http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.918591
BRIEF COMMUNICATION
The ORAC/kcal ratio qualifies nutritional and functional properties of fruit juices, nectars, and fruit drinks Paolino Ninfali, Andrea Chiarabini, and Donato Angelino
Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Chicago on 05/23/14 For personal use only.
Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo", Urbino, Italy
Abstract
Keywords
Fruit beverages are source of antioxidants, but their sugar content plays an important role in the epidemic of obesity. In this study, we considered 32 fruit beverages consumed in Italy (13 fruit juices, 11 nectars, and 8 fruit drinks), which were analyzed for caloric intake, total phenols (TP), ascorbic acid, and antioxidant capacity (oxygen radical absorbance capacity (ORAC) method). Results showed that the caloric intake was almost completely provided by the sugar content, ranging from 5.5 to 19%. The ORAC/kcal ratio was taken as an indicator of the antioxidant performance of fruit beverages. Fruit juices containing berries, red orange, and goji showed the best performances, together with berries or pears nectars and fruit drinks made with rose hips or tea extracts. The 95% of antioxidant capacity was provided by TP, which showed a significant linear correlation with the net ORAC values. Overall, the results indicate that the ORAC/kcal ratio is a suitable parameter to rank the quality of fruit beverages.
Calories, fruit beverages, microbial control, ORAC method, oxidative stress, total phenols
Introduction The consumption of fruit beverages in the European Union (EU) is sharply increasing due to higher internal and external product demand (Food Drink Europe, 2011). Three main categories of fruit beverages are used by the Italian families: fruit juices, nectars, and fruit drinks. The EU legislation (European Union, 2001) defines the fruit juice and the nectars, while the fruit drink definition is left to the EU member states. The Italian legislation defines a fruit drink as ‘‘product with a fruit content higher than 12%, in which are present one or more mixed fruits, tropical fruits or superfruits, vegetable extracts, aromas with the addition of vitamins and minerals’’ (Italian Ministry of Health, 2006). These three categories of beverages have been investigated as they are mostly consumed by teenagers, as well as because it demonstrates a correlation between these products and the increased incidence of obesity among young people, due to their high content of refined sugars (Te, 2013). Phenolic compounds are the most important source of antioxidants in fruit juices (Gu, 2004; Halliwell, 1995; Scalbert et al., 2005). The capacity of phenolic compounds to reduce oxygen radicals is measured by several methods (Prior et al., 2005), among which we underlie the oxygen radical absorbance capacity (ORAC) (Bacchiocca et al., 2006; Ninfali et al., 2009), which uses the peroxyl radical, the most common free radical in the human body and it is the only method that combines both the inhibition time and the degree of inhibition in a single quantity (Prior et al., 2003). In the last decade, some important papers define the antioxidant effect of fruit beverages (Scalbert et al., 2005; Correspondence: Prof. Paolino Ninfali, Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Via Saffi 2, 61029 Urbino (PU), Italy. Tel: +39 0722 305288. Fax: +39 0722 305324. E-mail: [email protected]. web: http://www.uniurb.it/orac/
History Received 26 February 2014 Revised 16 April 2014 Accepted 23 April 2014 Published online 19 May 2014
Stella et al., 2011). Perez-Jimenez et al. (2010) proposed a database with the polyphenol content of several non-alcoholic beverages, fruits, and fruits products as a reference guide for the epidemiologists to determine polyphenol intake in people and its associations with health and diseases. Mullen et al. (2007) investigated the composition of individual phenol compounds in several fruit juices consumed in the UK and they found as main components flavan-3-ols, anthocyanins, and hydroxycinnamates. Overall, data obtained on the antioxidant capacity suggested that combination of juices, rich in different varieties of phenol compounds, provide a better protection against chronic diseases (Mullen et al., 2007). Donnelly et al. (2010) explored a number of fruit and berry juices consumed in US and proposed the polyphenols on the total calories per serving, to rank the healthy level of the beverages. This ratio is particularly useful as the healthy effect of the antioxidants can be hampered by an increased amount of free radicals, produced during the sugar metabolism (Finley et al., 2011). In this light, producers and consumers should take into account both the antioxidant capacity and the energy intake of the fruit beverage. In this study, we compared the caloric intake, the TP, and the antioxidant capacity (ORAC) per serving of 32 fruit beverages, subdivided in fruit juices, nectars, and fruit drinks, in order to verify if the ORAC/kcal ratio can be used as a suitable index, to rank the healthy properties of fruit beverages.
Material and methods Chemicals The Folin–Ciocalteau reagent, fluorescein sodium salt, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). 2,20 -Azobis(2-amidinopropane)dihydrochloride (AAPH)
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P. Ninfali et al.
Int J Food Sci Nutr, Early Online: 1–5
was purchased from Polysciences Inc. (Warrington, PA) and ascorbic acid was furnished by Sveba s.r.l. (Como, Italy).
Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Chicago on 05/23/14 For personal use only.
Sample preparation Fruit juices, nectars, and fruit-based drinks were obtained from the local market and stored at +4 C. We considered samples from the most important commercial brands, which observe prescription of the EFSA legislation for nutrient concentrations in the product label (European Union, 2001). For each sample, 5 g aliquots were drawn, diluted (1:10 w/v) with 70% acetone, and used for the assays. Samples containing solid materials were completely homogenized before dilution with acetone, then centrifuged for 15 min at 1308 g. Ascorbic acid stock solution (1% w/v) was prepared in water and then working solutions were immediately prepared by dilutions with 0.2 M HCl–potassium chloride pH 2.0 (Jagota & Dani, 1982) to obtain concentrations in the range 2.5–60 mg/100 g (w/v). The diluted solutions were used for preparing calibration curves of the ORAC values as a function of the ascorbic acid concentrations.
of 0.33 M AAPH as a peroxyl radical generator. Fluorescence was read at 485 nm excitation and 520 nm emission until complete extinction. ORAC values are expressed as mmoles Trolox equivalents (TE)/100 g of juice. A calibration curve was made each time with the Trolox standard (400, 200, 150, 100, 50, and 25 mM) and the blank was 0.075 M Na–phosphate buffer (pH 7.0). Values were the means ± SD of eight independent measurements. The Net-ORAC was obtained by subtracting the ORAC contribute due to the ascorbic acid from the total ORAC of the sample. Other assays Total phenols (TP) were assayed according the Folin–Ciocolteau method (Singleton et al., 1999), and values were expressed as mg caffeic acid equivalents (CAE). The ascorbate concentration was assayed according to Jagota et al. (1982) and expressed as mg of ascorbic acid per 100 g sample. This assay also utilizes the Folin reagent, but due to the acidic pH, ascorbic acid can react with the Folin–Ciocalteau reagent without any interference by other possible substances.
ORAC assay Total antioxidant activity was carried out by the ORAC assay, according to Ninfali et al. (2005), using a Fluostar Optima plate reader fluorimeter (BMG Labtech, Offenburgh, Germany). Incubator temperature was set at 37 C. The reaction mixture for hydrophilic assay was the following: 200 mL of 0.096 mM fluorescein sodium salt in 0.075 M Na–phosphate buffer (pH 7.0), 20 mL of sample or Trolox. The reaction was initiated with 40 mL
Statistical analysis Polyphenol and ascorbate concentrations were measured in triplicate and results were the mean ± SD of three detection on four different samples of the same brand, i.e. of 12 determinations. Data of antioxidant capacity were performed with eight independent determinations for each sample; each value was the mean ± SD of 32 determinations. Statistical
Table 1. Microbial control, composition and nutritional characteristics of fruit juices. Sample number Fruit juices 1 2 3 4 5 6 7 8 9 10 11 12 13 Nectars 14 15 16 17 18 19 20 21 22 23 24 Fruit drinks 25 26 27 28 29 30 31 32
Microbial control
Main fruit and herb components
Retail temperature ( C)
CHO g/200 g
kcal/200 g
Pressure Pressure Pressure Pressure Pressure Pressure Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Orange, banana, mango Orange, banana, strawberry Orange, strawberry Orange Grapefruit Orange, carrot, banana, apple, grape Raspberry, grape, blueberry Blueberry, grape American red blueberry Grape, blackberry, apple, blueberry, banana Apple 1 Goji Grape
+4 +4 +4 +4 +4 +4 R.T. R.T. R.T. R.T. R.T. R.T. R.T.
22 18 14 14 14 15 30 24 27 27 22 30 29
94 84 64 68 62 84 128 104 120 116 92 132 120
Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Blueberry Apple 2 Peach Pear Orange Apple, banana Pineapple Strawberry, banana, grape, raspberry Pineapple Pear 2 Blueberry
R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T.
30 26 26 26 20 32 25 23 22 28 27
122 120 110 118 84 136 112 106 94 118 114
Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Berry, rose hips Pomegranate, green tea Blueberry, white tea Red orange, pomegranate, cherry and apple Mango, passion fruit, peach, apple, grape, orange Red grape, blueberry, elder, apple Red orange, pomegranate, apple Raspberry, grape, blueberry, green tea
R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T.
18 38 22 22 23 23 22 16
96 58 116 92 96 96 86 66
Values are those reported by the producers. R.T., room temperature; CHO, carbohydrates.
Fruit beverages and antioxidant/kcal ratio
DOI: 10.3109/09637486.2014.918591
significance was tested using Student’s t test with a p50.05. Linear regression analysis performed by means of Microsoft ExcelÕ was used to detect R2 values.
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Results and discussion Table 1 shows the characterization of fruit beverages on the basis of microbial control, component mixture, retail temperature, carbohydrate, and caloric intakes. The fruit juices, 1–6, were natural fruits squeezed, homogenized, bottled, and then processed by pressure based cold pasteurization (PBCP), by the application of high pressure (200–700 MPa) for few minutes (Alpa et al., 2000). The fruit juices, 7–13, were processed by UHT conventional technology treatment and stored at room temperature. Some of the fruit juices were done with one fruit only, whereas others with fruit mixtures. Two nectars were done with a combination of fruits, the others with one fruit only. Fruit drinks were all formulated with mixtures of different fruits, but in some of them, tea or rose hips were added. Overall, orange, grape, and berry were the most frequently used fruits, and in some cases, superfruits, like pomegranate, mango, passion fruit, and goji, were employed. The average caloric value was 38 ± 6 kcal/100 g and 58 ± 6.4 kcal/100 g for the PBCP fruit juices and UHT juices, respectively. The two values were found to be significantly different (p50.001). The average caloric intake of the nectars was
56 ± 6 kcal/100 g, whereas the fruit drinks average value was 49 ± 16 kcal/100 g. The average caloric intake of the PBCP fruit juices was significantly lower with respect to the average nectars’ value (p50.001), but not significantly different from that of the fruit drinks (p ¼ 0.192). The PBCP fruit juices maintained the same nutritional density of the original fruit mixture, whereas pasteurized juices, nectars, and fruit drinks were prepared with exogenously added sugars. Therefore, differences observed in the caloric intake were principally due to the carbohydrate content, which ranged from 5.5 to 19%; the maximum caloric value for one serving size (200 g) was 136 kcal. Table 2 shows TP, total ORAC, ascorbic acid ORAC, and Net ORAC, i.e. total ORAC–ascorbate ORAC per serving size (200 g). TP values ranged from 60 to 610 mg/200 g and ORAC from 312 to 8930 mmol TE/200 g. The linear regression analysis between the net ORAC values and the TP provided a straight line (y ¼ 13 281) with a significant correlation between the two parameters (R2 ¼ 0.625, p50.05). Regarding ascorbic acid, if one excludes the goji juice (sample 12), which showed individually 316 mg/200 g ascorbic acid, the other samples ranged from 12 to 120 mg/100 g. This range was similar to that observed in the fruit beverage survey recently performed in a Brazilian study (Stella et al., 2011). The linear regression analysis between ascorbic acid concentration and ORAC values gave a straight line (y ¼ 0.8017; R2 ¼ 0.995), which allowed us to draw the ORAC value due to the
Table 2. Total phenols, total ORAC, ascorbic acid, ORAC of ascorbic acid, and net ORAC of fruit juices, nectars and fruit drinks. Total ORAC (mmol TE/200 g)
Ascorbate (mg/200 g)
ORAC ascorbate (mmol TE/200 g)
Net ORACa (mmol TE/200 g)
136 ± 4 152 ± 10 176 ± 2 144 ± 6 64 ± 16 60 ± 2 218 ± 20 340 ± 18 210 ± 8 242 ± 14 120 ± 8 436 ± 18 86 ± 4
1890 ± 58 2544 ± 104 2400 ± 38 2430 ± 174 1688 ± 118 1682 ± 122 1644 ± 116 3928 ± 226 2294 ± 146 2942 ± 98 1186 ± 134 7376 ± 560 844 ± 132
120 ± 4 120 ± 4 120 ± 6 120 ± 2 42 ± 2 120 ± 6 18 ± 2 22 ± 2 112 ± 8 38 ± 2 56 ± 4 316 ± 12 22 ± 2
96 ± 8 96 ± 6 96 ± 4 96 ± 4 6±2 96 ± 4 14 ± 2 18 ± 2 44 ± 4 14 ± 2 46 ± 4 254 ± 12 18 ± 2
1794 ± 82 2448 ± 92 2304 ± 70 2334 ± 120 1682 ± 68 1586 ± 70 1630 ± 84 3910 ± 202 2250 ± 82 2928 ± 110 1140 ± 50 7122 ± 240 826 ± 42
260 ± 28 124 ± 10 168 ± 6 116 ± 8 132 ± 4 134 ± 10 84 ± 6 286 ± 24 70 ± 4 160 ± 10 268 ± 28
3876 ± 68 1402 ± 34 1752 ± 48 996 ± 36 748 ± 72 1264 ± 82 540 ± 14 3268 ± 188 312 ± 52 2328 ± 54 3758 ± 90
18 ± 2 56 ± 4 24 ± 2 30 ± 2 64 ± 6 12 ± 2 24 ± 2 48 ± 4 80 ± 4 80 ± 4 10 ± 2
14 ± 2 44 ± 2 8±2 22 ± 2 50 ± 4 10 ± 2 20 ± 2 38 ± 2 64 ± 4 64 ± 2 8±2
3862 ± 142 1358 ± 60 1744 ± 62 974 ± 62 698 ± 44 1254 ± 46 520 ± 30 3230 ± 142 248 ± 64 2264 ± 92 3750 ± 108
610 ± 30 56 ± 2 54 ± 4 212 ± 16 228 ± 8 260 ± 28 122 ± 6 170 ± 6
8930 ± 120 4120 ± 94 4722 ± 94 2732 ± 130 2712 ± 168 2700 ± 140 1578 ± 74 1780 ± 168
120 ± 8 20 ± 2 18 ± 2 80 ± 4 80 ± 6 80 ± 4 48 ± 2 72 ± 2
96 ± 4 16 ± 2 14 ± 2 64 ± 4 64 ± 6 64 ± 7 38 ± 2 58 ± 2
8834 ± 162 4104 ± 170 4708 ± 264 2668 ± 114 2648 ± 102 2636 ± 102 1540 ± 82 1722 ± 70
Total phenols (TP) (mg CAE/200 g) Fruit juices 1 2 3 4 5 6 7 8 9 10 11 12 13 Nectars 14 15 16 17 18 19 20 21 22 23 24 Fruit drinks 25 26 27 28 29 30 31 32
mg CAE/200 g, mg of caffeic acid equivalents per 200 g fresh sample; mmol TE/200 g, micromoles of trolox equivalents per 200 g fresh sample. Net ORAC, ORAC total phenols–ORAC ascorbate.
a
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Figure 1. Net ORAC (total ORAC–ascorbate ORAC)/kcal ratio of fruit juices, nectars, and fruit drinks. Dotted lines represent the average value of each category. Same letters indicate statistically significant different values (p50.05) from the average value in the respective group, but not among them. All other histograms, under the dotted line, indicate significantly lower values with respect to the average value. n.s., not significantly different with respect to the average value.
ascorbic acid. This contribution reached a maximum 5% fraction of the total ORAC. By subtracting the ascorbate ORAC from the total ORAC, it was possible to draw the net ORAC value per 200 g, which disclose the most potent antioxidant beverages in each category (Table 2). For instance, in food drinks, the sample 25 reached the maximum level, followed by samples 27 and 26. The antioxidant capacity as a function of the calories was calculated by the Net ORAC/kcal ratio. Figure 1 shows the histograms of the obtained ratios, with the dotted lines indicating the average value in each group. Overall, the net ORAC/kcal ratio (Figure 1) gives a different picture with respect to the ORAC value alone of Table 2. For instance, in the fruit drinks, sample 26 overcame sample 27, being the reverse on the basis of the net ORAC/200 g. The same happens for samples 3 and 4, which overcame sample 2 in ORAC/kcal ratio, being the reverse in the net ORAC/200 g scale.
Conclusions In this study, the ORAC/kcal ratio was proposed as a parameter of quality for ranking antioxidant potency as a function of energy density of fruit beverages, inside to their respective category. Although in vitro antioxidant potency does not prove in vivo biological activity, there are also valuable reasons to prefer fruit beverages with ORAC/kcal ratios at least near the indicated average values, which provides high concentration and quality of antioxidants with low caloric intake. Certainly, the true in ‘‘in vivo’’ effect depends on whether the phenol compounds are getting absorbed and metabolized in the body, but we can accept that beneficial antioxidant effects of a phenol rich antioxidant juice drink are exploited in the digestive tract. However, when used during the meals, all fruit beverages must be consumed in the suggested 200 g size, as the high sugar content adds caloric intake from food, without significantly affecting the satiety (DellaValle et al., 2005) and may
gradually bring to obesity (Allison & Mattes, 2009; Troiano et al., 2000). Although the marketing of several fruit beverages is based on their antioxidant potential, consumers still do not have enough knowledge to choose among the different categories of fruit beverages. The choice must be done keeping in mind that an excess of added carbohydrates and low phenols provide a scarce protection from the ROS oxidative injury. In this light, schools and media should educate towards the healthy choice by means of presenting scientific results in terms of simple reference data, such as the ratio of the antioxidant potency on the caloric value.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Allison DB, Mattes RD. 2009. Nutritively sweetened beverage consumption and obesity: the need for solid evidence on a fluid issue. J Am Med Assoc 301:318–320. Alpa H, Kalchayanand N, Bozoglu F, Ray B. 2000. Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. Int J Food Microbiol 60:33–42. Bacchiocca M, Biagiotti E, Ninfali P. 2006. Nutritional and technological motives for evaluating the antioxidant capacity of vegetable products. Italian J Food Sci 18:209–217. DellaValle DM, Roe LS, Rolls BJ. 2005. Does the consumption of caloric and non-caloric beverages with a meal affect energy intake? Appetite 44:187–193. Donnelly PE, Churilla TM, Coco Jr. MG, Vinson JA. 2010. Vitamin enhanced waters and polyphenol rich beverages analyzed for antioxidant capacity and antioxidants/calorie. Nutrients 2: 1290–1296. European Union. 2001. Council Directive 2001/112/EC of 20 December 2001. Relating to fruit juices and certain similar products intended for human consumption. Off J Eur Commun 12:58–66.
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DOI: 10.3109/09637486.2014.918591
Finley JW, Kong AN, Hintze KJ, Jeffery EH, Ji LL, Lei XG. 2011. Antioxidants in foods: state of the science important to the food industry. J Agric Food Chem 59:6837–6846. Food Drink Europe. 2011. Data & trends of the European food and drink industry. Available at: http://www.fooddrinkeurope.eu/publication/ data-trends-of-the-european-food-and-drink-industry-2011. Accessed on 24 February 2014. Gu L, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D, Gebhardt S, Prior RL. 2004. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J Nutr 134: 613–617. Halliwell B, Murcia MA, Chirico S, Aruoma OI. 1995. Free radicals and antioxidants in food and in vivo: what they do and how they work. Crit Rev Food Sci Nutr 35:7–20. Italian Ministry of Health. 2006. Applicazione della procedura di comunicazione prevista dal decreto del Presidente della Repubblica 2 agosto 2004, n. 230. Regolamento recante modifica al decreto del Presidente della Repubblica 19 maggio 1958, n. 719, in materia di disciplina della produzione e commercio delle acque gassate e delle bibite analcoliche gassate e non gassate confezionate in recipienti chiusi. Gazzetta Ufficiale Italiana 56:85–86. Jagota SK, Dani HM. 1982. A new colorimetric technique for the estimation of vitamin C using Folin phenol reagent. Anal Biochem 127:178–182. Mullen W, Marks SC, Crozier A. 2007. Evaluation of phenolic compounds in commercial fruit juices and fruit drinks. J Agric Food Chem 55:3148–3157. Ninfali P, Gennari L, Biagiotti E, Cangi F, Mattoli L, Maidecchi A. 2009. Improvement in botanical standardization of commercial freeze-dried herbal extracts by using the combination of antioxidant capacity and constituent marker concentrations. J AOAC Int 92:797–805.
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Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M. 2005. Antioxidant capacity of vegetables, spices and dressings relevant to nutrition. Br J Nutr 93:257–266. Perez-Jimenez J, Neveu V, Vos F, Scalbert A. 2010. Systematic analysis of the content of 502 polyphenols in 452 foods and beverages: an application of the phenol-explorer database. J Sci Food Agric 58: 4959–4969. Prior RL, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M, et al. 2003. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. J Agric Food Chem 51:3273–3279. Prior RL, Wu X, Schaich K. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53:4290–4302. Scalbert A, Manach C, Morand C, Remesy C, Jimenez L. 2005. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci 45: 287–306. Singleton VL, Orthofer R, Lamuela-Raventos RM. 1999. Analysis of total phenols and other antioxidant substrates and antioxidant by mean of Folin–Ciocalteu reagent. Meth Enzymol 299:152–178. Stella SP, Ferrarezi AC, dos Santos KO, Monteiro M. 2011. Antioxidant activity of commercial ready-to-drink orange juice and nectar. J Food Sci 76:C392–C397. Te ML, Mallard S, Mann J. 2013. Dietary sugars and body weight: systematic review and meta-analyses of randomised controlled trials and cohort studies. Br Med J 346:e7492–e7517. Troiano RP, Briefel RR, Carroll MD, Bialostosky K. 2000. Energy and fat intakes of children and adolescents in the united states: data from the national health and nutrition examination surveys. Am J Clin Nutr 72:1343S–1353S.
BRIEF COMMUNICATION
The ORAC/kcal ratio qualifies nutritional and functional properties of fruit juices, nectars, and fruit drinks Paolino Ninfali, Andrea Chiarabini, and Donato Angelino
Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Chicago on 05/23/14 For personal use only.
Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo", Urbino, Italy
Abstract
Keywords
Fruit beverages are source of antioxidants, but their sugar content plays an important role in the epidemic of obesity. In this study, we considered 32 fruit beverages consumed in Italy (13 fruit juices, 11 nectars, and 8 fruit drinks), which were analyzed for caloric intake, total phenols (TP), ascorbic acid, and antioxidant capacity (oxygen radical absorbance capacity (ORAC) method). Results showed that the caloric intake was almost completely provided by the sugar content, ranging from 5.5 to 19%. The ORAC/kcal ratio was taken as an indicator of the antioxidant performance of fruit beverages. Fruit juices containing berries, red orange, and goji showed the best performances, together with berries or pears nectars and fruit drinks made with rose hips or tea extracts. The 95% of antioxidant capacity was provided by TP, which showed a significant linear correlation with the net ORAC values. Overall, the results indicate that the ORAC/kcal ratio is a suitable parameter to rank the quality of fruit beverages.
Calories, fruit beverages, microbial control, ORAC method, oxidative stress, total phenols
Introduction The consumption of fruit beverages in the European Union (EU) is sharply increasing due to higher internal and external product demand (Food Drink Europe, 2011). Three main categories of fruit beverages are used by the Italian families: fruit juices, nectars, and fruit drinks. The EU legislation (European Union, 2001) defines the fruit juice and the nectars, while the fruit drink definition is left to the EU member states. The Italian legislation defines a fruit drink as ‘‘product with a fruit content higher than 12%, in which are present one or more mixed fruits, tropical fruits or superfruits, vegetable extracts, aromas with the addition of vitamins and minerals’’ (Italian Ministry of Health, 2006). These three categories of beverages have been investigated as they are mostly consumed by teenagers, as well as because it demonstrates a correlation between these products and the increased incidence of obesity among young people, due to their high content of refined sugars (Te, 2013). Phenolic compounds are the most important source of antioxidants in fruit juices (Gu, 2004; Halliwell, 1995; Scalbert et al., 2005). The capacity of phenolic compounds to reduce oxygen radicals is measured by several methods (Prior et al., 2005), among which we underlie the oxygen radical absorbance capacity (ORAC) (Bacchiocca et al., 2006; Ninfali et al., 2009), which uses the peroxyl radical, the most common free radical in the human body and it is the only method that combines both the inhibition time and the degree of inhibition in a single quantity (Prior et al., 2003). In the last decade, some important papers define the antioxidant effect of fruit beverages (Scalbert et al., 2005; Correspondence: Prof. Paolino Ninfali, Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Via Saffi 2, 61029 Urbino (PU), Italy. Tel: +39 0722 305288. Fax: +39 0722 305324. E-mail: [email protected]. web: http://www.uniurb.it/orac/
History Received 26 February 2014 Revised 16 April 2014 Accepted 23 April 2014 Published online 19 May 2014
Stella et al., 2011). Perez-Jimenez et al. (2010) proposed a database with the polyphenol content of several non-alcoholic beverages, fruits, and fruits products as a reference guide for the epidemiologists to determine polyphenol intake in people and its associations with health and diseases. Mullen et al. (2007) investigated the composition of individual phenol compounds in several fruit juices consumed in the UK and they found as main components flavan-3-ols, anthocyanins, and hydroxycinnamates. Overall, data obtained on the antioxidant capacity suggested that combination of juices, rich in different varieties of phenol compounds, provide a better protection against chronic diseases (Mullen et al., 2007). Donnelly et al. (2010) explored a number of fruit and berry juices consumed in US and proposed the polyphenols on the total calories per serving, to rank the healthy level of the beverages. This ratio is particularly useful as the healthy effect of the antioxidants can be hampered by an increased amount of free radicals, produced during the sugar metabolism (Finley et al., 2011). In this light, producers and consumers should take into account both the antioxidant capacity and the energy intake of the fruit beverage. In this study, we compared the caloric intake, the TP, and the antioxidant capacity (ORAC) per serving of 32 fruit beverages, subdivided in fruit juices, nectars, and fruit drinks, in order to verify if the ORAC/kcal ratio can be used as a suitable index, to rank the healthy properties of fruit beverages.
Material and methods Chemicals The Folin–Ciocalteau reagent, fluorescein sodium salt, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). 2,20 -Azobis(2-amidinopropane)dihydrochloride (AAPH)
2
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Int J Food Sci Nutr, Early Online: 1–5
was purchased from Polysciences Inc. (Warrington, PA) and ascorbic acid was furnished by Sveba s.r.l. (Como, Italy).
Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Chicago on 05/23/14 For personal use only.
Sample preparation Fruit juices, nectars, and fruit-based drinks were obtained from the local market and stored at +4 C. We considered samples from the most important commercial brands, which observe prescription of the EFSA legislation for nutrient concentrations in the product label (European Union, 2001). For each sample, 5 g aliquots were drawn, diluted (1:10 w/v) with 70% acetone, and used for the assays. Samples containing solid materials were completely homogenized before dilution with acetone, then centrifuged for 15 min at 1308 g. Ascorbic acid stock solution (1% w/v) was prepared in water and then working solutions were immediately prepared by dilutions with 0.2 M HCl–potassium chloride pH 2.0 (Jagota & Dani, 1982) to obtain concentrations in the range 2.5–60 mg/100 g (w/v). The diluted solutions were used for preparing calibration curves of the ORAC values as a function of the ascorbic acid concentrations.
of 0.33 M AAPH as a peroxyl radical generator. Fluorescence was read at 485 nm excitation and 520 nm emission until complete extinction. ORAC values are expressed as mmoles Trolox equivalents (TE)/100 g of juice. A calibration curve was made each time with the Trolox standard (400, 200, 150, 100, 50, and 25 mM) and the blank was 0.075 M Na–phosphate buffer (pH 7.0). Values were the means ± SD of eight independent measurements. The Net-ORAC was obtained by subtracting the ORAC contribute due to the ascorbic acid from the total ORAC of the sample. Other assays Total phenols (TP) were assayed according the Folin–Ciocolteau method (Singleton et al., 1999), and values were expressed as mg caffeic acid equivalents (CAE). The ascorbate concentration was assayed according to Jagota et al. (1982) and expressed as mg of ascorbic acid per 100 g sample. This assay also utilizes the Folin reagent, but due to the acidic pH, ascorbic acid can react with the Folin–Ciocalteau reagent without any interference by other possible substances.
ORAC assay Total antioxidant activity was carried out by the ORAC assay, according to Ninfali et al. (2005), using a Fluostar Optima plate reader fluorimeter (BMG Labtech, Offenburgh, Germany). Incubator temperature was set at 37 C. The reaction mixture for hydrophilic assay was the following: 200 mL of 0.096 mM fluorescein sodium salt in 0.075 M Na–phosphate buffer (pH 7.0), 20 mL of sample or Trolox. The reaction was initiated with 40 mL
Statistical analysis Polyphenol and ascorbate concentrations were measured in triplicate and results were the mean ± SD of three detection on four different samples of the same brand, i.e. of 12 determinations. Data of antioxidant capacity were performed with eight independent determinations for each sample; each value was the mean ± SD of 32 determinations. Statistical
Table 1. Microbial control, composition and nutritional characteristics of fruit juices. Sample number Fruit juices 1 2 3 4 5 6 7 8 9 10 11 12 13 Nectars 14 15 16 17 18 19 20 21 22 23 24 Fruit drinks 25 26 27 28 29 30 31 32
Microbial control
Main fruit and herb components
Retail temperature ( C)
CHO g/200 g
kcal/200 g
Pressure Pressure Pressure Pressure Pressure Pressure Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Orange, banana, mango Orange, banana, strawberry Orange, strawberry Orange Grapefruit Orange, carrot, banana, apple, grape Raspberry, grape, blueberry Blueberry, grape American red blueberry Grape, blackberry, apple, blueberry, banana Apple 1 Goji Grape
+4 +4 +4 +4 +4 +4 R.T. R.T. R.T. R.T. R.T. R.T. R.T.
22 18 14 14 14 15 30 24 27 27 22 30 29
94 84 64 68 62 84 128 104 120 116 92 132 120
Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Blueberry Apple 2 Peach Pear Orange Apple, banana Pineapple Strawberry, banana, grape, raspberry Pineapple Pear 2 Blueberry
R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T.
30 26 26 26 20 32 25 23 22 28 27
122 120 110 118 84 136 112 106 94 118 114
Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization Pasteurization
Berry, rose hips Pomegranate, green tea Blueberry, white tea Red orange, pomegranate, cherry and apple Mango, passion fruit, peach, apple, grape, orange Red grape, blueberry, elder, apple Red orange, pomegranate, apple Raspberry, grape, blueberry, green tea
R.T. R.T. R.T. R.T. R.T. R.T. R.T. R.T.
18 38 22 22 23 23 22 16
96 58 116 92 96 96 86 66
Values are those reported by the producers. R.T., room temperature; CHO, carbohydrates.
Fruit beverages and antioxidant/kcal ratio
DOI: 10.3109/09637486.2014.918591
significance was tested using Student’s t test with a p50.05. Linear regression analysis performed by means of Microsoft ExcelÕ was used to detect R2 values.
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Results and discussion Table 1 shows the characterization of fruit beverages on the basis of microbial control, component mixture, retail temperature, carbohydrate, and caloric intakes. The fruit juices, 1–6, were natural fruits squeezed, homogenized, bottled, and then processed by pressure based cold pasteurization (PBCP), by the application of high pressure (200–700 MPa) for few minutes (Alpa et al., 2000). The fruit juices, 7–13, were processed by UHT conventional technology treatment and stored at room temperature. Some of the fruit juices were done with one fruit only, whereas others with fruit mixtures. Two nectars were done with a combination of fruits, the others with one fruit only. Fruit drinks were all formulated with mixtures of different fruits, but in some of them, tea or rose hips were added. Overall, orange, grape, and berry were the most frequently used fruits, and in some cases, superfruits, like pomegranate, mango, passion fruit, and goji, were employed. The average caloric value was 38 ± 6 kcal/100 g and 58 ± 6.4 kcal/100 g for the PBCP fruit juices and UHT juices, respectively. The two values were found to be significantly different (p50.001). The average caloric intake of the nectars was
56 ± 6 kcal/100 g, whereas the fruit drinks average value was 49 ± 16 kcal/100 g. The average caloric intake of the PBCP fruit juices was significantly lower with respect to the average nectars’ value (p50.001), but not significantly different from that of the fruit drinks (p ¼ 0.192). The PBCP fruit juices maintained the same nutritional density of the original fruit mixture, whereas pasteurized juices, nectars, and fruit drinks were prepared with exogenously added sugars. Therefore, differences observed in the caloric intake were principally due to the carbohydrate content, which ranged from 5.5 to 19%; the maximum caloric value for one serving size (200 g) was 136 kcal. Table 2 shows TP, total ORAC, ascorbic acid ORAC, and Net ORAC, i.e. total ORAC–ascorbate ORAC per serving size (200 g). TP values ranged from 60 to 610 mg/200 g and ORAC from 312 to 8930 mmol TE/200 g. The linear regression analysis between the net ORAC values and the TP provided a straight line (y ¼ 13 281) with a significant correlation between the two parameters (R2 ¼ 0.625, p50.05). Regarding ascorbic acid, if one excludes the goji juice (sample 12), which showed individually 316 mg/200 g ascorbic acid, the other samples ranged from 12 to 120 mg/100 g. This range was similar to that observed in the fruit beverage survey recently performed in a Brazilian study (Stella et al., 2011). The linear regression analysis between ascorbic acid concentration and ORAC values gave a straight line (y ¼ 0.8017; R2 ¼ 0.995), which allowed us to draw the ORAC value due to the
Table 2. Total phenols, total ORAC, ascorbic acid, ORAC of ascorbic acid, and net ORAC of fruit juices, nectars and fruit drinks. Total ORAC (mmol TE/200 g)
Ascorbate (mg/200 g)
ORAC ascorbate (mmol TE/200 g)
Net ORACa (mmol TE/200 g)
136 ± 4 152 ± 10 176 ± 2 144 ± 6 64 ± 16 60 ± 2 218 ± 20 340 ± 18 210 ± 8 242 ± 14 120 ± 8 436 ± 18 86 ± 4
1890 ± 58 2544 ± 104 2400 ± 38 2430 ± 174 1688 ± 118 1682 ± 122 1644 ± 116 3928 ± 226 2294 ± 146 2942 ± 98 1186 ± 134 7376 ± 560 844 ± 132
120 ± 4 120 ± 4 120 ± 6 120 ± 2 42 ± 2 120 ± 6 18 ± 2 22 ± 2 112 ± 8 38 ± 2 56 ± 4 316 ± 12 22 ± 2
96 ± 8 96 ± 6 96 ± 4 96 ± 4 6±2 96 ± 4 14 ± 2 18 ± 2 44 ± 4 14 ± 2 46 ± 4 254 ± 12 18 ± 2
1794 ± 82 2448 ± 92 2304 ± 70 2334 ± 120 1682 ± 68 1586 ± 70 1630 ± 84 3910 ± 202 2250 ± 82 2928 ± 110 1140 ± 50 7122 ± 240 826 ± 42
260 ± 28 124 ± 10 168 ± 6 116 ± 8 132 ± 4 134 ± 10 84 ± 6 286 ± 24 70 ± 4 160 ± 10 268 ± 28
3876 ± 68 1402 ± 34 1752 ± 48 996 ± 36 748 ± 72 1264 ± 82 540 ± 14 3268 ± 188 312 ± 52 2328 ± 54 3758 ± 90
18 ± 2 56 ± 4 24 ± 2 30 ± 2 64 ± 6 12 ± 2 24 ± 2 48 ± 4 80 ± 4 80 ± 4 10 ± 2
14 ± 2 44 ± 2 8±2 22 ± 2 50 ± 4 10 ± 2 20 ± 2 38 ± 2 64 ± 4 64 ± 2 8±2
3862 ± 142 1358 ± 60 1744 ± 62 974 ± 62 698 ± 44 1254 ± 46 520 ± 30 3230 ± 142 248 ± 64 2264 ± 92 3750 ± 108
610 ± 30 56 ± 2 54 ± 4 212 ± 16 228 ± 8 260 ± 28 122 ± 6 170 ± 6
8930 ± 120 4120 ± 94 4722 ± 94 2732 ± 130 2712 ± 168 2700 ± 140 1578 ± 74 1780 ± 168
120 ± 8 20 ± 2 18 ± 2 80 ± 4 80 ± 6 80 ± 4 48 ± 2 72 ± 2
96 ± 4 16 ± 2 14 ± 2 64 ± 4 64 ± 6 64 ± 7 38 ± 2 58 ± 2
8834 ± 162 4104 ± 170 4708 ± 264 2668 ± 114 2648 ± 102 2636 ± 102 1540 ± 82 1722 ± 70
Total phenols (TP) (mg CAE/200 g) Fruit juices 1 2 3 4 5 6 7 8 9 10 11 12 13 Nectars 14 15 16 17 18 19 20 21 22 23 24 Fruit drinks 25 26 27 28 29 30 31 32
mg CAE/200 g, mg of caffeic acid equivalents per 200 g fresh sample; mmol TE/200 g, micromoles of trolox equivalents per 200 g fresh sample. Net ORAC, ORAC total phenols–ORAC ascorbate.
a
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Figure 1. Net ORAC (total ORAC–ascorbate ORAC)/kcal ratio of fruit juices, nectars, and fruit drinks. Dotted lines represent the average value of each category. Same letters indicate statistically significant different values (p50.05) from the average value in the respective group, but not among them. All other histograms, under the dotted line, indicate significantly lower values with respect to the average value. n.s., not significantly different with respect to the average value.
ascorbic acid. This contribution reached a maximum 5% fraction of the total ORAC. By subtracting the ascorbate ORAC from the total ORAC, it was possible to draw the net ORAC value per 200 g, which disclose the most potent antioxidant beverages in each category (Table 2). For instance, in food drinks, the sample 25 reached the maximum level, followed by samples 27 and 26. The antioxidant capacity as a function of the calories was calculated by the Net ORAC/kcal ratio. Figure 1 shows the histograms of the obtained ratios, with the dotted lines indicating the average value in each group. Overall, the net ORAC/kcal ratio (Figure 1) gives a different picture with respect to the ORAC value alone of Table 2. For instance, in the fruit drinks, sample 26 overcame sample 27, being the reverse on the basis of the net ORAC/200 g. The same happens for samples 3 and 4, which overcame sample 2 in ORAC/kcal ratio, being the reverse in the net ORAC/200 g scale.
Conclusions In this study, the ORAC/kcal ratio was proposed as a parameter of quality for ranking antioxidant potency as a function of energy density of fruit beverages, inside to their respective category. Although in vitro antioxidant potency does not prove in vivo biological activity, there are also valuable reasons to prefer fruit beverages with ORAC/kcal ratios at least near the indicated average values, which provides high concentration and quality of antioxidants with low caloric intake. Certainly, the true in ‘‘in vivo’’ effect depends on whether the phenol compounds are getting absorbed and metabolized in the body, but we can accept that beneficial antioxidant effects of a phenol rich antioxidant juice drink are exploited in the digestive tract. However, when used during the meals, all fruit beverages must be consumed in the suggested 200 g size, as the high sugar content adds caloric intake from food, without significantly affecting the satiety (DellaValle et al., 2005) and may
gradually bring to obesity (Allison & Mattes, 2009; Troiano et al., 2000). Although the marketing of several fruit beverages is based on their antioxidant potential, consumers still do not have enough knowledge to choose among the different categories of fruit beverages. The choice must be done keeping in mind that an excess of added carbohydrates and low phenols provide a scarce protection from the ROS oxidative injury. In this light, schools and media should educate towards the healthy choice by means of presenting scientific results in terms of simple reference data, such as the ratio of the antioxidant potency on the caloric value.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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