Influence of Fermentation Process on the Anthocyanin Composition of Wine and Vinegar Elaborated from Strawberry ´ lvarez-Fern´andez, Ana B. Cerezo, Isidoro Garcia-Garcia, Ana M. Troncoso, Ruth Hornedo-Ortega, M. Antonia A and M. Carmen Garcia-Parrilla
Anthocyanins are the major polyphenolic compounds in strawberry fruit responsible for its color. Due to their sensitivity, they are affected by food processing techniques such as fermentation that alters both their chemical composition and organoleptic properties. This work aims to evaluate the impact of different fermentation processes on individual anthocyanins compounds in strawberry wine and vinegar by UHPLC-MS/MS Q Exactive analysis. Nineteen, 18, and 14 anthocyanin compounds were identified in the strawberry initial substrate, strawberry wine, and strawberry vinegar, respectively. Four and 8 anthocyanin compounds were tentatively identified with high accuracy for the 1st time to be present in the beverages obtained by alcoholic fermentation and acetic fermentation of strawberry, respectively. Both, the total and the individual anthocyanin concentrations were decreased by both fermentation processes, affecting the alcoholic fermentation to a lesser extent (19%) than the acetic fermentation (91%). Indeed, several changes in color parameters have been assessed. The color of the wine and the vinegar made from strawberry changed during the fermentation process, varying from red to orange color, this fact is directly correlated with the decrease of anthocyanins compounds.
Abstract:
Food Chemistry
Keywords: anthocyanins, fermentation processes, strawberry, vinegar, wine
Reducing food waste is a challenge worldwide to prevent economic losses, reduce environmental impact, and contribute to a more sustainable agriculture. This work intends to reduce food waste of strawberry surpluses as this perishable fruit is harvested in a very short period of time and a large amount is discarded if not marketed. The strategy of transforming it into other products that last for a longer period of time is a way of improving value along the food chain. This work proposes to elaborate strawberry vinegar and strawberry wine that can be appreciated by consumers to accomplish this purpose.
Practical Application:
Introduction Strawberries and derived strawberry products are considered a rich sources of phytochemicals (ellagic acid, anthocyanins, quercetin, and catechin), vitamins C and E, and folic acid (Proteggente and others 2002; St¨urtz and others 2011). Although this fruit is widely consumed as fresh product, currently there are more and more processed products commercially available (spreads, jams, syrups, alcoholic and nonalcoholic beverages, teas, and so forth). Furthermore, innovative trends in designing food products are devoted to satisfy consumer’s demand of a higher diversity on the market. In addition to the fact that the transformation of a high perishable fruit into beverages permits to increase its conservation period and prevent food waste, fermentation has been proposed to increase the bioaccesibility of the polyphenolic compounds by their release from the vegetal matrix (Acosta-Estradaand others 2014). JFDS-2016-1529 Submitted 9/19/2016, Accepted 12/19/2016. Authors ´ Hornedo-Ortega, Alvarez-Fern´ andez, Cerezo, Troncoso, and Garcia-Parrilla are with Dept. of Nutrition and Food Science, School of Pharmacy, Universidad de Sevilla, C/P., Garc´ıa Gonz´alez n° 2, Sevilla 41012, Spain. Author Garcia-Garcia is with Dept. of Inorganic Chemistry and Chemical Engineering, School of Sciences, Campus of Rabanales, Universidad de Cordoba, Ctra. de Madrid, km 396, C´ordoba 14071, Spain. Direct inquiries to author M. Carmen Garcia-Parrilla (E-mail:
[email protected]).
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It is color that represents an aspect of major importance on strawberry quality. Indeed, attractive colors increase consumer’s preference and consequently, its price. Not only, does this parameter has that influence on the quality of the beverage but also on its chemical composition. In this context, anthocyanins compounds are relevant due to their contribution on color and bioactivity (Basu and others 2014). Furthermore, it is well known that pelargonidin 3-glucoside is the major anthocyanin in strawberry (153 to 652 mg/kg fresh weight) followed by pelargonidin 3-rutinoside and other pelargonidin and cyanidin derivatives (Lopes-da-Silva and others 2007; Cerezo and others 2010). Anthocyanins are known to be unstable compounds. Food processing, time, storage, and temperature are crucial factors that influence significantly the stability of these compounds (Clifford 2000). These factors can lead to several chemical and enzymatic reactions that produce a change or modification of their chemical structure (Cavalcanti and others 2011). In fact, changes in anthocyanin compounds during winemaking process and vinegar elaboration have been studied in depth. For example, pyranoanthocyanins constitute one of the most important classes of anthocyanin-derived pigments occurring naturally in red wine (Oliveira and others 2010; de Freitas and Mateus 2011). Moreover, it have been established that anthocyanins with acylating substituents are more stable during processing and storage (Giusti and Wrolstad 2003; Fossen and others 2003; Kamiloglu and R C 2017 Institute of Food Technologists
doi: 10.1111/1750-3841.13624 Further reproduction without permission is prohibited
Anthocyanins in strawberry wine & vinegar . . .
Materials and Methods
oxygen only at the beginning of the fermentation process before the inoculum was added (10% [w/v] glucose, 0.1% [w/v] MgSO4 , 0.2% [w/v] KH2 PO4 , 0.3% [w/v] (NH4 )2 SO4 , 0.4% [w/v] yeast extract and 0.36% [w/v] bacteriological peptone). The alcoholic fermentation process was stopped once sugars were depleted. In total, 3 fermentation cycles were performed in duplicate (strawberry initial sample and strawberry wine).
Acetic acid fermentation An active mixture of acetic acid bacteria from a Frings acetator (Heinrich Frings Gmb and Co., KG, Bonn, Germany; BaenaRuano and others 2006, 2010a, 2010b; Garcia-Garcia and others 2007; Maestre and others 2008; Jimenez-Hornero and others 2009a, 2009b) producing alcohol vinegar was used as started for the acetification process. The fermentation was operated in a semicontinuous mode under the following operational conditions: temperature, 31 °C; agitation, 500 rpm; and dissolved oxygen, 70%. Initially, the bioreactor was loaded with 3.6 L of alcoholic fermented strawberry; once ethanol was depleted, 2.6 L of acetic acid ferment were unloaded and subsequently replaced by the same volume of alcoholic fermented strawberry. Two fermentation cycles were performed in duplicate (strawberry vinegar). Samples Samples, which were supplied by Dept. of Inorganic Chemistry and Chemical Engineering, Faculty of Sciences, Univ. of Cordoba (Cordoba, Spain), were taken at the beginning (strawberry initial samples) and at the end of each fermentation cycle (final samples), in both fermentation processes.
Reagents and standards Folin-Ciocalteu reagent and sodium acetate were purchased from Merck (Darmstadt, Germany); potassium chloride was supplied by Panreac (Castellar del Vall`es, Barcelona); gallic acid was purchased from Fluka (Steinheim, Germany); Amberlite XAD7HP Sigma, (Steinheim, Germany); methanol, acetic acid, formic acid and acetonitrile were obtained from VWR Chemicals, (Llinars del Vall´es, Barcelona); pelargonidin 3-glucoside, cyanidin 3-glucoside, delphinidin 3-glucosise and peonidin 3-glucoside were purchased from Chromadex Inc. (Irvine, Calif., U.S.A.).
Sample preparation The preparation of anthocyanin fraction of fermented strawberry was carried out, according to Cerezo (2010). An Amberlite XAD7HP column (30 × 1.5 cm) was conditioned with 200 mL of methanol and then 200 mL of water. A total of 20 g of sample was diluted with water (1:1 w/v). The column was loaded with the diluted sample and cleaned with water. Subsequently, the anthocyanin fraction was eluted with methanol: acetic acid (19:1); flow rate 1 drop/s. This fraction was collected and concentrated with a rotary evaporator under vacuum (B¨uchi Rotavapor, R-200/205, Flawil, Switzerland). Finally the extracts were reconstituted in 2 Raw material mL of acidified water (5% formic acid) and stored at -20 °C until Strawberry pur´ee elaborated by Hudisa, S.A. (Lepe, Spain) was analysis. used as substrate. The pur´ee (2012 harvest) was industrially pasteurized at 92 °C for 90 to 120 s and stored at 0 °C to 4 °C, con- Determination of total phenolic index (TPI) taining 34.00 ± 1.25 g sugars/L (45% glucose and 55% fructose). The TPI was determined by the Folin–Ciocalteu method Samples were stored at this temperature for 2 or 3 d. The necessary (Klopotek and others 2005). First, 50 μL of each sample were time to transport it from the Hudisa Comp. to the Universidad of diluted in 1 mL of distilled water. Then, 20 μL of sample solution Cordoba where they were frozen until they were fermented. was mixed with 1.58 mL of distilled water in a glass cuvette. Subsequently, 100 μL of Folin-Ciocalteu’s reagent were added 4 min Fermentation processes after we put in 300 μL of Na2 CO3 (20%). After the incubation for R Fermentation runs were conducted in a 5 L Biostat fermen- 30 min in a bath at 40 °C, the absorbance was recorded at 750 nm. tation tank equipped with pH, agitation, dissolved oxygen, and The TPI was expressed as milligrams of gallic acid/L using this temperature controls. compound as standard. Samples were analyzed in quadruplicate. Alcoholic fermentation A Saccharomyces cerevisiae strain (CECT 13057), isolated from native strawberry yeast (Hidalgo and others 2013) was used as a starter for the alcoholic fermentation process. The fermentation was operated in a batch mode, using a loading volume of 3.6 L of strawberry pur´ee. The operational conditions were: temperature, 29 °C; and agitation, 250 rpm. The medium was saturated with
Determination of total anthocyanin (TA) content The TA content was estimated by a pH differential method (Giusti and Wrolstad 2001). First, samples were filtered and diluted (1/5). Then, 600 μL of each sample were mixed with 2.4 mL of sodic acetate (pH 4.5) or potassium chloride buffer (pH 1). After 15 min, absorbance (A) was measured at 520 and 700 nm in both buffers (pH 1.0 and 4.5). Samples were analyzed in quadruplicate. Vol. 82, Nr. 2, 2017 r Journal of Food Science 365
Food Chemistry
others 2015). This fact has been attributed to the stacking of the acyl groups with the pyrilium ring of the flavylium cation, reducing the susceptibility of a nucleophile attack of water and therefore the formation of intramolecular copigmentation of a pseudobase or a chalcone (Brouillard and others 2003). Despite that the characterization of anthocyanin composition of strawberry has been accomplished lately, studies devoted to their profile in beverages made from strawberry are rather scarce (Hornedo-Ortega and others 2016a, 2016b). Most of the studies of anthocyanins composition in strawberry derived products have been conducted to determine their total anthocyanin content expressed as a chemical index. For example, Ubeda and others (2013) reported a decrease in total anthocyanins after alcoholic and acetic fermentation process of strawberry. Klopotek and others (2005) showed a loss of total anthocyanins when comparing the strawberry mash with the strawberry wine. However, very limited information about the individual anthocyanin composition of wines and vinegars made from strawberry is available. Consequently, it is relevant to gain knowledge on anthocyanin characterization to understand the effect of fermentation on this class of bioactive compounds, which in turn exert a role on sensory properties, color, in particular. The aim of this paper is to characterize the individual anthocyanins composition of strawberry-derived products obtained by alcoholic and acetic fermentation processes by means of (UHPLCMS/MS)-Q Exactive and to evaluate the influence of these processes on the color of the resulting products.
Anthocyanins in strawberry wine & vinegar . . . Results were calculated as follows: A =(A515 −A700 )pH1.0 −(A515 −A700 )pH4.5 C (mg/L) = (A x molecular weight x dilution factor x 1000) /εl The molecular weight of pelargonidin 3-glucoside is 433.0 g/ mol and ɛ is 22400 mol−1 . 1 = path length (1 cm).
Food Chemistry
Color measurements Color measurements were determined using a Konica Minolta CM-3600d spectrophotometer (Minolta Co. Ltd., Osaka, Japan) in the CIELab color space, with the D65 illuminant and 10° observer. Color results were expressed as tristimulus parameters (Lࢩ , aࢩ , bࢩ , Hࢩ , Cࢩ ). Hue angle (Hࢩ = tan−1 bࢩ /aࢩ ) indicates sample color (0° or 360° = red, 90° = yellow, 180° = green, 270° = blue), and chroma (Cࢩ = [aࢩ2 + bࢩ2 ]1/2 ) indicates color purity or saturation (high values are more vivid); aࢩ and bࢩ chromaticity coordinates indicate color directions green (−aࢩ )/red (+aࢩ ) and blue (−bࢩ )/yellow (+bࢩ ) (Bakker and others 1986). Samples were analyzed in quadruplicate. Analysis of individual anthocyanin compounds An UHPLC Dionex Ultimate 3000 system (Thermo Scientific, San Jose, Calif., U.S.A.), coupled to a Thermo Scientific Q-ExactiveTM hybrid quadrupole-orbitrap mass spectrometer (Bremen, Germany), was used. The UHPLC system consisted of consisting of a quaternary Rs Pump Dionex Ultimate 3000 (serial number: 8077352) and Rs autosampler Dionex Ultimate 3000 (serial number 8077399), connected to a quadrupole-orbitrap (Q Exactive) hybrid mass spectrometer with heated-electrospray ionization probe (HESI-II, Thermo Fisher Scientific). The analytical method was previously published (Nati´c and others 2015). Separation was performed on a column SB-C18 (2.1 × 100 mm, 1.8 μm) set at 40 °C (Agilent, Santa Clara, CA). Injection volume was 1 μL and flow rate was 0.4 mL/min. The solvents used as a mobile phase were: Solvent A (water/formic acid 95:5 v/v) and solvent B (acetonitrile/formic acid 95:5 v/v), scheduled in the following gradient: 0.0 to 2.0 min 5% B, 2.0 to 12.0 min from 5% to 100 % B, 12.0 to 13.0 min from 100% to 5% B, then 5% B up to 15.0 min. Anthocyanin identification and quantitation were acquired in positive mode by full-range acquisition covering m/z 100 to 1500 at 35000 resolution and by targeted MS2 normalized Higher Energy Collision Dissociation (HCD). HESI source parameters were as follows: cell at 20 eV, source voltage 3.5 kV, tube lens voltage 50 V, capillary temperature 263 °C, and sheath and auxiliary gas flow rate (N2 ) 50 and 13 (arbitrary units). Xcalibur software (version 3.0.63) was used for instrument control, data acquisition, and data analysis. Compounds were identified according to their, calculated mass, accurate mass, mass spectra, characteristic fragmentation data, and retention time. Pelargonidin 3-glucoside, cyanidin 3-glucoside, delphinidin 3-glucosise, and peonidin 3-glucoside available standards were used both for positive identification and quantification purposes. Anthocyanin compounds were quantified using the areas of the aglycone counterparts. Statistical analysis One-way analysis of variance (ANOVA test) (P
