Employment of different processes for the production of strawberry vinegars: Effects on antioxidant activity, total phenols and monomeric anthocyanins

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in LWTFOOD SCIENCE AND TECHNOLOGY on 2013, available at: https://doi.org/10.1016/j.lwt.2012.04.021.” Employment of different processes for the production of strawberry vinegars: 1 Effects on antioxidant activity, total phenols and monomeric anthocyanins 2 C. Ubeda1, R. Callejón1, C. Hidalgo2, M.J. Torija2, A.M. Troncoso1, M.L. Morales1* 3 1Área de Nutrición y Bromatología. Facultad de Farmacia. Universidad de Sevilla. 4 C/ P. García González nº2, E- 41012. Sevilla. Spain. 5 2Departamento de Bioquímica y Biotecnología. Facultad de Enología. Universitat 6 Rovira i Virgili. C/ Marcel·lí Domingo s/n. E- 43007. Tarragona. Spain. 7 *Corresponding author: [email protected]; Tel.: 34-954-556760; Fax.: 34-954-233765 8 Abstract 9 The use of strawberry surpluses for the production of added value products seems to be 10 a good solution choice to avoid the waste of this fruit. We produced strawberry vinegars 11 through double fermentation (alcoholic and acetous) from three different harvests of 12 Fragaria x ananassa var. Camarosa. The objective was to study the evolution of 13 antioxidant activity, total phenols and monomeric anthocyanins during the vinegar 14 production process. These parameters increased when sulphur dioxide and pectolytic 15 enzymes were added to substrates. Inoculation with the Saccharomyces cerevisiae strain 16 RP1 produced wines with half the anthocyanins with respect to the spontaneous 17 fermentations. The use of wood barrels, particularly cherry wood barrels, had a positive 18 effect on all the parameters determined. All measured parameters decreased during the 19 double fermentation process. In general, the acetification stage led to a high loss of 20 antioxidant compounds. Moreover, the production of these vinegars at a semi-pilot scale 21 yielded final commodities with the best values for antioxidant activity, total phenols and 22 monomeric anthocyanins comparing with the vinegars obtained in 2008 and 2009 23 harvest. 24 Keywords: antioxidant activity; monomeric anthocyanins; strawberry; vinegar; wine. 25 2 1. Introduction 26 Strawberries are a widely researched fruit for their nutritional and health benefits as 27 well as their organoleptic properties. This fruit is rich in vitamins, minerals, fibre and 28 phytochemicals. In addition, strawberries contain potentially bioactive compounds and 29 are a great source of phenolic compounds such as flavonoids and phenolic acids (Aaby, 30 K., Skrede, G., & Wrolstad, R. E, 2005; Määttä-Riihinen, K. R., Kamal-Eldin, A., & 31 Törrönen, A. R, 2004; Seeram, N. P., Lee, R., Scheuller, H. S., & Heber, D, 2006). All 32 of these phenolic compounds have been shown to prevent oxidative processes, 33 particularly those caused by reactive oxygen species (ROS) (Aaby, K., Ekeberg, D., & 34 Skrede, G, 2007; Cerezo, A. B., Cuevas, E., Winterhalter, P., Garcia-Parrilla, M. C., & 35 Troncoso, A. M, 2010a). These compounds make strawberries a highly antioxidant fruit 36 (Aaby et al., 2005; Wolfe, K. L., Kang, X., He, X., Dong, M., Zhang, Q., & Liu, R. H, 37 2008) with potential health benefits. Among the numerous healthy properties described 38 in the literature are anti-proliferative effects on cancer cells (Meyers, K. J., Watkins, C. 39 B., Pritts, M. P., & Liu, R. H, 2003; Olsson, M. E., Andersson, C. S., Oredsson, S., 40 Berglund, R. H., & Gustavsson, K, 2006) and the antioxidant and anti-inflammatory 41 effects that have been shown to reduce cardiovascular disease risk factors in several 42 prospective cohort studies (Hannum, 2004). 43 According to the latest data from the FAO (FAOStat, FAO 2011), Spain is the second- 44 largest strawberry producer in the world; a large portion of this production is harvested 45 in Huelva (Andalucía). Every year, part of the crop is discarded for various reasons, 46 including size or deformations of the berries, or overproduction which leads to 47 surpluses. Because vinegar is generally an inexpensive product, its production requires 48 low-cost raw materials, such as sub-standard fruit and seasonal agricultural surpluses 49 (Solieri & Giudici, 2009). In addition, there is a growing demand for fruit vinegars, 50 3 which are sold as a health food (Shau-mei & Chang, 2009). The use of strawberries of 51 second quality, which are still suitable for human consumption, to production healthy 52 vinegars with special organoleptic nuances may be a good method to reduce losses due 53 to discarding the fruit. 54 For this purpose, we have produced strawberry vinegars using second-quality 55 strawberries employing two-stage fermentation and assessed different conditions and 56 treatments. The aim of this work was to evaluate the changes in the antioxidant activity 57 (AA), total phenols index (TPI) and total monomeric anthocyanins (TA) during the 58 production process of strawberry vinegar. In addition, an adequate extraction method to 59 perform these determinations was designed. 60 2. Materials and methods 61 2.1. Chemicals 62 The reagents acetone, methanol, Folin-Ciocalteu reagent, ethanol, di-potassium 63 hydrogen phosphate (anhydrous), sodium di-hydrogen phosphate 1-hydrate, potassium 64 chloride, sodium acetate and sodium carbonate (anhydrous) were purchased from Merck 65 (Darmstadt, Germany). Fluorescein sodium and gallic acid were supplied from Fluka 66 (Madrid, Spain). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 67 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH) and 2,2’-diphenyl-1- 68 picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Steinheim, Germany). 69 2.2. Samples 70 For the optimisation of the extraction process, we used strawberries (Fragaria ananassa 71 var. camarosa) acquired at the market. The fruit was crushed in our laboratory, 72 distributed into amber glass flasks and frozen at -20º C. 73 For the production of the vinegars, we employed three different batches of strawberries 74 (Fragaria ananassa var. camarosa) from the Huelva area (Spain), corresponding to 75 4 three harvests: 2008, 2009 and 2010. The production processes were performed in the 76 laboratories of the Dept of Biochemistry and Biotechnology, Faculty of Oenology, Univ 77 Rovira i Virgili (Tarragona). In 2008 and 2009, the substrate employed were purees 78 prepared in the laboratory using a beater. In 2010, we used a commercial puree provided 79 by the Hudisa Company (Huelva). Sulphur dioxide (60 mg/L), sucrose and two types of 80 pectolytic enzymes (Depectil extra-garde FCE® and Depectil clarification® from Martin 81 Vialatte Oenologie, Epernay, France), both at a concentration of 15 mg/L, were added 82 to the puree. After this point, the procedures were slightly different in each harvest. 83 2008 harvest 84 One portion of the strawberry puree was pressed to study the effect of two types of 85 starting substrates (semi-solid and liquid) (Table 1). Six glass containers were filled 86 with 6 L of fruit substrate (four purees and two liquids). Half of the containers of each 87 type of substrate were inoculated with the yeast Saccharomyces cerevisiae QA23 at a 88 concentration of 2x106 cells/mL, and spontaneous alcoholic fermentation was allowed 89 to occur in the other half. All wines were spontaneously acetified keeping it in the same 90 containers. Two final treatments were tested in vinegars: pasteurization or 91 centrifugation. The average acetic degrees in the 2008 strawberry vinegars were 4.8. 92 2009 harvest 93 For the vinegar production in 2009, eight glass vessels were filled with 6 L of 94 strawberry puree each. Half of these vessels were inoculated with the yeast strain 95 Saccharomyces cerevisiae RP1, isolated during the 2008 spontaneous alcoholic 96 fermentation, and spontaneous alcoholic fermentation was allowed to occur in the other 97 half. All of the wines obtained from the inoculated alcoholic fermentation were mixed 98 and dispensed in three different types of containers: a glass vessel and oak or cherry 99 wood barrels. Samples were then inoculated with a strain of acetic acid bacteria isolated 100 5 from the 2008 acetification. Wines from the spontaneous alcoholic fermentation were 101 processed in the same way and left to acetify spontaneously. The vinegars obtained 102 were pasteurised. Inoculated vinegars from the 2009 harvest reached an acetic degree of 103 5.5 (glass container), 6.6 (oak barrel) and 6.3 (cherry barrel). 104 A portion of the puree from the 2009 strawberries was concentrated by heating in a 105 water bath at 80ºC during 10 hours, to test another method of increasing the sugar 106 content; the resulting product was a cooked must (Table 1). The sucrose final 107 concentration was 140 g/L. One litre of this substrate was fermented by a spontaneous 108 process and one litre was inoculated with the RP1 strain of yeast. The inoculated wines 109 (IWs) were acetified with the same acetic acid bacteria isolated in 2008, and the 110 spontaneous wines (SWs) were left to acetify spontaneously. 111 2010 harvest 112 In this harvest, the pectolytic enzymes added were Rohapect® (12 mg/hL) and the pH 113 was adjusted to 3.5 with 2 g/L CaCO3. In this case, 45 L of puree were fermented in a 114 stainless steel container on a semi-pilot scale, after inoculation with S. cerevisiae RP1. 115 The acetous fermentation was performed in a cherry wood barrel. The vinegar had an 116 acetic degree of 6.3. 117 All vinegars from 2009 and 2010 harvest were pasteurized as final treatment. 118 Forty-one samples, taken throughout these production processes, were analysed. The 119 codes and characteristics of the samples are shown in Table 1. In addition, five 120 commercial vinegars were also analysed to carry out comparative studies: Aceto 121 Balsamico, red wine and white wine vinegars, apple vinegar and sherry vinegar. 122 2.3. Sample-extraction procedure 123 The consistency of the samples (purees, wines and vinegars) made it necessary to 124 establish an extraction system prior to analysis. The method employed was based on the 125 6 extraction procedures designed and optimised previously by Ubeda, Hidalgo, Torija, 126 Mas, Troncoso & Morales (2011a). Twenty grams of sample were mixed in a beaker 127 with 40 ml of extract for 10 min while shaking at 800 rpm. The sample was then 128 subjected to ultrasonication followed by a centrifugation at 4000 rpm for 15 min. The 129 supernatant was recovered, and the pellet was re-extracted with 40 ml of solvent 130 following the same procedure. Both extracts were subsequently mixed, and the organic 131 solvent was removed under vacuum. Finally, the extract was filtered, and MilliQ water 132 was added to a final volume of 15 ml. Every extraction was performed in duplicate. We 133 tested different condition to get the maximum values of AA, TPI and TA as well as 134 economy of solvent used and time. Thus, the parameters studied to select the best 135 extraction conditions were: type of solvent (acetone, methanol or ethanol), percentage 136 of solvent (80% or 100%) and ultrasonic extraction time (15, 25, 35 or 50 min). 137 2.4. Assays and Methods 138 2.4.1. ORAC-FL assay 139 The Oxygen Radical Absorbance Capacity assay (ORAC-FL) was performed in a Black 140 96-well microplate, following the procedure described in Ubeda et al. (2011a). This 141 assay was conducted in a Multi-detection plate reader (Synergy HT, Vermont, USA) 142 located at the Centre for Research, Technology and Innovation at the University of 143 Seville (CITIUS). All reaction assays were performed in triplicate. Results were 144 expressed as µmol Trolox equivalents (TE)/kg of sample. 145 2.4.2. DPPH radical scavenging assay 146 To determine the radical scavenging capacity, the DPPH assay described by Brand- 147 Williams, Cuvelier, & Berset (1995) was used. For this test, we used an UV/Vis 148 spectrophotometer U-2800 Digilab coupled to a Peltier themostatic system (Hitachi, 149 7 Tokyo, Japan). Results were expressed as µmol Trolox equivalents (TE)/kg of sample. 150 The assays were performed in triplicate. 151 2.4.3. Total Phenols Index 152 This parameter was determined in triplicate, using the Folin-Ciocalteu method 153 following the procedure described in Waterhouse (2001). Results were expressed as mg 154 gallic acid/L. 155 2.4.4. Total monomeric anthocyanins 156 The determination of total monomeric anthocyanin content (TA) was measured 157 following the pH-differential method described in Giusti & Wrolstad (2001). TA was 158 expressed as pelargonidin-3-glucoside (Plg-3-glu), which is the major anthocyanin in 159 strawberry fruit with a λ vis-max at 510 nm (Swain, 1965). Two buffers were prepared: 160 potassium chloride buffer pH=1 (0.025 M), and sodium acetate buffer pH=4.5 (0.4 M). 161 We measured the absorbance at 510 and 700 nm against a cuvette filled with distilled 162 water as a blank. 163 We then calculated the absorbance of the diluted sample (A) as follows: 164 A= (A 510 -A 700) pH 1.0 - (A 510 -A 700) pH 4.5 165 The monomeric anthocyanin concentration in the original sample was calculated using 166 the following formula: 167 TA[Plg-3-glu (mg/L)] = (A × MW × DF × 1000) / (ε×1) 168 Where 169 A = Sample absorbance 170 MW= Molecular weight of Plg-3-glu (487.5) 171 DF= Dilution factor 172 ε= Absorption coefficient of Plg-3-glu (17330) 173 8 The results were expressed as mg Plg-3-glu/kg of sample. 174 2.5. Statistical analysis 175 All statistical analysis was performed using the Statistica version 7.0 software package 176 (Statsoft, Tulsa, USA). 177 3. Results and discussion 178 3.1. Selection of the best extraction conditions 179 Several factors, such as solvent composition, time of extraction, temperature, pH, solid- 180 to-liquid ratio and particle size, may significantly influence solid-liquid extractions 181 (Azizah, A. H., Ruslawati, N. M. N., & Tee, T. S, 1999; Pinelo, M., Del Fabbro, P., 182 Manzocco, L., Nunez, M J., & Nicoli, M. C, 2005). In our case, the parameters that 183 were evaluated to determine the best extraction conditions were the type of solvent, the 184 solvent-water ratio and ultrasonication time. The criteria used to select the extraction 185 parameters were the maximum values of antioxidant activity, total phenols, 186 anthocyanins and time and solvent savings. 187 The type of solvent is one of the most influential variables in the extraction process. We 188 tested acetone, ethanol and methanol. The extraction with methanol gave the worst 189 results in all the assays. 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L., Kang, X., He, X., Dong, M., Zhang, Q., & Liu, R. H. (2008). Cellular 429 antioxidant activity of common fruits. Journal of Agricultural and Food Chemistry, 56, 430 8418-8426. 431 Xu, Q., Tao, W., & Ao, Z. (2007). Antioxidant activity of vinegar melanoidins. Food 432 Chemistry, 102, 841-849. 433 434 435 436 437 438 439 440 441 442 443 444 445 446 19 447 448 Figure captions 449 Figure 1. ORAC, DPPH (left axis) and TPI (right axis) values for the different 450 extraction solvents tested in strawberries acquired at the market. The bars in the same 451 assay with different letters show significant differences (p<0.05) (ORAC assay: a, b, c; 452 IPT: A, B, C; DPPH test: α, β, γ). 453 Figure 2. Effect of solvent percentages. a) ORAC and DPPH values. b) TPI and 454 TA values of strawberries acquired at the market. The bars in the same assay with 455 different letters show significant differences (p<0.05) (ORAC and TPI assays: a, b; 456 DPPH and TA tests: A, B). 457 Figure 3. Effect of different ultrasonication times a) ORAC and DPPH 458 values. b) TPI and TA values of strawberries acquired at the market. The 459 markers in the same assay with different letters show significant differences (p<0.05) 460 (ORAC and TPI assays: a, b, c; DPPH and TA tests: A, B, C). 461 Figure 4. Comparison of ORAC, DPPH (left axis) and TPI (right axis) values of 462 strawberry vinegars with commercial varieties. Sample codes: F9MCV (mean value of 463 all vinegars from cooked must), F9V (mean value of all vinegars from 2009 harvest) 464 and F8V (mean value of all vinegars from 2008 harvest). 465 466 467 468 469 470 471 20 Figure 1. 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 Figure 2. 487 488 489 Figure 3. 490 491 492 493 494 495 496 497 a b a α ß α C B A 0 5000 10000 15000 20000 25000 30000 Acetone Methanol Ethanol µmol TE/kg R 0 500 1000 1500 2000 2500 mg gallic acid/kg b a B A 0 5000 10000 15000 20000 25000 80% 100% µmol TE/Kg 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 µmol TE/Kg a b A B 0 500 1000 1500 2000 2500 80% 100% mg gallic acid/Kg 0 5 10 15 20 25 30 mg Plg-3-glu/Kg a) b) a) b) b b a a C B A A 22000 23000 24000 25000 26000 27000 28000 29000 15 25 35 50 µmol TE/Kg 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 µmol TE/Kg a b c d A B A A 2000 2100 2200 2300 2400 2500 2600 2700 15 25 35 50 mg gallic acid/Kg 40 45 50 55 60 65 70 mg Plg-3-glu/Kg a) b) 21 Figure 4. 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Balsamic F9MCV F10VI F9V F8V Apple Sherry wine Red wine White wine µmol TE/kg 0 500 1000 1500 2000 2500 3000 mg gallic acid/kg 32 Table 1. Samples description. Harvest Treatment Puree Sample Treatment Sample substrate Alcoholic fermentation (time) Wine Sample Acetification (time) Treatment or Recipient Vinegar sample 2008 Crushed F8P1 SO2 Pectolytic enzymes Sucrose (50 g/L) F8P2 Inoculated (4 days) F8WI1- F8WI4 Spontaneous (2 months) Centrifugation F8VIC1-F8SVIC2 Pasteurization F8SVIP1-F8SVIP2 Spontaneous (5 days) F8WE1- F8WE4 Centrifugation F8SVEC1-F8SVEC2 Pasteurization F8SVEP1-F8SVEP2 - F8P2 Pressing F8L Inoculated (4 days) F8LWI - - - Spontaneous (5 days) F8LWE 2009 Crushed F9P1 SO2 Pectolytic enzymes Sucrose (75 g/L) F9P2 Inoculated (5 days) F9WI1- F9WI4 Inoculated (2 months) glass vessel F9SVIG oak barrel F9SVIO cherry barrel F9SVIX Spontaneous (8 days) F9WE1- F9WE4 Spontaneous (2 months) glass vessel - oak barrel - cherry barrel - Heating Concentrated F9MC Inoculated (7 days) F9MCWI1-F9MCWI2 Inoculated (5 months) glass vessel F9MCVI1-F9MCVI2 Spontaneous (7 days) F9MCWE1-F9MCWE2 Spontaneous (2.5 months) glass vessel F9MCVE1-F9MCVE2 2010 Crushed F10P1 SO2 Pectolytic enzymes Sucrose (65 g/L) CaCO3 F10P2 Inoculated (4 days) F10WI Inoculated (1.5 months) cherry barrel F10VI 33 Table 2. Changes in 2008 samples on ORAC, DPPH, TPI and TA during strawberry vinegar production (average±standard deviation). 1 Samples ORAC (μmol TE/kg) DPPH (μmol TE/kg) TPI (mg gallic acid /kg) TA (mg plg-3-glu/kg) Substrates F8P1 21792 ± 221 8327 ± 99 2090 ± 10 26.3 ± 0.8 F8P2 26714 ± 910a 10116 ± 88a 2298 ± 0a 69 ± 0a F8L 20642 ± 111b 5907 ± 516b 1615 ± 33b 43 ± 0b Wines F8LWE 12757 ± 267b,c 2837 ± 59b,c 868 ± 29b,c 12.2 ± 0.2b F8LWI 13497 ± 227b,c 2898 ± 129b,c 858 ± 13b,c 17.9 ± 0.2b,d F8SWE1 25314 ± 650 8200 ± 58b 1907 ± 26 13.1 ± 0.7b F8SWE2 24696 ± 70 7879 ± 70b 1773 ± 32 12.9 ± 0.6b F8SWE3 25458 ± 403 7689 ± 82b 1757 ± 45 12.4 ± 0.7b F8SWI1 27987 ± 1227b 7241 ± 35b,d 1670 ± 9b,d 16 ± 0b,d F8SWI2 25451 ± 429b 8004 ± 35b,d 1584 ± 19b,d 18.0 ± 0.3b,d F8SWI3 23745 ± 15b 6515 ± 67b,d 1548 ± 6b,d 17.3 ± 0.6d Vinegars F8SVE1C 9202 ± 390b 3256 ± 205b 769 ± 13b 0.4 ± 0.0b F8SVE1P 9849 ± 413b 3368 ± 352b 774 ± 23b 0.5 ± 0.1b F8SVE2C 9215 ± 338b 3210 ± 129b 781 ± 0b 1.1 ± 0.2b F8SVE2P 10869 ± 190b 3252 ± 234b 683 ± 10b 0.6 ± 0.0b F8SVI1C 10139 ± 341b,e 3227 ± 117b 751 ± 16b 1.3 ± 0.0b F8SVI1P 11611 ± 89b,e 3388 ± 64b 744 ± 6b 0.9 ± 0.1b F8SVI2C 11054 ± 40b,e 3260 ± 246b 694 ± 16b 0.8 ± 0.1b F8SVI2P 11082 ± 86b,e 3380 ± 76b 712 ± 9b 1 ± 0b 34 Sample codes are located in Table 1. a Significant differences (p<0.05) with respect to the initial fruit puree (ANOVA). b Significant differences (p<0.05) with respect to the sample from which was produced (ANOVA). c Significant differences (p<0.05) with respect to semisolid wines obtained with similar alcoholic process (spontaneous or inoculated) (ANOVA). d Significant differences (p<0.05) with respect to spontaneous process (ANOVA). e Significant differences (p<0.05) with respect to the vinegars obtained from spontaneous wines (ANOVA). 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17