Visual and instrumental evaluation of orange juice color: A consumers' preference study

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: FERNÁNDEZVÁZQUEZ, R., STINCO, C.M., MELÉNDEZ-MARTÍNEZ, A.J., HEREDIA, F.J. and VICARIO, I.M. (2011), VISUAL AND INSTRUMENTAL EVALUATION OF ORANGE JUICE COLOR: A CONSUMERS' PREFERENCE STUDY. Journal of Sensory Studies, 26: 436444. , which has been published in final form at https://doi.org/10.1111/j.1745459X.2011.00360.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." VISUAL vs INSTRUMENTAL EVALUATION OF ORANGE JUICE COLOR. A 1 CONSUMER’S PREFERENCE STUDY 2 3 Rocío FERNÁNDEZ-VÁZQUEZ, Carla M. STINCO, Antonio J. MELÉNDEZ4 MARTÍNEZ, Francisco J. HEREDIA, Isabel M. VICARIO(*) 5 6 Food Colour & Quality Lab., Dept. Nutrition & Food Science. Universidad de Sevilla 7 Facultad de Farmacia, 41012 Sevilla, Spain. www.color.us.es 8 9 (*) Email: [email protected] 10 11 12 Abstract 13 This study determine the instrumental evaluation of color, color attributes (lightness, 14 chroma and hue) quantified by trained assessors and the color preferences evaluated by 15 consumers, in orange juices from five varieties. The samples were evaluated by image 16 analysis (DiyiEye System) and spectroradiometer. The trained panel consisted of 17 eighteen panellists, with normal color vision and experience in both visual assessments 18 of color and Tristimulus Colorimetry concepts. All of them were asked to classify the 19 samples in increasing order of hue (yellowish-reddish), chroma (dull-vividness) and 20 lightness (clear-dark). They were also asked to score the colorimetric parameters on a 21 continuous scale of 10 cm, anchored at the ends. Results showed that judges were able 22 to order the orange juices correctly based on hue and lightness and significant score 23 differences (p<0.05) were found for hue and lightness, but not for chroma. The 24 consumer panel consisted of 111 panelists. They were asked to order the samples 25 according to their colour preferences. A significant preference (p<0.05) was observed 26 for the sample with intermediate hue and lightness values (Valencia Midnight), while 27 the least preferred was the variety with the lowest value for lightness and the highest 28 hue value (the most yellowish) (Navel Foyos). Cluster analyses showed consumers’ 29 segmentation. 30 Practical applications 31 Instrumental colour measurement in orange juice is very useful for industry because is 32 an easy, fast and cheap measurement of one important quality parameter: color. 33 Correlation between visual evaluation and instrumental measurement of color has 34 demonstrated that trained panels can value effectively orange juice color. 35 Consumers’ preference suggests that preferred hues are the most orangish against other 36 more reddish or yellowish. Anyway, there are different segments of population that 37 showed preference for varieties more reddish. 38 Key words: color, orange juice, visual evaluation, consumers’ preference. 39 40 Introducción 41 Citrus juices are among the most heavily consumed fruit juices worldwide due to their 42 combination of desirable flavor, appealing color, and health benefits (Rouseff et al., 43 2009). The natural bright color of the citrus juices have been considered traditionally as 44 one of their main advantages over other juices (Barron R.W. et al., 1967). 45 Orange juice (OJ) color is due to carotenoids which belong to one of the main classes of 46 natural pigments. OJ has a complex carotenoid profile that comprises carotenes and 47 xanthophylls. Some of these compounds (-carotene, -carotene and -cryptoxanthin) 48 has provitamin A activity and they may exhibit other biological activities, like 49 antioxidant and anticarcinogenic activity (Krinsky et al., 2004). 50 OJ color may range from pale yellow at the beginning of the season to red-orange at the 51 end, but besides the stage of maturity, other factors, such as, species, variety and 52 climate, among others may affect the color of orange juices (Casas A and Mallent D, 53 1988). In industrial orange juices, changes in color can be used as a quality indicator 54 related to carotenoids deteriorations during the thermal process (Fernández-Vázquez R. 55 et al., 2010;Meléndez-Martínez et al., 2009). 56 The relevance of color as a quality attribute in the food industry is undoubtedly. The 57 color of citric beverages in general, is related to the consumer’s perception of flavour, 58 sweetness and other quality characteristics of these products (Huggart et al., 59 1977;Tepper B.J., 1993;Rose Marie Pangborn, 1960). 60 It is long known that the color of orange juice directly prejudices the consumers’ 61 opinion about such taste factors as sweetness, thickness, and other quality 62 characteristics. However, when studying consumer preferences´ for OJ, color has not 63 usually been included as a sensory attribute to consider (Birdsall L, 1955;Joandre 64 Hoegg and Joseph W.Alba, 2007;Luckow and Delahunty, 2004). 65 The most natural way of improving the color of orange juice is to add other juices, 66 which provide a more intense coloration. Certain varieties of oranges, whose pulp has a 67 peculiar reddish color, are becoming increasingly important (Lee, 2001;Lee, 2002). In 68 this sense, Valencia orange juices are worldwide appreciated due to their deep orange 69 color (Francis and Clydesdale, 1975;Robards and Antolovich, 1995), however few 70 studies have been conducted to characterize the color of other varieties. Moreover, color 71 besides a sensory attribute is also related to the nutritional value of OJs, since studies in 72 our group (Meléndez-Martínez et al., 2007) have proved a relationship between color 73 measured by tritimulus colorimetry and the Vitamin A activity. 74 Color measurement can be done by visual evaluation or instrumental analysis. The 75 visual evaluation of color is included within the sensory analysis. Comprehensive 76 information concerning the sensory evaluation of food color, including guidelines for 77 panel selection, physical requirements for visual assessments and types of sensory tests, 78 can be found in the literature (Hutchings JB, 2011). As a result of the visual analysis, a 79 particular description of color is obtained, for which there is a certain vocabulary. 80 Nevertheless, it must be always taken into account that this kind of analysis is 81 subjective, since it is influenced by several factors (illumination, type of container, 82 volume...). For that reason, implementation of instrumental measurement of color 83 within the orange juice industry is very important for quality control purposes 84 (Meléndez-Martínez et al., 2005). In certain countries, such as the United States, color 85 is one of the attribute visually evaluated for the commercial quality classification of 86 orange juices (Tepper B.J., 1993) by comparison with a collection of six standard plastic 87 tubes. A more precise evaluation of colour can be done by instrumental methods as 88 reviewed in (Meléndez-Martínez et al., 2005). 89 Traditional instrumental colour measurement of food products is done by using a 90 colorimeter or spectrophotometer. In these methods, only a limited area of the product is 91 measured, with subsequent data being an average color of the selected area, though 92 these are restrictive measurements. Besides total appearance of food consist of visual 93 structure, surface texture and distribution of color (Hutchings et al., 2002). When 94 correlating with visually perceived attributes, it has been suggested that 95 spectroradiometric rather than spectrophotometric measurement should be used 96 (Martínez et al., 2001). However, recent advances in image acquisition technology offer 97 the possibility of using technically sophisticated apparatus available at relatively low 98 cost to evaluate color in terms of millions of pixels. The advantage, in comparison with 99 the traditional light sensors, is that they allow to make a detailed evaluation of a wider 100 area of a food products, with inhomogeneous color possible, and every different color 101 present in the image of the analyzed food matrix can be accounted for by one or more 102 pixels (Antonelli et al., 2004). This is based upon digital cameras which can quickly 103 capture images in digital format without film processing. Application of digital image 104 (DigiEye analysis) offers a more reliable measurement of the food colour, which can be 105 more related to sensory analysis. Up to its utility to evaluate OJ colour in comparison 106 with other traditional methods and how it correlates with visual color attributes. 107 The objectives of this study were to characterize the color of the juice from five orange 108 varieties and to explore the relationship between instrumental and sensory evaluation of 109 the color attributes (lightness, chroma and hue) quantified by trained assessors. The 110 instrumental measurement techniques used were a spectroradiometer and a calibrated 111 digital camera. CIELAB color space was used for color specifications. Besides, 112 consumer’s preferences for color were also evaluated. The relationship between color 113 preferences in relation to the nutritional value (provitamin A activity) of the OJs was 114 also explored. 115 116 Material and Methods 117  Samples 118 Five orange varieties were harvest in an agricultural experimental field situated in the 119 south of Spain in November 2009. At the harvesting date, a sample of about three kg of 120 each variety of oranges was taken randomly from several trees of each variety. Orange 121 juices were obtained at the laboratory using a kitchen juicer. The main characteristics of 122 the OJs are summarized in Table 1. The samples were kept at -21ºC until its analysis in 123 the laboratory. Thawing was carried out at room temperature (23ºC) for 24 h. Acidity 124 and total soluble solids were measured according to AOAC methods (1997). The ratio 125 was calculated by dividing the total soluble solids by the acidity. 126  Colour Instrumental Measurements 127 For colour specifications, the OJs were placed in 75 mL capacity, transparent plastic 128 bottles. The samples were measured against a grey surround and white background 129 using two different techniques: spectroradiometer and image analyses. Reflection 130 measurement were done in a CAS 140 B spectroradiometer (Instrument Systems, 131 Munich, Germany) with an external incandescent lamp, equipped with a Top 100 132 telescope optical probe (Instrument Systems, Munich, Germany) and a Tamron zoom 133 mod. SP 23A (Tamron USA, Inc., Commack, NY, USA). All the instrumental 134 measurements were carried out in a dim ambient illumination to avoid possible 135 interferences from other external sources. In addition to this, the bottles were placed 136 inside a cabin with grey walls to which the external illumination source of the 137 spectroradiometer was attached. The zoom, to which the probe was attached, was held 138 at a fixed distance of 50 cm in a straight line from the sample. As far as geometry of 139 presentation, 45º incident illuminations were used throughout the experiment. The 140 spectroradiometer was set to take three consecutive measurements of each sample, so 141 colour coordinates obtained were averages of three measurements. The whole visible 142 spectrum (380–770 nm) was recorded and Illuminat D65 and 10º Observer were 143 considered as references. Blank measurements were made using distilled water. 144 Digital images were made in order to obtain the total appearance of juice at depths 145 observed by consumers. The DigiEye imaging system (Luo et al., 2001) was used to 146 capture digital images. The latter system includes a digital camera Nikon D-80, a 147 computer (provided with appropriate software), a colour sensor for calibrating displays, 148 and an illumination box designed by DigiEye Plc. The computer software included the 149 functions of camera characterisation, colour measurement, monitor characterisation and 150 various specialised functions such as colour texture mapping, colour selection and 151 fastness grading (Hutchings et al., 2002). In these measurements, the samples were 152 illuminated by a diffused D65 simulator. A GretagMacbeth ColourChecker DC chart 153 was used for calibration purposes (Li.C. et al., 2003). 154 From the uniform colour space, the psychological parameters of chroma (C*ab) and hue 155 (hab) are defined: 156 157 Chroma (Cab) is used to determine the degree of difference of a hue in comparison to a 158 grey colour with the same lightness, and is considered the quantitative attribute of 159 colourfulness. Hue (hab) is the attribute according to which colours have been 160 traditionally defined as reddish, greenish, etc and is used to define the difference of a 161 colour with reference to a grey colour with the same lightness. This attribute is related 162 to the differences in absorbance at different wavelengths and is considered the 163 qualitative attribute of colour. 164 Colour differences, which are very important to evaluate relationships between visual 165 and numerical analyses (Melgosa et al., 1997), are calculated as the Euclidean distance 166 between two points in the three-dimensional space defined by L*, a* and b*: 167 168  Assessment of vitamin A activity trough objective colour measurements 169 The vitamin A activity of the samples was expressed in terms of retinol activity 170 equivalents (RAE), considering the equivalences 1 RAE = 12 g of dietary all-trans-  - 171 carotene = 24 g of other dietary provitamin A carotenoids (dietary) (TRUMBO et al., 172 2001). 173 Estimation was done as explained elsewhere (Meléndez-Martínez et al., 2007) 174 according to the following equation: 175 RAE = -8.8961 x hab +781.8687 176  Sensory analysis 177 A trained panel, consisting of eighteen panellists aged between 30 and 45 with normal 178 colour vision (which was verified using the Farnsworth-Munsell 100 Hue Test) and 179 experienced in both visual assessments of colour and Tristimulus Colorimetry concepts, 180 was used to establish the correlation between instrumental and sensory evaluation of 181 orange juice color. 182 Visual analyses of the samples were carried out within a well-illuminated room 183 provided with a VeriVide CAC Portable cabinet with D65 source to control 184 illuminantion and observation conditions. Five bottles of transparent plastic fulfilled 185 with 75 ml of the OJs were placed in each cabin randomly. Before each visual analysis 186 all the bottles were vigorously shaken to avoid pulp sedimentation and randomly 187 distributed for. All of them were asked to classify the samples in increasing order of hue 188 (yellowish-reddish), chroma (dull-vividness) and lightness (clear-dark). They were also 189 asked to score the colorimetric parameters on a continuous scale of 10 cm, anchored at 190 the ends. 191  Consumer preference study 192 Consumer test was performed by 111 panelists recruited among students and personnel 193 of the Faculty of Pharmacy in Seville. They were grouped in six categories based on 194 gender (male and female) and age (<20 years old, 20–29 years old, and over 30 years 195 old) (Table 2). The consumer test was carried out in sensory boots under white light 196 (ISO 1988). The consumers were given two sets of questionnaires. Prior to sample 197 presentation, they answered a questionnaire designed to collect demographic data and 198 consumer habits related to orange juice consumption. 199 In the second questionnaire consumers were asked to order the OJs samples for the 200 overall liking related to color. The ranking decision was based only on the color, 201 without further information. 202  Data analysis 203 Instrumental color measurements data were subjected to analysis of variance (ANOVA) 204 and the Tukey least significant difference multicomparison test to determine significant 205 differences among orange juice samples. 206 To analyse differences in the color parameters evaluated by the panellists and the 207 consumer preference-ranking test, the Friedman rank sum was performed (O'Mahony, 208 1986), using a significance level (p<0.05) to determine whether the panellists were able 209 to discriminate between samples. Then, Fisher Test served to determine whether 210 significant differences (p<0.05) existed between orange juice samples. Cluster analysis 211 was applied to determinate segmentations in the consumers’ panel. 212 These analyses were performed using the Statistica program for Windows (StatSoft, 213 2007;StatSoft, 2007). 214 215 Results and Discussion 216 The orange varieties included in this study were collected in the optimum maturity 217 stage. As shown in Table 1 the mean value for soluble solid content was very close and 218 no adjustment was needed. Due to the influence of the pulp content in the final color, 219 the OJs were sieved in order to have a similar pulp content (mean = 4.65% ± 1.63). 220  Instrumental color characterization 221 In Figure 1 the CIELAB color space (a*b* and Cab*L* planes) is used to illustrate the 222 color of the 5 orange varieties included in this study and measured by both methods 223 (spectroradiometer and DigiEye). The values of the coordinate L* ranged from 56.09 in 224 the lighter OJ to 61.34 in the darker one; C*ab ranged from 54.03 in the dullest OJ to 225 60.29 in the most vivid, and hab ranged from 66.43 for the most reddish to 81.99 in the 226 most yellow, measured by image analyses. 227 In the spectroradiometer analyses, values ranged from 56.58 to 60.66 for L*, from 63.11 228 to 66.02 for C*ab and from 66.43 to 81.99 for hab. ANOVA (Table 3) showed that the 229 five varieties were significantly different in hab, the qualitative component of color, but 230 not in C*ab, the quantitative component of color, or in ligthness. In CIELAB, considered 231 as the most uniform color space recommended by CIE, the samples presented 232 significant differences (p<0.01) for the rectangular chromaticity coordinate a*, but not 233 for the coordinate b*, indicating that color differences were more related to the 234 proportion of red (represented by the positive axis a*), than to the proportion of yellow 235 (represented by positive axis b*). Slight variations were observed in the color 236 TABLES 426 TABLE 1. CHARACTERISTICS OF THE ORANGE VARIETIES STUDIED 427 Code Variety Acidity1 Total Soluble Solids2 Ratio RAE 3 NF Navel Foyos 0.56 10.76 19.12 77.83 F Fisher 0.52 10.76 20.51 115.02 NP Navel Powell 0.49 10.12 20.27 99.72 VM Valencia Midknight 0.77 8.16 10.62 129.79 RL Rohde Late 0.92 11.41 12.37 185.30 Mean 0.66 10.24 16.58 121.53 1grams of citric acid/100 ml of orange juice 428 2 expressed as ºBrix 429 3 Retinol Activity Equivalent (RAE)/L 430 431 TABLE 2. COMPOSITION OF THE CONSUMER POPULATION SAMPLED 432 Under 20 20-29 Over 30 Total Female 15 50 13 78 (70%) Male 4 24 5 33 (30%) 19 (17%) 74 (67%) 18 (16%) 111 (100%) 433 TABLE 3: COLOUR COORDINATES AND MEAN E*ab VALUES (OF EACH VARIETY IN 434 RELATION TO THE REST) OF THE VARIETIES MEASURED IN THE SPECTRORADIOMETER 435 (a) AND BY IMAGE ANALYSIS (b) 436 Varieties L* a* b* C*ab hab E*ab NF 60.66 ± 0.121a 12.40 ± 0.476a 64.66 ± 0.356a 65.84 ± 0.260a 79.14 ± 0.465a 6.518 F 57.99 ± 0.212b 16.61 ± 0.180b 61.80 ± 0.076b 63.99 ± 0.027b 74.96 ± 0.174b 5.015 NP 59.96 ± 0.230a 15.21 ± 0.238c 64.24 ± 0.560a 66.02 ± 0.490a 76.68 ± 0.313c 5.814 VM 57.41 ± 0.090bc 18.29 ± 0.096d 60.98 ± 0.143b 63.66 ± 0.109b 73.30 ± 0.120d 4.484 RL 56.48 ± 0.723c 24.60 ± 0.141e 58.12 ± 0.150c 63.11 ± 0.084b 67.06 ± 0.171e 8.388 (a) 437 (b) 438 439 TABLE 4. MEAN SCORES FOR THE COLORIMETRIC PARAMETERS GIVEN BY THE PANEL 440 RL NF F VM NP hab 7.88a 1.55b 4.27c 5.63d 2.97e L* 7.98a 2.34b 4.39c 6.12d 3.92c C*ab 5.65a 5.89a 4.76ab 3.64b 4.67ab a-e Different superscripts within row indicates statistically significant differences (p<0.05) 441 Varieties L* a* b* C*ab hab E*ab NF 61.34 ± 0.297a 8.40 ± 0.513a 59.70 ± 0.501a 60.29 ± 0.529a 81.99 ± 0.460a 8.508 F 60.43 ± 0.284a 10.48 ± 0.891b 56.87 ± 0.484b 57.84 ± 0.577bc 79.57 ± 0.828b 6.163 NP 59.77 ± 0.280ab 12.77 ± 0.380c 57.73 ± 0.365b 59.12 ± 0.364ac 77.53 ± 0.370c 5.852 VM 58.75 ± 0.240b 14.17 ± 0.487d 55.01 ± 0.628b 56.81 ± 0.605b 75.55 ± 0.519d 6.295 RL 56.09 ± 0.388c 21.61 ± 0.483e 49.52 ± 0.559c 54.03 ± 0.626d 66.43 ± 0.418e 13.430 442 443 TABLE 5. SIMPLE REGRESSION COEFFICIENTS (r) AND SIGNIFICANCE LEVELS (p) 444 BETWEEN INSTRUMENTAL AND SENSORY EVALUATION OF COLOUR 445 Parameter Spectroradiometer measurement Image analyses Lightness (L*) -0.94 (0.018) -0.96 (0.008) Chroma (C*ab) 0.22 (0.717) 0.069 (0.911) Hue (hab) -0.98 (0.002) -0.92 (0.026) 446 TABLE 6. PREFERENCE DATA: RANK SUMS GROUPED BY AGE SEX AND CONSUMPTION 447 HABITS 448 n NF F NP VM RL All 111 2.05 a 3.35 b 2.89 c 3.66 b 3.05 bc By gender Women 78 2.10 a 3.46 b 2.92 c 3.67 b 2.85 c Men 33 1.91 a 3.09 bc 2.82 c 3.67 b 3.51 b By gender and sex W < 20 15 2.53 a 2.93 a 2.87 a 3.40 a 3.27 a M < 20 4 2.00 a 2.75 a 2.75 a 3.50 a 4.00 a W 20-29 50 2.00 a 3.62 b 2.78 c 3.72 b 2.88 c M 20-29 24 2.04 a 3.13 bc 2.88 c 3.67 b d 3.29 cd W > 30 13 2.00 a 3.46 bc 3.54 bc 3.77 c 2.23 a M > 30 5 1.20 a 3.20 bc 2.60 b 3.80 bc 4.20 c By consumption habits Regular Consumers of OJ 86 2.16 a 3.28 b 2.90 b 3.71 c 2.95 b Commercial OJ 19 2.37 a 3.00 ab 3.11 ab 3.47 b 3.05 ab Fresh Home squeezed OJ 63 2.17 a 3.37 b 2.84 c 3.78 b 2.84 c Any 4 1.00 a 3.25 b 2.75 b 3.75 b 4.25 b No regular consumer 25 1.64 a 3.60 b 2.88 c 3.52 bc 3.36 bc Ranking preference: 1 the lowest, 5 the highest. a-dDifferent superscripts in column indicates statistically 449 significant differences (p<0.05) 450 451 TABLE 8. RANK SUMS FOR DIFFERENT SEGMENTS OF CONSUMERS 452 453 454 455 456 457 458 459 460 461 462 SEGMENTS Samples 1 (n=24) 21.62% 2 (n=50) 45.05% 3 (n=37) 33.33% NF 4.33 a 1.20 a 1.70 a F 3.58 a 2.78 b 3.97 b NP 3.25 b 2.14 c 3.68 b VM 2.13 c 4.10 d 4.08 b RL 1.71 d 4.78 e 1.57 a 463 FIGURES 464 FIG. 1. COLOR COORDINATES OF OJ SAMPLES IN THE a*b* AND C*ab L* PLANES. MEASUREMENTS MADE BY SPECTRORADIOMETRY AND IMAGE ANALYSES TECHNIQUES a* b* 6 9 12 15 18 21 24 27 30 45 48 51 54 57 60 63 66 69 NF F NP VM RL C*ab L* 50 52 54 56 58 60 62 64 66 68 70 54 56 58 60 62 64 NF F NP VM RL 465 466 FIG. 2.CONTRIBUTION OF C*ab, L* AND hab TO COLOR DIFFERENCES AMONG SAMPLES DIGIEYE (a) AND SPECTRORADIOMETER (b) 0% 20% 40% 60% 80% 100% NF-F NF-NP NF-VM NF-RL F-NP F-VM F-RL NP-VM NP-RL VM-RL % DC*ab % DL % DH (a) 0% 20% 40% 60% 80% 100% NF-F NF-NP NF-VM NF-RL F-NP F-VM F-RL NP-VM NP-RL VM-RL %DC*ab %DL* %DH* (b) 467 468 469 470 FIG. 4. FREQUENCY DISTRIBUTION OF OVERALL PREFERENCES FOR JUICE COLOR IN 471 DIFERENT ORANGE VARIETIES. CHI-SQUARE VALUES OF PEARSON TEST TO CHECK THE 472 FIT OF THE OBSERVED FREQUENCIES TO THE NORMAL DISTRIBUTION. SAMPLES CODES 473 IN TABLE 1 474 475 476 FIG. 5. DENDOGRAM OF CONSUMERS (N=111) 477 Dendrograma 73 11 69 32 48 52 40 64 68 91 38 107 30 6 58 28 111 45 42 100 50 47 82 62 39 74 55 110 87 89 43 23 51 53 95 96 101 71 13 76 60 1 102 105 2 25 15 10 19 26 33 34 57 63 78 12 97 61 46 20 85 5 3 16 18 27 36 44 49 54 56 67 70 79 80 81 83 84 90 93 94 103 104 108 109 31 21 35 59 65 92 98 72 8 106 22 66 24 99 7 9 4 14 17 29 37 41 75 77 86 88 0 50 100 150 200 250 Disimilitud 478 VARIETY RANK:1 RANK:2 RANK:3 RANK:4 RANK:5 FNP VM RL NF 0 10 20 30 40 50 60 70 X2=123.4 P<0.001 X2=123.4 P<0.001 X2=123.4 P<0.001 X2=123.4 P<0.001 X2=123.4 P<0.001