Comparative Study of Red Berry Pomaces (Blueberry, Red Raspberry, Red Currant and Blackberry) as Source of Antioxidants and Pigments

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s00217-018-3135-z” 1 Comparative study of red berries pomaces (blueberry, red raspberry, red currant and blackberry) as source of antioxidants and pigments. Abbreviated title: Red berry pomaces as antioxidant and pigment sources M. José Jara-Palacios1, Adela Santisteban1, Belén Gordillo1, Dolores Hernanz2, Francisco J. Heredia1, M. Luisa Escudero-Gilete1* 1Food Colour and Quality Laboratory, Área de Nutrición y Bromatología, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. 2Department of Analytical Chemistry, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. * Corresponding author: M. Luisa Escudero Gilete Food Colour & Quality Laboratory, Área de Nutrición y Bromatología, Universidad de Sevilla. Facultad de Farmacia, 41012 Sevilla, Spain. Tel.: +34 954556761 Fax: +34 954556110 e-mail: g[email protected] 2 Abstract Anthocyanins are phenolic compounds with important technological applications due to its bioactive and color properties. In this study, pomaces from four red berries (blueberries, red raspberries, red currants and blackberries) have been analyzed as sources of anthocyanins. Anthocyanins were determined by high performance liquid chromatography/mass spectrometry (HPLC/MS), total phenolic content (TPC) by Folin-Ciocalteu method, antioxidant activity (AA) by ABTS assay, and color by Tristimulus Colorimetry. A total of fifteen anthocyanins were identified and quantified in the different pomaces from red berries. Pomaces exhibited different qualitative and quantitative anthocyanin profile and antioxidant activity, depending on type of red berry. The highest amounts of anthocyanins were found in blueberries (1188 mg/100 g), however red currant pomaces exhibited the highest TPC (3447 mg/100 g) and AA (61 mmol/100 g). Color of extracts was different depends on individual and total content of anthocyanins. Results indicate that these berries pomaces are a natural source of antioxidants and pigments, and they may be useful for industrial purposes. Therefore, the exploitation of these pomaces, like possible byproducts for their reuse in the food, cosmetics, and drug industries, could be of great interest, considering either the whole pomaces or its individual components. Keywords: Red berries; pomaces; anthocyanins; antioxidant activity; pigments; color Abbreviations: AA: antioxidant activity, ABTS: 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), BB: blueberry, RR: red raspberry, RC: red currants, BK: blackberry, TA: Total anthocyanins, TPC: total phenolic content, DM: dry matter, TE: Trolox equivalent, SLDA: stepwise linear discriminant analysis 3 Introduction Phenolic compounds are secondary plant metabolites, which constitute one of the most numerous and widely distributed groups of natural products in the plant kingdom, and can in general be classified into two main groups: flavonoids (flavanols, flavonols and anthocyanins) and non-flavonoids (phenolic acids and stilbenes) [1]. These compounds are important determinants in the sensory and nutritional properties of fruits and vegetables, and they have received attention because of their antioxidant activity. Specifically, anthocyanins are a subgroup of flavonoids that are found in nature as conjugated anthocyanidins. There are six anthocyanidins distributed throughout the plant kingdom: cyanidin, malvidin, delphinidin, peonidin, petunidin and pelargonidin. These compounds are one the most common pigments in nature and they are widely distributed in fruits and vegetables, being responsible for their color [2]. Anthocyanins display a great diversity of colors, from orange and red through purple and blue hues. In addition, anthocyanins have been associated with positive health effects, such as protection against oxidative stress and coronary heart disease, antimicrobial, anti-inflammatory and anticarcinogenic activities, control of obesity and diabetes, or improvement of vision [3, 4]. Red berries such as blueberries, blackberries, raspberries, strawberries, elderberries, blackcurrants, red currants and cranberries have been postulated to be a good source of phenolic compounds, chiefly anthocyanins [5-11]. In Spain the cultivation of these fruits has increased in recent years and approximately 80% is used for fresh consumption. Also, these fruits are used in food industry for the elaboration of some products such as juices [12]. In this processing, berries are crushed and pressed and solid parts are discarded after extracting juice, which generates an intensive accumulation of byproducts consisting of seeds, peels and pulp rests. Through common processing, only a part of the phenolic compounds is transferred to juice and then the generated byproducts are rich in anthocyanins and other compounds [7]. In this sense, the recovery of anthocyanins from solid wastes (pomaces) represents an attractive, sustainable and cost-effective source of these compounds, which could be incorporated into foods to improve their biological value, and also could be used as natural colorants [6, 13]. Actually, consumers demand functional foods and healthy products, therefore food industry has been more conscious of the importance to use natural additives which provide the final product with a healthy added value. Due to the increasing demand for nutraceutical, antioxidant compounds and natural colorants, the study of pomaces like byproducts from fruits may be useful for industrial purposes [14]. 4 Although red berries have been extensively investigated, as far as we know, no many studies regarding red berries byproducts have been published. Therefore, the aim of this work was to carry out the determination of anthocyanin profile, total phenolic content, and antioxidant activity, and the colorimetric characterization of the extracts of pomaces from blueberries, red raspberries, red currants and blackberries. Materials and methods Chemicals and reagents Sodium carbonate, potassium persulfate and phosphate-buffered saline (PBS) were purchased from Sigma- Aldrich (Madrid, Spain). 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Fluka (Madrid, Spain). Acetonitrile, Folin reagent, methanol and formic acid were obtained from Panreac (Barcelona, Spain). Samples Samples used in this study were berries from four species: blueberries (Vaccinium myrtillus) (BB), red raspberries (Rubus idaeus) (RR), red currants (Ribes rubrum) (RC) and blackberries (Rubus fructicosus) (BK). They were collected during a period of suitable ripening from the 2015 harvest in Almonte, Huelva (southwestern Spain). Immediately after collecting, berries were pressed to mix the peel, the seeds and the pulp. The obtained juice was discarded, and the pomace samples were obtained. The pomaces were weighed, stored at -20 °C and lyophilized for 48 h (lyophilizer Cryodos-80, Telstar Varian DS 102); finally, the samples were ground and stored until phenolic extraction. After lyophilizing 100 g of each fresh pomace, 15.5, 26.1, 28.8 and 20.9 g of dry pomace from blueberries, red raspberries, red currants and blackberries were obtained, respectively. Preparation of extracts The extraction was carried out with 75% methanol (1% 1N HCl) according to the methodology described as follows: the dry pomace (1 g) was homogenized in 5 mL of the solvent kept under shaking for 12 h in an incubating minishaker at 25 ºC, and further centrifuged at 4190g for 10 min at 10 ºC; the supernatant was collected, and the residue submitted to the same process until loss of color. Finally the supernatants were combined and were adjusted to 25 mL with 75% methanol. Total phenolic content 5 The total phenolic content (TPC) was determined using the Folin-Ciocalteu assay with some modifications [15]. Gallic acid was employed as a calibration and results were expressed as gallic acid equivalents (mg GAE/100 g of dry matter (DM)). Determination of anthocyanins by HPLC/MS analysis The anthocyanins were determined by HPLC/MS following the method described by Fernández-Lara et al. [16]. Analyses were carried out in a Hewlett-Packard 1200 series liquid chromatograph (Agilent Technologies, PaloAlto, CA, USA) equipped with a diode-array detector, and a Zorbax SB C18 column (4.6 mm x 250 mm, 4.6 μm particle size) using an injection volume of 50 μL. Detection was carried out at 525 nm and was also performed in a micrOTOF-QII High Resolution Time-of-Flight mass spectrometer (UHR-TOF) with Q-TOF geometry (Bruker Daltonics, Bremen, 194 Germany) equipped with an electrospray ionization (ESI) interface. The instrument was operated in positive ion mode (ESI+) using a scan range from m/z 50-1200. Nitrogen was used as the dry gas at a flow rate of 8 mL/min with nebulizing (1.2 bar), and nebulized temperature was set at 200 ºC. Anthocyanins were identified by their retention time, UV-vis spectra and mass spectra, as well as by comparison with our data library. The quantification of the compounds was carried out by external calibration from the areas of the chromatographic peaks obtained by UV detection at 525 nm using a calibration curve of cyaniding-3-O-glucoside. All analyses were performed in triplicate and the results were expressed as milligrams per 100 g of DM (mg/100 g DM). Total anthocyanins (TA) were also estimated by summing the content of all of individual anthocyanins quantified. Antioxidant activity by ABTS assay The antioxidant activity (AA) was determined by the ABTS assay according to the methodology described previously [17]. The results (ABTS values) were expressed as millimol of Trolox equivalents (TE) per 100 grams of DM (mmol TE/100 g DM). This result indicates the Trolox-equivalent antioxidant capacity (TEAC): mmol of Trolox with the same antioxidant activity as 100 g of the pomace). Colorimetric measurements A Hewlett-Packard UV-vis HP8452 spectrophotometer (Palo Alto, CA) was used to carry out color measurements. Spectrophotometric measurement of the visible absorption spectrum (380-770 nm) at constant intervals (Δλ = 2 nm) was made, using 2 mm path length glass cells and distilled water as reference. The CIELAB parameters (L*, a*, b*, C*ab, and hab) were determined by using the original software CromaLab© [18] following the Commission Internationale de L’Eclariage’s recommendations [19]: the CIE 1976 10° Standard Observer and the Standard Illuminant D65. 6 Statistical analysis These statistical analyses of the data were performed using the Statsoft Statistica® V 8.0 software [20]. One way analysis of variance (ANOVA) was applied to determine whether significant differences (p < 0.05) exist among the different pomaces. A stepwise linear discriminant analysis (SLDA) was applied on experimental standardized data in order to classify red berries pomaces. Results and discussion Anthocyanin profile and total phenolic content Table 1 shows retention times, and mass spectrometric data of each anthocyanin identified in the HPLCDAD-MS analysis. A total of fifteen anthocyanins were identified and quantified in the different pomaces, according to their mass and chromatographic data and also by comparison with literature [21-24]. Figure 1 shows the HPLC chromatogram with the anthocyanin profile of blueberry (a), red raspberry (b), red currant (c) and blackberry (d) pomaces. Peak 1, 2 and 5 were corresponded to delphinidin-3-O-galactoside (m/z = 465, MS2 = 303), delphinidin-3- O-glucoside (m/z = 465, MS2 = 303) and delphinidin-3-O-arabinoside (m/z = 435, MS2 = 303), respectively. Peaks 3, 4, 6, 7, 8 and 10 with main MS fragmentation ions at m/z 287 were attributed to cyanidin derivatives. These peaks were identified as cyanidin-3-O-sophoroside (Peak 3: m/z = 611, MS2 = 287, loss of 324 amu, corresponding to a sophorosyl unit), cyanidin-3-O-glucosyl-rutinoside (Peak 4: m/z 757, MS2 = 287, loss of 146 amu corresponding to rhamnosyl group, and 324 amu corresponding to two glucosyl moieties), cyanidin-3-O-sambubioside (Peak 6: m/z 581 and MS2 = 287, loss of xylose or arabinose (132 amu) and glucose (162 amu)), cyanidin-3-O-glucoside (Peak 7: m/z 449, MS2 = 287), cyanidin-3-O- rutinoside (Peak 8: m/z 595, MS2 = 287, loss of a rutinosyl unit (308 amu)) and cyaniding-3-O-arabinoside (Peak 10: m/z 419, MS2 = 287). Peaks 9 and 11 were identified as petunidin-3-O-galactoside (m/z 479 and MS2 = 317) and petunidin-3-O-arabinoside (m/z 449 and MS2 = 317), respectively. Peaks 12, 13 and 15 were identified as malvidin-3-O-galactoside (m/z 493 and MS2 = 331), malvidin-3-O-glucoside (m/z 493 and MS2 = 331), and malvidin-3-O-arabinoside (m/z 463 and MS2 = 331), respectively. Finally, peak 14 was corresponded to peonidin-3-O-arabinoside (m/z 433 and MS2 = 301). Glycosides were assigned based on the characteristics losses of fragments, i.e., 162 amu (glucosides and galactosides), and 132 amu (arabinosides). 7 . Different qualitative and quantitative anthocyanin profiles were found in the pomaces (Table 1). As can be seen in this table, eleven different anthocyanins were identified in blueberry pomaces (delphinidin-3- O-galactoside, delphinidin-3-O-glucoside, delphinidin-3-O-arabinoside, cyanidin-3-O-glucoside, petunidin-3-O-galactoside, cyanidin-3-O-arabinoside, petunidin-3-O-arabinoside, malvidin-3-O- galactoside, malvidin-3-O-glucoside, peonidin-3-O-arabinoside, malvidin-3-O-arabinoside) whereas in red raspberry (cyanidin-3-O-sophoroside, cyanidin-3-O-(2”-O-glucosyl)rutinoside, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside) , red currant (cyanidin-3-O-sambusoside, cyanidin-3-O-glucoside, cyanidin-3-O- rutinoside) and blackcurrant pomaces (cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside) were identified four, three and two anthocyanins, respectively. In blueberry pomaces were identified delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-, based anthocyanins, while than in the rest of pomaces only cyanidin derivates. The most abundant anthocyanins in blueberry pomaces were malvidin-3-O-galactoside and malvidin-3-O- arbinoside (30 and 29 % of the anthocyanin content, respectively). The percentages of these two compounds are consistent with previous reports: in a work about ripe samples of blueberries (Vaccinium corymbosum) represented by four varieties, these anthocyanins represented between 28 and 36 % and between 11 and 16 % of the anthocyanin content, respectively [22]. In other study [21], a total of fifteen anthocyanins were identified and quantified in blueberries (Vaccinium corymbosum), and malvidin-3-O-galactoside and malvidin-3-O-arabinoside were also the most abundant anthocyanins (21 and 18 % of total content, respectively). According literature [25, 26], malvidin glycosides are the main anthocyanins in blueberries and they constitute between 30 and 50% of the total anthocyanin content in blueberries (Vaccinium myrtillus), being malvidin-3-O-galactoside the predominant anthocyanin (15%). Other compounds as delphinidin-3-O-galactoside, petunidin-3-O-galactoside, delphinidin-3-O-arabinoside and petunidin-3-O-arabinoside also showed relevant contribution to the levels of total anthocyanins in blueberry pomaces (10, 9, 8 and 7 %, respectively). However, the concentration of cyanidin-3-O- arabinoside and cyanidin-3-O-glucoside in blueberry was low, representing the 1 and 0.4 % of total anthocyanins, respectively. These anthocyanins have also been identified by other authors in blueberries [21, 22, 25, 26]. The main anthocyanins in the red raspberry pomaces were cyanidin-3-O-sophoroside and cyanidin-3-O- glucoside (32 and 31 %, respectively). Cyanidin-3-O-sophoroside, has been previously described as the 8 major anthocyanin (42 % of total anthocyanins) in red raspberries of the same specie as the one analyzed in this study [21]. Therefore, this compound has been described as an important and distinctive anthocyanin in red raspberries [21, 27]. However, in a study on raspberries grown in USA, this compound was the less abundant among the four anthocyanins identified [28]. Cyanidin-3-O-sophoroside was not identified in blueberry, red currant and blackberry pomaces.. Cyanidin-3-O-(2”-O-glucosyl)-rutinoside and cyanidin-3- O-rutinoside were also quantified in high concentrations (around 20 and 17 % of total anthocyanin content, respectively), the last one has been identified in black raspberry seeds as principal anthocyanin [29]. The predominant compound in red currant extracts was cyanidin-3-O-sambubioside (82 % of the anthocyanin content), which only was identified in pomace of this fruit. This compound has been also identified in red currants of the same studied specie (Ribes rubrum), although in lower proportion (around 12 %) [22]. In blackberry pomaces, cyanidin-3-O-glucoside was the main anthocyanin (166 mg/100 g DM), which represented 86 % of anthocyanin content. Cyanidin-3-O-glucoside was the only compound identified in all pomaces, with highest concentration in blackberry pomaces followed by red raspberry, blueberry and red currant pomaces (59, 5 and 4 mg/100 g DM, respectively), and significant differences (p < 0.05) were found (Table 1). Significant differences were found in the content of cyanidin-3-O-rutinoside in red raspberry, red currant and blackberry pomaces (32, 22 and 26 mg/100 g DM, respectively). The different pomaces (blueberry, red raspberry, red currant and blackberry) showed significant differences (p < 0.05) in their contents of TA (Table 2). The highest amounts were found in blueberries (1188 mg/100 g DM), that was around 6-fold the concentration of blackberries (192 mg/100 g DM) and red raspberries (190 mg/100 g DM) and 8-fold the content of red currants, which presented the lowest one (147 mg/100 g DM). The TPC values for pomaces from blueberries, red raspberries, red currants and blackberries are shown in Table 2. The comparison among the four studied type of pomaces revealed that red currant extracts exhibited higher TPC (3447 mg GAE/100 g; around 2-fold the concentration of the other samples) followed by red raspberry (2015 mg GAE/100 g), blueberry (1955 mg GAE/100 g) and blackberry (1699 mg GAE/100 g) pomaces. The ANOVA test applied to the set of data indicated that significant differences were assigned between red currants and rest of the pomaces. Other authors analyzed the whole berries from blueberries (Vaccinium corymbosum) and red raspberries (Rubus idaeus) and they indicated that have higher phenolic content that red currants (Ribes rubrum) [21, 15 13. Paes J, Dotta R, Barbero GF, Martínez J (2014) Extraction of phenolic compounds and anthocyanins from blueberry (Vaccinium myrtillus L.) residues using supercritical CO2 and pressurized liquids. J Supercrit Fluids 95:8-16. 14. Jara-Palacios MJ, Hernanz D, Cifuentes-Gomez T, Escudero-Gilete ML, Heredia FJ, Spencer JPE (2015) Assessment of white grape pomace from winemaking as source of bioactive compounds, and its antiproliferative activity. Food Chem 183:78-82. 15. Jara-Palacios MJ, Hernanz D, Gonzalez-Manzano S, Santos-Buelga C, Escudero-Gilete ML, Heredia FJ (2014) Detailed phenolic composition of white grape by-products by RRLC/MS and measurement of the antioxidant activity. Talanta 125:51-57. 16. Fernández-Lara R, Gordillo B, Rodríguez-Pulido FJ, González-Miret ML, Villar-Martínez AA, Dávila- Ortiz G, Heredia FJ (2015) Assessment of the differences in the phenolic composition and color characteristics of new strawberry (Fragaria x ananassa Duch.) cultivars by HPLC–MS and Imaging Tristimulus Colorimetry. Food Res Int 76:645-653. 17. Stinco CM, Baroni MV, Di Paola Naranjo RD, Wunderlin DA, Heredia FJ, Meléndez-Martínez AJ, Vicario IM (2015) Hydrophilic antioxidant compounds in orange juice from different fruit cultivars: Composition and antioxidant activity evaluated by chemical and cellular based (Saccharomyces cerevisiae) assays. J Food Compos Anal 37:1-10. 18. Heredia FJ, lvarez C, Gonzlez-Miret ML, Ramírez A (2004) CromaLab, Anlisis de Color; Registro General de la Propiedad Intelectual. 19. CIE (2004). Technical Report Colorimetry; Commission Internationale de l’Eclairage Central Bureau: Vienna, Austria. 20. StatSoft (2007) S. v. 8. 0. StatSoft. (Computer software). 21. Borges G, Degeneve A, Mullen W, Crozier A (2010) Identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries. J Agr Food Chem 58:3901-3909. 22. Gavrilova V, Kajdzanoska M, Gjamovski V, Stefova M (2011) Separation, characterization and quantification of phenolic compounds in blueberries and red and black currants by HPLC-DAD-ESI- MSn. J Agr Food Chem 59:4009-4018. 23. Joaquín-Cruz E, Dueñas M, García-Cruz L, Salinas-Moreno Y, Santos-Buelga C, García-Salinas C (2015) Anthocyanin and phenolic characterization, chemical composition and antioxidant activity of 16 chagalapoli (Ardisia compressa K.) fruit: A tropical source of natural pigments. Food Res Int 70:151- 157. 24. Mertz C, Cheynier V, Günata Z, Brat P (2007) Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ion Trap Mass Spectrometry. J Agr Food Chem 55:616-8624. 25. Huang W, Zhang H, Liu W, Li C (2012) Survey of antioxidant capacity and phenolic composition of blueberry, blackberry, and strawberry in Nanjing. J Zhejiang Univ-SC B 13:94-102. 26. Li D, Meng X, Li B (2016) Profiling of anthocyanins from blueberries produced in China using HPLCDAD-MS and exploratory analysis by principal component analysis. J Food Compos Anal 47:1-7. 27. Beattie J, Crozier A, Duthie GG (2005) Potential health benefits of berries. Curr Nutr Food Sci 1:71- 86. 28. Wang SY, Chen CT, Wang CY (2009) The influence of light and maturity on fruit quality and flavonoid content of red raspberries. Food Chem 112:676-684.29. Choi MH, Shim SM, Kim GH (2016) Protective effect of black raspberry seed containing anthocyanins against oxidative damage to DNA, protein, and lipid. J Food Sci Technol 53:1214-1221. 30. Paes J, Dotta R, Barbero GF, Martínez J (2014) Extraction of phenolic compounds and anthocyanins from blueberry (Vaccinium myrtillus L.) residues using supercritical CO2andpressurized liquids. J. Supercrit. Fluids 95:8-16. 31. Reátegui JLP, Machado APF, Barbero GF, Rezende CA, Martínez J (2014) Extraction of antioxidant compounds from blackberry (Rubus sp.) bagasse using supercritical CO2assisted by ultrasound. J. Supercrit. Fluids 94:223-233.32. Castrejón AD, Eichholz I, Rohn S, Kroh LW, Huyskens-Keil S (2008) Phenolic profile and antioxidant activity of highbush blueberry (Vaccinium corymbosum L.) during fruit maturation and ripening. Food Chem 109:564-572. 33. Koca I, Karadeniz B (2009) Antioxidant properties of blackberry and blueberry fruits grown in the Black Sea Region of Turkey. Sci Hort 121:447-450. 34. Mazur SP, Nes A, Wold AB, Remberg SF, Aaby K (2014) Quality and chemical composition of ten red raspberry (Rubus idaeus L.) genotypes during three harvest seasons. Food Chem 160:233-240. 35. Reque PM, Steffens RS, Jablonski A, Flôres SH, Rios ADO, de Jong EV (2014) Cold storage of blueberry (Vaccinium spp.) fruits and juice: Anthocyanin stability and antioxidant activity. J Food Compos Anal 33:111-116. 17 36. Lee SG, Vance TM, Nam TG, Kim DO, Koo SI, Chun OK (2015) Contribution of anthocyanin composition to total antioxidant capacity of berries. Plant Foods Hum Nutr 70:427-432. 37. Giusti MM, Wrolstad RE (2003) Acylated anthocyanins from edible sources and their applications in food systems. Biochem Eng J 14:217-225 38. Hernanz D, Recamales AF, Meléndez-Martínez AJ, González-Miret ML, Heredia FJ (2008) Multivariate statistical analysis of the color-anthocyanin relationships in different soilless-grown strawberry genotypes. J Agr Food Chem 56:2735-2741. 39. Meléndez-Martínez AJ, Vicario IM, Heredia FJ (2003) Application of Tristimulus Colorimetry To Estimate the Carotenoids Content in Ultrafrozen Orange Juices. J Agr Food Chem 51:7266-7270. 40. Nemzer B, Vargas L, Xia X, Sintara M, Feng H (2018) Phytochemical and physical properties of blueberries, tart cherries, strawberries, and cranberries as affected by different drying methods. Food Chem 262: 242-250. 41. Gomes Rocha JC, Ramalho Procópio F, Corrêa Mendoça A, Marques Vieira L, Tuler Perrone I, Ribeiro de Barros FA, Cesar Stringueta P (2018) Optimization of ultrasound-assisted extraction of phenolic compounds from jussara (Euterpe edulis M.) and blueberry (Vaccinium myrtillus) fruits. Food Sci Tech 38:45-53. 18 Figure captions Fig. 1 HPLC chromatograms recorded at 525 nm of blueberry (a), red raspberry (b), red currant (c) and blackberry (d) pomace extracts. Peaks: 1, delphinidin-3-O-galactoside; 2, delphinidin-3-O-glucoside; 3, cyanidin-3-O-sophoroside; 4, cyanidin-3-O-(2”-O-glucosyl)rutinoside; 5, delphinidin-3-O-arabinoside; 6, cyanidin-3-O-sambusoside; 7, cyanidin-3-O-glucoside; 8, Cyanidin-3-O-rutinoside; 9, petunidin-3-O- galactoside; 10, cyanidin-3-O-arabinoside; 11, petunidin-3-O-arabinoside; 12, malvidin-3-O-galactoside; 13, malvidin-3-O-glucoside; 14, peonidin-3-O-arabinoside; 15, malvidin-3-O-arabinoside. Fig. 2 Representation of blueberry, red raspberry, red currant and blackberry pomace extracts in the (a*b*) plane. Two different groups: red raspberry, red currant and blackberry pomaces in a red hue area, and blueberry pomaces in a red-orange region. Fig. 3 Scatterplot of the four types of samples (blueberry, red raspberry, red currant and blackberry pomaces) in the plane defined by the canonical function when phenolic composition, antioxidant activity and color parameters are considered for discrimination by applying a stepwise linear discriminant analysis (SLDA). 1 Figure 1. 1 Figure 2. 1 Figure 3. Root 1 Root 2 -40 -20 0 20 40 60 80 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 Blueberry Red raspberry Red currant Blackberry 1 Table 1. HPLC retention times (TR), mass spectrometry data of anthocyanins detected and quantitative anthocyanin profile (mg/100g DM) in blueberry (BB), red raspberry (RR), red currants (RC) and blackberry (BK) pomaces. Peak TR (min) MS+ (m/z) MS2 Compound BB RR RC BK 1 5.55 465 303 Delphinidin-3-O- galactoside 130.08 ± 2.16 nd nd nd 2 5.89 465 303 Delphinidin-3-O-glucoside 4.40 ± 0.16 nd nd nd 3 6.12 611 287 Cyanidin-3-O-sophoroside nd 60.55 ± 4.25 nd nd 4 6.23 757 287 Cyanidin-3-O-(2”-O- glucosyl)rutinoside nd 37.32 ± 2.73 nd nd 5 6.41 435 303 Delphinidin-3-O- arabinoside 97.20 ± 1.33 nd nd nd 6 6.62 581 287 Cyanidin-3-O- sambusoside nd nd 124.33 ± 16.71 nd 7 6.79 449 287 Cyanidin-3-O-glucoside 4.89 ± 1.32a 59.47 ± 3.59b 3.58 ± 0.48a 166.39 ± 19.11c 8 6.93 595 287 Cyanidin-3-O-rutinoside nd 31.71 ± 2.27a 22.00 ± 2.05b 26.01 ± 1.76c 9 6.99 479 317 Petunidin-3-O-galactoside 106.76 ± 2.28 nd nd nd 10 7.29 419 287 Cyanidin-3-O-arabinoside 14.25 ± 0.76 nd nd nd 11 7.85 449 317 Petunidin-3-O-arabinoside 89.17 ± 1.72 nd nd nd 12 8.33 493 331 Malvidin-3-O-galactoside 363.14 ± 6.18 nd nd nd 13 8.69 493 331 Malvidin-3-O-glucoside 11.25 ± 0.23 nd nd nd 14 8.78 433 301 Peonidin-3-O-arabinoside 11.39 ± 0.25 nd nd nd 15 9.23 463 331 Malvidin-3-O-arabinoside 355.79 ± 7.72 nd nd nd nd: not detected Values in the same row followed by different letters are significantly different (p< 0.05). 1 Table 2. Total phenolic content, total anthocyanins and antioxidant activity of blueberry (BB), red raspberry (RR), red currants (RC) and blackberry (BK) pomaces. BB RR RC BK TPC (mg GAE/100 g DM) 1954.54 ± 177.82a,c 2014.66 ± 100.91a 3446.59 ± 805.17b 1699.62 ± 174.50c TA (mg/100 g DM) 1188.34 ± 19.35a 188.05 ± 12.55b 149.91 ± 19.90c 192.41 ± 20.83b AA (mmol TE/100 g DM) 26.98 ± 0.35a 29.75 ± 0.73a 60.83 ± 5.29b 22.54 ± 4.45c Each value represents mean (n=9) ± SD. Values in the same row followed by different letters are significantly different by ANOVA test (p< 0.05). 1 Table 3. Color parameters of extracts from blueberry (BB), red raspberry (RR), red currants (RC) and blackberry (BK) pomaces. L* C*ab hab BB 24.08 ± 1.25 a 65.24 ± 1.51 a 32.11 ± 0.92 a RR 62.05 ± 1.96 b 54.18 ± 1.75 b 17.96 ± 2.08 b RC 65.03 ± 1.44 c 51.46 ± 1.68 c 19.77 ± 0.91 c BK 52.44 ± 0.92 d 58.14 ± 1.21 d 21.41 ± 0.99 c Each value represents mean (n=9) ± SD. Values in the same column followed by different letters are significantly different by ANOVA test (p< 0.05).