruth Hornedo-Ortega and Tristan Richard. We would also 375 like to thank M.L. Iglesias, E. Pedrot and H. Temsamani for technical assistance and 376 HUDISA Desarrollo Industrial S.A., in Lepe, Spain, for providing the strawberry purée 377 samples. We would like to thank Professor Isidoro García García (Dept. Chemical 378 Engineering, University of Córdoba) for providing the fermenting products, as well as 379 Biology services (CITIUS) of the University of Seville, for the multi-detector micro-plate 380 18 reader. MS experiments were performed at the Plateforme Métabolome-Fluxome, 381 Centre de Génomique Fonctionnelle de Bordeaux, France. 382 383 19 Figure captions 384 Figure 1. Chromatogram and chromatogram (zoom) recorded at 520 nm showing the 385 anthocyanin profile of the strawberry samples. 1: Catechin-(4-8)-pelargonidin 3386 glucoside; 2: Epicatechin-(4-8)-Pelargonidin 3-glucoside; 3: Delphinidin 3-glucoside; 4: 387 (epi)Afzelechin-pelargodinin 3-glucoside; 5: Cyanidin 3-glucoside; 6: Pelargonidin 3,5388 diglucoside; 7: Pelargonidin 3-glucoside; 8: Pelargonidin 3-rutinoside; 9: 5389 carboxypyranopelargonidin 3-glucoside; 10: Pelargonidin 3-(6’’-malonylglucoside); 11: 390 Pelargonidin 3-acetylglucoside. 391 392 Figure 2 (A-B). Cells viability determination. Cells were treated 24h by strawberry 393 extracts (A) or pelargonidin 3-glucoside (B), in presence or absence of 10 µM of Aβ25-35. 394 Results are expressed as mean SEM of four replicates (n=4). ♯ P < 0.05 Aβ (25-35) versus 395 control, * P < 0.05 extract versus Aβ (25-35). 396 397 Figure 3. ROS (Fluorescence intensity). Production of ROS in PC12. 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