PaPer Ital. J. Food Sci., vol. 27 - 2015 375 - Keywords: fruit, grape must, health, juice, sensory analysis - EffEct of thE addition of fruit juicEs on grapE must for natural bEvEragE production l. chiusano1,2 *, m. c. cravEro1, d. borsa1, c. tsolaKis1, g. ZEppa2 and v. gErbi2 1CREA Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria Centro di Ricerca per l’Enologia, Via Pietro Micca 35, 14100 Asti, Italia 2Università degli Studi di Torino, Dipartimento di Scienze Agrarie, Forestali e Alimentari, L.go P. Braccini 2, 10095 Grugliasco, Torino, Italy *Corresponding author: Tel. + 39 0141 433814, Fax + 39 0141 436829, e-mail: mariacarla.cravero@entecra.it AbstrAct the consumer attention for products with healthy properties is increased in time, and fruit juic- es, for their ease of consumption, can satisfy this demand providing them bioactive compounds. the grape juice has numerous health benefits demonstrated by several studies such as, among other, the antioxidant activities and the positive functions of their phenolic compounds. this work is aimed at blending grape and others fruits in a new fruit juice made only with natural ingredi- ents of local production. the grape juice (cv barbera) has substituted water and its percentage was fixed (70%). It was mixed with apple (cv Golden delicious), pear (cv Williams) and peach (cv red Haven) juices to obtain 25 different prototypes. In each of these at least two fruit juices were present and added in a percentage variable from 0 to 25%, with a step of 5%. the objectives of this study were to check the feasibility of the mixing process and the evaluation of the samples overall pleasantness. Other sensory aspects of samples were also evaluated by consumers with a JAr (just-about- right) structured scale. the results didn’t reveal particular technological problems regarding the blending process. the brix mean value of the samples was about 15.3, with a significant reduc- tion compared to that of the grape juice (about 19). the pH mean value of the samples (3.44) was significantly higher than that of the grape juice (3.36). the titrable acidity and the antioxidant ca- pacity mean value of the samples was, namely, 6.22 g L-1 and 535.18 mg L-1. the penalty analy- sis of the liking test pointed out the importance of the persistence in mouth. the overall pleasant- ness was significantly (p≤0,01) positively correlated with the °brix/acid ratio (r=0.54) and sam- ples with the highest percentage of pear juice were generally preferred. 376 Ital. J. Food Sci., vol. 27 - 2015 IntrOductIOn Fruit consumption has a positive impact on health (O’nEIL et al., 2011) and, including also vegetables, five are their daily servings (FsA, 2010), though this advice is generally ignored (WOOttOn-bEArd and rYAn, 2011). In this re- gard, the “dietary Guidelines for Americans” consider the 100% fruit juice as alternative to whole fruit (usdA, 2010). Indeed, fruit juic- es in general are deemed as one of the main sources of bioactive compounds for diet (rOd- rÍGuEZ-rOQuE et al., 2014). Even if the link between weight and sweetened beverages, in- cluding fruit juice 100%, must to be taken into account, referring to these latters, there is no consistent association (O’nEIL et al., 2011) and, actually, these have demonstrated to improve nutrient adequacy among children and adoles- cents of 2-18-year-olds (O’nEIL et al., 2012). Also grape has proved to have numerous health benefits, such as antioxidant activity and the functions of flavonoid compounds (VIsLOcKY and FErnAndEZ, 2010; WOOttOn-bEArd and rYAn, 2011). Grape-based products may pre- vent cardiovascular deseases, decrease oxida- tive stress and protect against atherosclero- sis. results from animal models suggest that especially purple grape juice more effective- ly improves blood lipids (VIsLOcKY and FEr- nAndEZ, 2010). From a organoleptic and sen- sory perspective, grape juice is characterised by a high concentration of sugars and acids, a low pH and, generally, a very poor odour/ aroma. thus, grape juice has a high-energy value, which reduces the nutritional, while its high acidity and low odour/aroma intensi- ty can reduce consumer preference. OJEdA et al. (2009) highlighted the too high sugar con- tent of the pure grape juice and, for this rea- son, it is important to reach a right sugar/ac- ids balance to develop appreciable grape juice. to reach this result it is necessary to use the optimal grape variety and/or mixing it with other fruit. the blending, indeed helps to im- prove flavour, taste, and nutritive value and it reduces the cost of production, improves stor- ability and inhibits microbial growth (bHArd- WAJ and PAndEY, 2011). As reported by bAtEs and MOrrIs (2001), the reasons for producing blends are many and all attributable to ad- just and improve acceptability. the aim of this work was to develop an innovative concept of fruit juice obtained by mixing grape with oth- er fruit juices to reduce its sugar concentra- tion, acidity and to improve its olfactory pro- file. the tested fruit juices (peach, pear and ap- ple) were chosen based on their appreciation by consumers, low acidity and sugar content, high antioxidant activity and high odour/aro- ma intensity. the use of grape must would also help to reduce the wine surplus that, current- ly, amounts to, approximately, 30 million hec- tolitres world wide (rAMOs et al., 2012; AYL- WArd, 2012). MAtErIALs And MEtHOds Juice production the grape juice (cv barbera) was provided by terre dei santi (castelnuovo don bosco, Asti, Italy), while the other fruit juices were provided by Valter Valle Farm (san damiano d’Asti, Asti, Italy). the apple, pear and peach juices were ob- tained from the Golden delicious, Williams and red Haven cultivars, respectively. For juice pro- duction, fruits were directly pressed, and the juice was filtered and stored at +1°c until use. because the aim of this study was to develop a new grape-based juice, the percentage of grape juice was fixed (70%) and the other fruit juices were added in percentages from 0 to 25%, with a step of 5% (table 1). table 1 - Experimental plan of blending. Sample code Barbera juice (%) Fruit juices (%) Pear Peach Apple S-1 70 0 5 25 S-2 70 0 10 20 S-3 70 0 15 15 S-4 70 0 20 10 S-5 70 0 25 5 S-6 70 5 0 25 S-7 70 5 5 20 S-8 70 5 10 15 S-9 70 5 15 10 S-10 70 5 20 5 S-11 70 5 25 0 S-12 70 10 0 20 S-13 70 10 5 15 S-14 70 10 10 10 S-15 70 10 15 5 S-16 70 10 20 0 S-17 70 15 0 15 S-18 70 15 5 10 S-19 70 15 10 5 S-20 70 15 15 0 S-21 70 20 0 10 S-22 70 20 5 5 S-23 70 20 10 0 S-24 70 25 0 5 S-25 70 25 5 0 this ratio was defined taking into account that, generally, in a fruit juice, the fruit/wa- ter ratio is approximately 35:65 (FÜGEL et al., 2005) and in this study water was replaced by grape juice. because for each beverage at least two fruit juices must be present, a total of 25 mixed juices were obtained. the proto- types were then bottled, pasteurised (105°c, 25 min) and stored at ambient temperature. Ital. J. Food Sci., vol. 27 - 2015 377 three replicates for each of the 25 recipes are been prepared. Reagents Folin-ciocalteu reagent, sodium hydroxide, glucose, fructose, phosphoric acid, methanol, sulphuric acid, caesium chloride, tartaric, mal- ic and citric acids were purchased from sigma- Aldrich (Milano, Italy). ultrapure water was ob- tained from a Milli-Q gradient A10 instrument (Millipore corporation, billerica, usA). Analyses density, extract, pH, sulphur dioxide, titrable acidity, total sugars, glucose, fructose, ashes and potassium of grape must were determined in accordance with the commission regula- tion (EEc) no. 2676/90 of 17 september 1990, while tartaric, malic and citric acids were de- termined by HPLc (cAnE, 1990). the polyphe- nolic composition of the grape must and fruit juices (total polyphenols, anthocyanin and fla- vonoid contents) was determined by spectro- photometry (dI stEFAnO et al., 1989). the glu- cose, fructose, total sugars, ashes, titrable acidity, pH, tartaric acid, malic acid, citric acid and potassium of the fruit juices and beverag- es were determined in accordance with Italian standard Methods (dM 03/02/1989). the fruit juice antioxidant capacity, expressed as Vita- min-c Equivalent Amount or VEAc Index, was determined according to KIM et al. (2002). the colour was measured using a Konica Minolta spectrophotometer cM-5 (Minolta corp, Osa- ka, Japan) in the cIELab colour system with a d65 illuminant. the parameters measured were L* (whiteness or brightness/darkness), a* (redness/greenness) and b* (yellowness/blue- ness). Each sample was evaluated in a 40-mL cuvette (1-cm thickness). All evaluations were performed in triplicate. Liking test As reported by MAMMAssE and scHLIcH (2014), literature recommend a range from 50 to 100 consumers in hedonic tests and gener- ally no replication are needed. taking into ac- count this and the limited quantity of samples, the liking test was executed once by recruiting 50 consumers (22 males and 28 females, aged 26-65 years). they have received an invitation and volun- tarily have participated to the tests. All tests were conducted individually, and social interac- tion was not permitted. the test was performed inside an air-conditioned meeting room with white light. the temperature was approximately 21 °c, and the relative humidity was approxi- mately 50%. tests were performed from 11 a.m. over 5 days. For each session, five experimental beverage samples (approximately 30 mL each) were presented in a completely randomised and balanced order. the samples were offered to the consumers in coded plastic cups. natural bottled water was provided to each participant for palate cleansing. to decrease fatigue, there was a 5 minutes break between each sample. during each break, the consumers rinsed their mouths with water. All beverages were evalu- ated for specific parameters by consumers on a Just-about-right (JAr) structured scale, and then the consumers were asked to express the overall pleasantness of each product. For JAr evaluation, consumers rated the samples on a 5-point JAr scale (1 = much too low, 2 = a little too low, 3 = just about right-JAr, 4 = a little too much, and 5 = much too much) for five sensory parameters: colour, odour, aro- ma, sweet taste and persistence in the mouth. For the overall pleasantness evaluation, a seg- ment of known length (100 mm), limited to the extremes of two adjectives of opposite mean- ing (bipolar scale) was used. consumers were asked to mark the line that corresponded to their degree of overall pleasantness. the data were collected on a paper card. According to PAGÈs et al. (2014), the 5 JAr variables were reduced to 3 for data evaluation: “not enough” (by grouping the “much too low” and “a little too low” responses), “JAr” and “too much” (by grouping the “much too much” and “a little too much” responses). this grouping of variables leads to simpler analyses, and it allows for ob- taining more stable results because non-JAr categories are associated with higher frequen- cies (PAGÈs et al., 2014). Statistical analysis compositional data and overall pleasantness were examined by one-way analysis of variance (AnOVA) with tukey’s test (p≤0.05) as a multiple range test with XLstAt 2011 (Addinsoft sArL, california, usA) and then used for a Principal component Analysis, also performed with XL- stAt 2011 (Addinsoft sArL, california, usA). the °brix/acid ratio and the overall pleasant- ness were subjected to Pearson’s test (r). the JAr data were subjected to a penalty analysis with XLstAt-MX 2014.2.07 (Addinsoft sArL, california, usA). rEsuLts And dIscussIOn Compositional aspects the compositions of grape and fruit juices used for beverage production are reported in ta- ble 2, while the composition of the obtained bev- erages are reported in tables 3 and 4. As highlighted by MOrALEs-dE LA PEnA et al. (2010), the overall quality of a fruit juice 378 Ital. J. Food Sci., vol. 27 - 2015 is evaluated by a few parameters such as sol- uble solids, pH and acidity. the grape juice displayed a total soluble solids content of 19 °brix with approximately 170 g L-1 of sugars, while the peach, apple and pear juices exhibit- ed 11.5, 11.0 and 13.5 °brix, respectively. the mean value of °brix for new beverages was ap- proximately 15.3, with a significant reduction with respect to grape juice, approximately 19. the obtained value is similar to that of a fruit juice (GunAtHILAKE et al., 2014) and ideal for the formulation of nutraceutical food beverages (sArAVAnAn and ArAdHYA, 2011a). the content of fructose in apple juice (approximately 62 g L-1) was higher than that reported by Wu et al. (2007) but lower than that reported by WILL et al. (2008) and MArKOWsKI et al. (2009). Addi- tionally, the fructose content of pear juice (ap- proximately 75 g L-1) was higher than that re- ported by cOLArIc et al. (2006). Acidity is one of the most important quality parameters for fruit juices (bHArdWAJ and PAndEY, 2011), as confirmed by AL bIttAr et al. (2013), who in- cluded this factor in the sensory analysis of an innovative grape juice enriched in polyphe- nols. nevertheless, LIu et al. (2006) highlight- ed that: “high acidity has a negative influence on the palatability of table grapes, as well as the suitability for wines”. the value of the total acidity, expressed as tartaric acid, of the grape juice used in this study (6.26 g L-1) is compa- rable to that reported for juices made with dif- ferent grape cultivars (MArsELLÉs-FOntAnEt et al., 2013; LIu et al., 2006; sOYEr et al., 2003). the main organic acid in grape is tartaric acid, which has a pK 1 of 3.04, followed by malic acid, which has a pK 1 of 3.40 (LIu et al., 2006). the table 2 - composition of grape and fruit juices used for beverages production. data are expressed as mean ± sd. Fruit juices Grape juice Pear Peach Apple Glucose (g L-1) 19.08±0.2 39.78±0.2 20.41±0.2 Glucose (g L-1) 86.94 ±0.2 Fructose (g L-1) 75.43±0.4 41.59±0.5 61.94±0.2 Fructose (g L-1) 94.82 ±0.09 Ashes (g Kg-1) 2.6±0.03 4.3±0.02 2.7±0.02 Ashes (g L-1) 3.4 ±0.1 Potassium (mg Kg-1) 1720±0.2 3542±6 1540±0.4 Potassium (mg Kg-1) 1223±5 °Brix 13.5±0.4 11.5±0.3 11±0.2 °Brix 19±0.3 Total Acidity (g L-1) 4.3±0.2 4.85±0.3 4.13±0.1 Total Acidity (g L-1) 6.26±0.04 pH 3.73±0.03 3.79±0.01 3.76±0.01 pH 3.36±0.05 Tartaric acid (g L-1) 0.281±0.01 0.233±0.02 0.26±0.03 Tartaric acid (g L-1) 2.73±0.02 Malic acid (g L-1) 0.312±0.03 0.892±0.02 1.028±0.01 Malic acid (g L-1) 2.35±0.03 Citric acid (g L-1) 0.588±0.03 0.788±0.04 nd Citric acid (g L-1) 0.1 ±0.01 Polyphenols (mg Kg-1) 126.7±5 81.9±4 96.5±4 Polyphenols (mg L-1) 446 ±6 Density (g L-1) 1.07715 ±0.0004 Extract (g L-1) 206.6±0.4 Free Sulphur Dioxide (mg L-1) nd Total Sulphur Dioxide (mg L-1) 11.2±0.4 Anthocyanins (mg L-1) 228 ±1.74 (nd – not determined). grape juice had a tartaric acid content of 2.73 g L-1, similar to juice reported by LIu et al. (2006). the pH value also plays an important role in the preparation of beverages (bHArdWAJ and PAn- dEY, 2011). the blending process here studied is aimed to increase the pH value of grape must (3.36). Our obtained results indicated that the addition of fruit juice with pH values of 3.79 (peach), 3.76 (apple) and 3.73 (pear) increased the pH of grape juice so that it reached a mean value of 3.44 in the prepared beverages. In his research on the properties of fruit juices used for functional beverages, GunAtHILAKE et al. (2014) reported a pH of 3.60 for apple juice, while AndrÉs et al. (2014) in their evaluation of the bioactive compounds in non-fermented beverages highlighted that the pH ranged be- tween 3.20 and 4.01, in agreement with sAArE- LA et al. (2011). typically, the pH values of fruit juices are below 4, or even 3, depending on the fruits used. the amount of organic acids in the fruit juices depended on the cultivar: apple dis- played the highest amount of malic acid, with a content of 1.028 g L-1, while pear juice had the highest citric acid content (0.588 g L-1). AGuI- LAr-rOsAs et al. (2007) reported a malic acid content below 0.35 g L-1 for the same cultivar, whereas burOn-MOLEs et al. (2014) reported a malic acid content of 1.4 g L-1. For beverag- es, the most abundant organic acid was malic acid, with a mean content of 2.84 g L-1, while the mean tartaric acid amount in these samples was 2.39 g L-1. For this compound, the concen- tration was similar among all of the beverages because the same quantity of grape must was used and because the quantity of tartaric acid is very low for fruit juice. the highest values were Ital. J. Food Sci., vol. 27 - 2015 379 t a b le 3 - c o m p o si ti o n o f sa m p le s o b ta in ed b y m ix in g g ra p e ju ic e a n d f ru it j u ic es o f p ea r, p ea ch a n d a p p le a n d r es u lt s o f A n o va w it h t u k ey ’s t es t. d a ta a re e x p re ss ed a s m ea n ± s d . F o r sa m p le s co d e se e t a b le 1 . V a lu es i n e a ch c o lu m n h a vi n g d if fe re n t le tt er s a re s ig n ifi ca n tl y d if fe re n t a t p < 0 .0 5 . Sa m pl e co de Ex tra ct °B rix G lu co se Fr uc to se Ac id ity pH O rg an ic a ci ds (g L -1 ) As he s Po ta ss iu m °B rix / (g L -1 ) (g L -1 ) (g L -1 ) (g L -1 ) (g k g- 1 ) (m g kg -1 ) ac id ra tio ta rta ric m al ic ci tri c S- 1 16 7.8 5± 0. 21 15 .7 5± 0. 35 b 66 .1 9± 0. 01 d ef 80 .3 ±0 .7 1 cd 6. 00 ±0 .0 0 j 3. 44 ±0 .0 1 fg h 2. 52 ±0 .0 0 ab 3. 45 ±0 .0 0 ab 0. 55 ±0 .0 3 cd ef g 2. 6± 0. 01 b c 12 43 ±1 .6 8 bc de fg 26 .2 5 S- 2 17 7.9 0± 0. 00 17 .0 0± 0. 00 a 71 .4 0± 1.4 1 ab cd 85 .1 0± 1.5 6 bc 6. 15 ±0 .0 0 h 3. 45 ±0 .0 1 cd ef g 2. 51 ±0 .0 0 ab 3. 32 ±0 .0 9 ab 0. 73 ±0 .0 2 de fg 2. 7± 0. 02 b c 12 98 ±4 .1 5 ab cd ef 27 .6 4 S- 3 18 1.8 0± 0. 00 17 .0 0± 0. 00 a 75 .5 3± 0. 62 a b 87 .4 4± 0. 55 a b 6. 49 ± 0. 06 e f 3. 43 ±0 .0 1g hi 2. 69 ±0 .0 3 ab 3. 54 ±0 .0 5 a 0. 94 ±0 .0 4 ab cd e 2. 8± 0. 01 b c 13 85 ±9 .7 2 ab c 26 .1 9 S- 4 18 3. 10 ±0 .0 0 17 .0 0± 0. 00 a 76 .1 8± 0. 81 a 85 .9 5± 0. 78 a bc 6. 75 ±0 .0 0 c 3. 45 ±0 .0 0 de fg 2. 76 ±0 .3 4 a 3. 60 ±0 .2 6 a 1.3 2± 0. 30 a b 3. 1± 0. 01 a bc 14 24 ±2 .6 4 a b 25 .1 9 S- 5 18 1.4 5± 0. 21 17 .2 0± 0. 00 a 75 .4 7± 0. 18 a b 83 .0 1± 0. 09 b c 6. 94 ±0 .0 0 b 3. 44 ±0 .0 1 fg h 2. 66 ±0 .1 8 ab 3. 47 ±0 .2 0 ab 0. 28 ±0 .0 2 fg 3. 2± 0. 01 a b 14 64 ±2 .5 1 a 24 .7 8 S- 6 17 6. 25 ±0 .2 1 16 .8 0± 0. 00 a 68 .4 5± 0. 64 c de 85 .7 8± 0. 82 b c 7.2 0± 0. 00 a 3. 43 ±0 .0 0 gh i 2. 47 ±0 .0 1a b 3. 22 ±0 .0 2 ab c 0. 18 ±0 .0 0 g 2. 6± 0. 01 c 11 61 ±5 .6 6 cd ef g 23 .3 3 S- 7 17 4. 30 ±0 .0 0 16 .8 0± 0. 00 a 70 .3 9± 1.4 2 bc d 85 .0 2± 1.5 6 bc 6. 45 ±0 .0 0 f 3. 45 ±0 .0 1 de fg 2. 51 ±0 .0 4 ab 3. 21 ±0 .0 6 ab c 0. 20 ±0 .0 2 g 2. 7± 0. 01 b c 12 33 ±0 .6 9 bc de fg 26 .0 5 S- 8 17 8. 05 ±0 .2 1 16 .9 0± 0. 14 a 73 .6 2± 3. 69 a bc 87 .1 8± 2. 48 a b 6. 60 ±0 .0 0 d 3. 45 ±0 .0 1 de fg 2. 37 ±0 .1 8 ab 3. 18 ±0 .0 1 ab cd 0. 22 ±0 .0 3 g 3. 2± 0. 00 a b 12 75 ±0 .6 5 ab cd ef 25 .6 1 S- 9 18 0. 50 ±0 .0 0 16 .8 ±0 .0 0 a 73 .3 3± 1.8 2 ab c 86 .1 5± 0. 62 a b 6. 51 ±0 .0 3 e 3. 43 ±0 .0 1 gh i 2. 64 ±0 .0 5 ab 3. 30 ±0 .0 7 ab 1.1 8± 0. 00 a b 3. 1± 0. 01 a bc 13 04 ±0 .1 6 ab cd ef 25 .8 1 S- 10 18 1.7 0± 0. 14 17 .0 0± 0. 00 a 75 .7 0± 0. 82 a b 86 .5 4± 0. 41 a b 6. 64 ±0 .0 0 d 3. 40 ±0 .0 0 j 2. 47 ±0 .1 3 ab 3. 22 ±0 .0 1 ab c 0. 27 ±0 .0 3 fg 3. 2± 0. 03 a b 13 70 ±3 .0 8 ab cd 25 .6 0 S- 11 18 2. 10 ±2 .2 3 17 .0 0± 0. 00 a 76 .3 3± 1.1 4 a 84 .1 2± 1.2 0 bc 6. 75 ±0 .0 0 c 3. 40 ±0 .0 0 j 2. 60 ±0 .0 0 ab 3. 09 ±0 .0 9 ab cd e 0. 21 ±0 .0 0 g 3. 5± 0. 01 a 13 28 ±5 .8 3 ab cd e 25 .1 9 S- 12 14 8. 60 ±0 .0 0 14 .6 5± 0. 21 c 51 .4 6± 0. 10 g 75 .8 7± 0. 57 d e 5. 55 ± 0. 00 o 3. 39 ±0 .0 0 j 2. 30 ±0 .0 4 ab 2. 87 ±0 .0 4 bc de f 0. 35 ±0 .2 7 ef g 2. 6± 0. 01 c 10 59 ±9 .9 2 g 26 .4 0 S- 13 13 4. 00 ±0 .0 0 13 .1 0± 0. 14 e 45 .2 1± 0. 16 h i 68 .3 1± 0. 29 fg 5. 70 ± 0. 00 m 3. 48 ±0 .0 1 ab c 2. 21 ±0 .0 4 ab 2. 60 ±0 .0 6 cd ef g 0. 79 ±0 .1 4 bc de fg 2. 6± 0. 01 c 11 56 ±9 .7 2 ef g 22 .9 8 S- 14 13 2. 90 ±0 .0 0 13 .0 0± 0. 00 e 45 .6 8± 0. 25 h i 66 .8 6± 0. 44 g 5. 63 ±0 .0 0 n 3. 50 ±0 .0 0 a 2. 38 ±0 .0 9 ab 2. 65 ±0 .0 8 cd ef g 0. 93 ±0 .0 7 ab cd e 2. 8± 0. 01 b c 11 57 ±1 .5 9 ef g 23 .0 9 S- 15 13 6. 00 ±0 .0 0 13 .1 0± 0. 14 e 48 .3 3± 0. 66 g hi 67 .9 2± 0. 91 fg 5. 93 ±0 .0 0 k 3. 48 ±0 .0 1 ab c 2. 33 ±0 .0 4 ab 2. 62 ±0 .0 7 cd ef g 1.1 4± 0. 06 a bc 3± 0. 00 a bc 12 22 ±3 .0 8 bc de fg 22 .0 9 S- 16 13 8. 50 ±0 .1 4 13 .2 0± 0. 00 e 50 .7 6± 0. 04 g h 68 .9 2± 1.0 2 fg 6. 00 ±0 .0 0 j 3. 48 ±0 .0 0 ab 2. 29 ±0 .0 3 ab 2. 59 ±0 .0 3 cd ef g 1.5 1± 0. 20 a 3. 1± 0. 01 a bc 12 45 ±3 .9 4 bc de fg 22 .0 0 S- 17 13 4. 60 ±0 .1 4 13 .2 0± 0. 00 e 44 .5 1± 0. 35 i 70 .6 3± 0. 42 e fg 5. 55 ±0 .0 0 o 3. 46 ±0 .0 0 bc de f 2. 27 ±0 .0 2 ab 2. 66 ±0 .1 3 de fg 0. 72 ±0 .0 3 bc de fg 2. 6± 0. 03 b c 11 18 ±1 .5 7 ef g 23 .7 8 S- 18 13 5. 9± 0. 14 13 .2 0± 0. 00 e 45 .6 9± 0. 09 h i 69 .9 0± 0. 37 fg 5. 55 ±0 .0 0 o 3. 47 ±0 .0 0 bc d 2. 19 ±0 .0 2 ab 2. 38 ±0 .0 1 fg 0. 85 ±0 .0 4 b cd ef 2. 6± 0. 03 b c 11 21 ±0 .4 9 ef g 23 .7 8 S- 19 13 8. 75 ±0 .2 1 13 .3 0± 0. 14 e 46 .9 8± 0. 12 g hi 69 .4 1± 0. 31 fg 6. 08 ±0 .0 0 i 3. 47 ±0 .0 1 bc de 2. 11 ±0 .1 2 ab 2. 25 ±0 .1 3 g 1.0 0± 0. 02 a bc 2. 9± 0. 01 a bc 11 71 ±3 .6 6 de fg 21 .8 8 S- 20 13 9. 55 ±0 .2 1 13 .2 0± 0. 00 e 47 .2 65 ±0 .3 2 gh i 67 .7 7± 0. 11 fg 6. 08 ±0 .0 0 i 3. 46 ± 0. 00 b cd ef 2. 02 ±0 .2 9 b 2. 08 ±0 .3 6 g 1.0 6± 0. 10 a bc 3± 0. 01 a bc 12 01 ±3 .6 4 cd ef g 21 .7 1 S- 21 14 1.1 0± 0 .0 0 13 .8 0± 0. 00 d 45 .3 8± 0. 10 h i 72 .9 2± 0. 30 e f 5. 78 ±0 .0 0 l 3. 44 ±0 .0 0 ef gh 2. 04 ±0 .1 8 b 2. 08 ±0 .1 8 g 0. 76 ±0 .2 1 bc de fg 2. 7± 0. 03 b c 10 98 ±1 .9 6 fg 23 .8 8 S- 22 16 8. 20 ±0 .0 0 15 .8 0± 0. 00 b 60 .8 6± 1.2 1 f 82 .5 4± 1.0 5 bc 6. 15 ±0 .0 0 h 3. 44 ±0 .0 0 ef gh 2. 33 ±0 .0 4 ab 2. 51 ±0 .0 9 ef g 0. 72 ±0 .0 4 bc de fg 2. 8± 0. 01 b c 11 22 ±1 0. 78 e fg 25 .6 9 S- 23 16 9. 80 ±0 .0 0 15 .8 0± 0. 00 b 62 .9 7± 0. 71 e f 82 .5 8± 0. 52 b c 6. 3± 0. 00 g 3. 42 ±0 .0 1 hi j 2. 40 ±0 .0 0 ab 2. 49 ±0 .1 0 ef g 1.1 9± 0. 05 a b 3± 0. 01 a bc 12 42 ±2 .3 9 bc de fg 25 .0 8 S- 24 17 2. 25 ±0 .2 1 16 .1 0± 0 .1 4 b 62 .6 4± 0. 49 f 85 .6 5± 0. 63 b c 6. 30 ±0 .0 0 g 3. 44 ±0 .0 0 ef gh 2. 37 ±0 .0 3 ab 2. 41 ±0 .0 2 fg 1.1 2± 0. 17 a bc 3± 0. 01 a bc 11 95 ±0 .9 9 cd ef g 25 .5 6 S- 25 17 6. 50 ±0 .1 4 17 .0 0± 0. 00 a 70 .3 4± 4. 38 b cd 91 .74 ±5 .4 1 a 6. 49 ±0 .0 0 ef 3. 41 ±0 .0 1 ij 2. 32 ±0 .0 8 ab 2. 35 ±0 .0 6 fg 0. 21 ±0 .0 5 g 2. 9± 0. 02 a bc 12 51 ±8 .6 8 ab cd ef g 26 .1 9 found for beverages s-3 and s-4, which contained higher quantities of apple juice. the brix/acid ratio (table 3) is an important parameter usual- ly used to control fruit quali- ty. In this study a positive cor- relation (r = 0.54, p≤0.01.) re- sulted between it and the over- all pleasantness in accordance with JAYAsEnA And cAMEr- On (2007). these authors re- ported that the °brix/acid ra- tio compared with the °brix alone demonstrated a higher degree of association with the consumer acceptability and it appeared a very useful matu- rity indicator. the peach juice exhibited the highest potassi- um content. this characteris- tic determined an increase in the content of this important component in the beverages containing high percentages of peach, e.g., sample s-5. the lowest value was determined for sample s-12, which was obtained without peach juice. the total polyphenols content ranged between 265.5 mg L-1 for beverage s-5 and 407 mg L-1 for beverage s-24, with a mean value of 359.30 mg L-1. According to the total poly- phenol contents of fruit juic- es, higher values were exhib- ited by beverages with high percentages of pear juice. the same beverage also displayed some of the highest values for the flavonoid content (627.5 mg L-1 , the highest) and an- tioxidant capacity (585 mg L-1 , the second highest one). For this parameter, the result for s-24 was similar to that of beverage s-1 (593 mg L-1). the lowest value for the anti- oxidant capacity (464.41 mg L-1) was displayed by beverage s-5. these results highlight- ed that the most interesting findings were obtained with a high quantity of apple or pear juice in the beverage, while a high content of peach juice led to a reduction of this param- eter. the AnOVA and tukey’s test performed for each pa- rameter of the beverages dis- played high variability among all samples and strictly corre- 380 Ital. J. Food Sci., vol. 27 - 2015 table 4 - Polyphenol composition (PHEn - total polyphenols; tAI - anthocyanins; tFI - flavonoids; VcEAc - antioxidant ca- pacity) and cIELab values of samples obtained by mixing grape juice and fruit juices of pear, peach and apple and results of AnOVA analysis with tukey’s test. data are expressed as mean ± sd. For sample code see table 1. Values in each column having different letters are significantly different at p<0.05. Sample PHEN TAI TFI VCEAC L*(D65) a*(D65) b*(D65) code (mg L-1) (mg L-1) (mg L-1) (mg L-1) S-1 384.5±4.95 cd 84±1.41 abc 540.5±44.55 cdefgh 593.83±10.40 a 68.14±2.86 ab 32.18±0.46 11.20±0.43 S-2 389.5±2.12 bcd 84.5±2.12 abc 478±4.24 ghijkl 578.38±1.04 ab 64.75±0 .01 b 33.9±0.10 11.675±0.15 S-3 366±1.41 efg 82±2.82 abcd 407.5±20.51 l 526.17±4.16 abcd 66.28±0.45 ab 33.56±0.19 11.36±0.26 S-4 362±0.7 gh 82.5±1.06 abc 497±4.95 fghijk 468.09±20.28 cd 66.63±0.19 ab 32.72±0.51 10.52±0.27 S-5 265.5±6.36 o 80±0.00 abcde 447±2.83 ijkl 464.41±10.40 d 66.65±1.98 ab 34.84±1.72 11.23±0.30 S-6 402.5±4.95 ab 88.5± 0.71 a 612.5±0.71 abc 534.26±48.87 abcd 64.89±0.25 b 32.94±0.40 11.31±0.28 S-7 405.5±3.54 a 88±0.00 ab 576±1.41 abcd 476.91±7.28 cd 64.13±0.93 b 32.75±0.07 10.89±0.01 S-8 385.5±2.12 cd 80.5±0.71 abcde 546.5±7.78 bcdefg 492.35±47.83 bcd 63.85±1.52 b 33.52±2.33 10.97±1.19 S-9 380.5±2.12 de 81.5±0.71 abcd 545±5.66 cdefg 530.59±29.12 abcd 65.63±1.25 ab 33.85±0.76 11.46±0.59 S-10 362.5±2.12 fgh 80.5±0.71 abcde 517±1.41 defghi 480.59±10.40 cd 66.21±3.21 ab 34.4±2.77 11.25±1.00 S-11 338.5±4.95 ijk 79.5±0.71 abcde 490.5±.71 fghijk 537.94±18.72 abcd 67.96±2.23 ab 33.8±1.33 10.9±0.72 S-12 377±1.41 def 82±0.00 abcd 581±1.41 abcd 577.65±0.00 ab 66.62±0.40 ab 33.49±1.94 9.45±0 .47 S-13 334±0.00 jkl 67.5±0.71 gh 515.5±4.95 defghi 551.18±0.00 abcd 67.5±2.21 ab 31.66±0.46 9.81±0.01 S-14 325±4.24 klm 63.5±2.12 h 493.5±4.95 fghijk 510±2.08 abcd 68.51±1.62 ab 32.66±1.77 10.04±0.04 S-15 314.5±0.71 mn 68.5±2.12 gh 430±15.56 kl 506.32±11.44 abcd 70.09±0.14 ab 30.79±1.81 9.77±0.95 S-16 309.5±0.71 n 65.5±0 .71 gh 436±9.90 jkl 510.74±15.60 abcd 74.03±4.72 a 31.21±1.16 9.26±1.27 S-17 350±4.24 hi 72±1.41 efgh 552.5±6.36 bcdef 583.53±12.48 a 66.6±0.62 ab 32.86±1.14 11.09±0.09 S-18 342±1.42 ij 69.5±2.12 fgh 514±2.83 defghi 551.17±33.28 abcd 68±2.18 ab 33.46±1.28 10.93±0.60 S-19 333±1.14 jkl 67±0.00 gh 503±0.00 efghij 540.88±6.24 abcd 69.48±1.60 ab 32.71±1.05 10.76±0.69 S-20 321±1.41 lmn 64.5±2.12 gh 469±8.49 hijkl 551.18±29.12 abcd 67.82±2.71 ab 33.63±2.10 9.95±0.09 S-21 365±4.24 fg 68±0.00 gh 571±1.41 abcde 546.76±12.48 abcd 67.49±0.49 ab 32.6±1.43 10.56±1.41 S-22 395.5±0.71 abc 80±7.07 abcde 618±49.5 ab 576.91±7.28 ab 63.95±1.63 b 34.42±0.86 11.88±0.30 S-23 376±2.83 defg 73±1.41 defg 556±0.00 abcdef 548.24±0.00 abcd 70.73±5.76 ab 32.09±1.22 10.05±1.86 S-24 407±7.07 a 78±1.41 cdef 627.5±4.95 a 585±37.43 a 65.57±1.23 ab 32.27±0.23 11.68±0.23 S-25 390.5±7.78 bcd 79±5.66 bcde 576±46.67 abcd 556.32±1.04 abcd 66.51±0.28 ab 32.82±0.91 10.89±0.59 lated with the composition of each single fruit juice and the different percentages used for bev- erage production. In fact, there were no differ- ences between the beverages for the cIELab pa- rameters a* and b* only, and this is due to the high grape must percentage used. Sensory aspects concerning the overall pleasantness, the AnO- VA highlighted significant differences among the 25 experimental beverages (table 5). Even if the content of grape juice was kept constant in all of the beverages at 70%, the dif- ferent percentages of other fruit juices can in- fluence the acceptability. the most appreciated samples (s-22, s-24 and s-23) had the highest pear juice percentages, while the least appre- ciated (s-14, s-12, s-1 and s-16) had the low- est pear juice concentrations. the least appre- ciated was beverage s-14, which was obtained with a mix of apple, pear and peach juices at the same percentage (10%). Penalty analysis was used because with this test it is possi- ble to identify the sensory attributes that have the largest influence on consumer liking and provides directions for product reformulation (ArEs et al., 2014) and also allows one to de- termine if a specific product attribute is “just about right” (tAYLOr, 2013). Penalty analysis combines JAr variables and overall liking tests to find correlations between a decrease in con- sumer acceptance and attributes not at the JAr level. this analysis, based on multiple compar- isons, is aimed to identify and determine if the rankings on the JAr scale are related to sig- nificantly different results in the liking scores for each sensory attribute studied on the JAr scale. this can be achieved by evaluating the mean decrease in overall liking versus percent- age of not-JAr variables (i.e., the low percentage of not-JAr evaluation determines a low mean decrease in overall liking). When some not-JAr categories receive at least 20% (Pareto princi- ple) responses for an attribute, this becomes a candidate for penalty analysis. Penalty analy- sis uses the 20% cut-off theory on the percent- age of not-JAr consumers based on the Pareto principle (i.e., the Pareto principle recognises that “80% of effects occur from 20% of causes” or the 80-20 rule) and signifies several common occurrences in everyday phenomena. therefore, the 20% cut-off is used as a general rule for pen- alty analysis (nArAYAnAn et al., 2014). In Fig. 1 are reported the JAr scores for each param- eter used in the beverage evaluation. the colour was judged “just right” by 50% of the consumers, odour by 37%, aroma by 38% and persistence in the mouth by 39%. In gen- eral, the “JAr” value was chosen by a high- er number of assessors: the higher frequen- cy was highlighted by the “a little too low” val- Ital. J. Food Sci., vol. 27 - 2015 381 table 5 - Mean values of overall pleasantness and results of AnOVA and tuckey’s test. data are expressed as the mean ±sd. Values with different letters are significantly different at p<0.05. Sample code Fruit juices Overall Tuckey test pleasantness (p< 0.05) pear (%) peach (%) apple (%) S-22 20 5 5 56.98 a S-24 25 0 5 55.3 ab S-23 20 10 0 55.14 ab S-8 5 10 15 54.18 abc S-9 5 15 10 54.08 abc S-2 0 10 20 54.04 abc S-25 25 5 0 53.07 abcd S-5 0 25 5 53.36 abcde S-11 5 25 0 52.36 abcdef S-3 0 15 15 50.82 abcdefg S-4 0 20 10 50.56 abcdefg S-6 5 0 25 50.54 abcdefg S-7 5 5 20 49.72 abcdefg S-10 5 20 5 48.9 abcdefg S-21 20 0 10 48.38 abcdefg S-15 10 15 5 46.36 abcdefgh S-20 15 15 0 45.7 bcdefgh S-17 15 0 15 43.8 cdefgh S-18 15 5 10 42.9 defgh S-19 15 10 5 42.68 efgh S-13 10 5 15 42.6 efgh S-16 10 20 0 42.46 fgh S-1 0 5 25 41.96 fgh S-12 10 0 20 40.86 gh S-14 10 10 10 36.36 h Fig. 1 - distribution of JAr scores for each sensory attribute evaluated. ue for only the odour. Fig. 1 also demonstrates that the “much too much” and “much too low”, although they may affect the overall pleasant- ness, do not weigh significantly on it because of their low frequency in the responses of con- sumers. the variables can then be grouped into two main groups with “a little too much” or “a little too low”. the first group corresponds to “much too much”, while the second corre- sponds to “not enough” for the parameters of 382 Ital. J. Food Sci., vol. 27 - 2015 colour, aroma, sweet taste and persistence in the mouth. In Fig. 2 are displayed the distribu- tion of frequency and then their effect on the mean drop in overall pleasantness. sweet taste, aroma and persistence in mouth exhibited a higher effect on the overall pleasant- ness if classified as “not enough”. Also impor- tant for determining the overall pleasantness was the odour, if classified as “not enough”. Fig. 2 - Penalty analysis from JAr data. not-JAr data with a frequency <20% of total re- sponses are not considered signif- icant. When the sensory parameters were classified as “too much”, they had less impact on the over- all pleasantness. A principal component analy- sis was also performed to highlight the correla- tion between chemical-physical parameters and overall pleasantness. the first two components explained 72.82% of the variance (Fig. 3). the first component explained 50.74% of the variance and was mainly correlated with the to- Fig. 3 - distri- bution on plane defined by the first two compo- nents of chemi- cal-physical pa- rameters, over- all pleasantness and beverage samples. Ital. J. Food Sci., vol. 27 - 2015 383 tal soluble solids, glucose and fructose contents, which corresponded to 12.1, 11.8 and 11.2%, respectively, of the total variance explained by this axis. the second component that explained 22.08% of the total variance is associated with the flavonoids, total polyphenols and antiox- idant capacity, accounting for 20.7, 16.7 and 13.5%, respectively, of the total variance ex- plained by this axis. the overall pleasantness was positively correlated with the contents of the total soluble solids and fructose and nega- tively correlated with the pH, citric acid content and L*. because the overall pleasantness is lo- cated in the upper right graph quadrant, all of the beverages placed in the same quadrant are the most appreciated. In particular, the highest appreciation was found for the s-22, s-24 and s-23 samples, as also demonstrated by table 5. the less appreciated samples are on the lower left side of the graph. they can be grouped into two groups: s-16, s-15, s-14, s-20 and s-19 in the lower left quadrant of the PcA graph and s-13, s-18, s-17, s-21 and s-12 in the upper left quadrant. the first group exhibited a more transparent colour, with a high value for L* and higher pH and citric acid contents. the second group demonstrated a low overall pleasantness but a high antioxidant capacity. In this group, it must be highlighted that beverage s-14, with the same percentages of fruit juice (10-10-10), had the lowest appreciation and the highest pH. cOncLusIOns One of the first internationally accepted de- scriptions of functional food has been provided by dIPLOcK et al. (1999) according to which: “a food product can be considered functional if together with the basic nutritional impact it has beneficial effects on one or more functions of the human or- ganism...”. taking this into account, and also of the scientific evidence regarding the benefits of the products based on grapes to human health, the re- sults obtained in this study have shown that these experimental fruit juices have functional charac- teristics. Additionally, as reported by bHArdWAJ and PAndEY (2011), it may be concluded that the formulation of mixed beverages can satisfy con- sumer tastes and preferences. In particular, the overall pleasantness results indicate a tenden- cy of consumers to prefer samples with the high- est percentage of pear juice, followed by samples containing mixtures of peach and apple juices. 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