Food Chemistry 126 (2011) 1749–1758
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RP-HPLC–DAD analysis of phenolic compounds in pomace extracts from five grape cultivars: Evaluation of their antioxidant, antiradical and antifungal activities in orange and apple juices Osman Sagdic a,⇑, Ismet Ozturk a, Gulcan Ozkan b, Hasan Yetim a, Lutfiye Ekici a, Mustafa Tahsin Yilmaz c a b c
Erciyes University, Engineering Faculty, Food Engineering Department, 38039 Kayseri, Turkey Suleyman Demirel University, Engineering Faculty, Food Engineering Department, 32260 Isparta, Turkey Erciyes University, Safiye Cikrikcioglu Vocational College, Food Technology Department, 38039 Kayseri, Turkey
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 26 October 2010 Accepted 15 December 2010 Available online 19 December 2010 Keywords: Grape pomace extracts Phenolic compounds RP-HPLC Antioxidant activity Antifungal activity in vitro and in situ
a b s t r a c t Phenolic compounds, related to antioxidative and antifungal properties of ethanolic extracts from five commercial grape cultivars (three red and two white) grown in Turkey were determined. A reversedphase high performance liquid chromatography (RP-HPLC) procedure was developed, and a total 18 different phenolic compounds were identified. Total phenolic contents of the extracts were determined using Folin–Ciocalteau method. Antioxidant activities of the extracts were evaluated by using DPPH radical scavenging and phosphomolybdenum methods. All extracts exhibited strong antioxidant and antiradical activity. Phenolic compounds and antioxidant activities of the extracts were variety dependent. Antifungal activities of the pomaces and extracts were screened by both in vitro agar-well diffusion assay and antifungal activity in apple and orange juices in situ using Zygosaccharomyces rouxii and Z. bailii. Antifungal activities revealed that the pomaces and extracts of Gamay and Kalecik karasi could be more effective antifungal agents than those of Emir, Narince and Okuzgozu grape cultivars. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Winary by-products are of particular interest since grape (Vitis vinifera) is amongst the world’s largest fruit crop, with an annual production of more than 61 million tonnes according to the data of 2005 year (FAO, 2010). At present; however, only minimum amounts of these wastes are up-graded or recycled, which is particularly true in Europe, where vegetable wastes are generally dumped or used as animal feed and compost, without any pretreatment (Ruberto et al., 2007). This is also case for Turkey, where 4 million metric tons are processed per year, and 15–25% of the processed grapes is left over as pomace and not revalued anyway (Anonymous, 2008). Grape pomace is a waste product of grape juice and wine industry (Gokturk-Baydar, Sagdic, Ozkan, & Cetin, 2006; Ozkan, Sagdic, Gokturk-Baydar, & Kurumahmutoglu, 2004). These products may contain high phenolic compounds because of poor extraction during the winemaking processes; hence it makes their utilisation worthwhile and support sustainable agricultural production (Arvanitoyannis, Ladas, & Mavromatis, 2005). From the nutritional point of view, these compounds are polyphenols and the most
⇑ Corresponding author. Tel.: +90 352 4374937x32726; fax: +90 352 437 54 84. E-mail address: [email protected] (O. Sagdic). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.075
important constituents of grape pomace (Kammerer, Claus, Schieber, & Carle, 2005). In addition, many recent studies have highlighted the beneficial effects of dietary phenolic compounds in grape for human health (Ruberto et al., 2007). Grape seed procyanidins have been reported to be potent free radical scavengers and were found to exhibit novel cardio-protective properties (Bagchi et al., 2003). It has also been indicated that the growth of pathogen fungi such as Penicillium chrysogenum, Penicillium expansum, Aspergillus niger and Trichoderma viride could be inhibited by grape pomace extracts (Corrales, Butz, Tauscher, & Cabrejas, 2010). Therefore, pomace extracts can show their antifungal effects on some yeast too. Grape extracts contain large quantities of monomeric phenolic compounds such as (+)-catechins, ()-epicatechin and ()-epicatechin-3-o-gallate, and dimeric, trimeric and tetrameric procyanidins (Saito, Hosoyama, Ariga, Kataoka, & Yamaji, 1998). For instance, trans-resveratrol is produced from grape berries in response to fungal infection and UV irradiation. Some red grape varieties may be genetically richer in this compound than the others (Kallithraka, Arvanitoyannis, El-Zajouli, & Kefalas, 2001). In addition, quantitative and qualitative distribution of polyphenols in grape pomace may show significant differences, depending on other factors, such as varietal differences of the grapes, climate, location of the cultures and wine-making procedures (Ruberto
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et al. 2007). Therefore, several grape varieties should be analysed to differ them with respect to their antioxidative activities. The distribution of total extractable phenolics of fresh grape was reported to be approximately 10% in pulp, 60–70% in seeds, and 28–35% in skin (Shi, Yu, Pohorly, & Kakuda, 2003). The pomace consist of seeds, skins and stems; thus, separating and using grape seeds alone as a raw material would give lower results in total phenolics than grape pomace (Ozcan, 2006). Therefore, antioxidant and antimicrobial properties of pomace extract should also be studied. However, there are very limited reports on the pomace extract of V. vinifera species, especially belonging to Turkish flora (Ozcan, 2006) even though phenolic composition and antioxidant activity of V. vinifera species have been investigated in different countries. Furthermore, no study has appeared to report in vitro and in situ antifungal effect of these grape species on some yeast; for example osmophilic yeasts Z. rouxii and Z. bailii. Therefore, this work was aimed to determine the total phenolic content, phenolic composition, antioxidant and in vitro and in situ antifungal activities of five different Turkish grape pomace extracts. 2. Materials and methods 2.1. Grape cultivars and preparation of pomaces and extracts The pomace samples of wine grape ( V. vinifera L) cultivars grown in Turkey were collected from local wine processing plants. The grape pomace cultivars were Emir (white grape cultivar) grown in Cappadocia district of Nevsehir province, Gamay (red grape cultivar) grown in Sarkoy-Murefte district of Trakya Region, Kalecik karasi (red grape cultivar) grown in Ankara province, Narince (white grape cultivar) grown in Tokat province and Okuzgozu (red grape cultivar) grown in Elazig province. The grape pomaces were only used in the antifungal assays in this study. The grape pomaces were used as powder form; therefore, they were dried at 70 °C for 72 h, and ground to obtain fine powder using a laboratory type grinder (Faema MPN, Italy). To obtain the pomace extracts, the powdered pomace was extracted in a Soxhlet apparatus with petroleum ether (Merck, Darmstadt, Germany) at 60 °C for 6 h to remove fatty materials. The defatted pomace powder (100 g) was re-extracted in the Soxhlet extraction apparatus with 100 mL of ethanol:water (95:5) at 60 °C for 8 h. After filtered through Whatman filter paper (Whatman No:1), all extracts were concentrated by a rotary evaporator (Buchi, R 200, Switzerland) under vacuum conditions at 50 °C to get crude pomace extracts. The pomace extracts were freeze-dried using a lyophilizator (Labconco FreeZone 2.5, USA) and then stored in an ultra high deep freeze (Hettich HS4486, Germany) at 80 °C until analysis. 2.2. Determination of total phenolic content Total phenolics in grape pomace extracts were determined using the Folin–Ciocalteau reagent (Singleton & Rossi, 1965). The reaction mixture was prepared by mixing 40 lL of methanolic solution of extract, 2400 lL of distilled water, 200 lL of the Folin–Ciocalteau’s reagent and 600 lL sodium carbonate (20% Na2CO3). After the mixture was kept for 2 h at room temperature, the reduction of the Folin–Ciocalteau reagent by phenolic compounds under alkaline conditions, which resulted in the development of a blue colour, was recorded at 765 nm (spectrophotometer, Shimadzu UV–visible 1700, Tokyo-Japan). The absorbance was measured against blank that had been prepared in a similar manner, by replacing the extract with distilled water. The absorbance was compared to a gallic acid calibration curve. The total phenolic content was calculated as mg of gallic acid equivalents (GAE)/g extract.
2.3. Antioxidant activity determination by phosphomolybdenum method The antioxidant activities of extracts were evaluated using the phosphomolybdenum method as outlined (Prieto, Pineda, & Aguilar, 1999). An aliquot of 0.4 mL of the sample solution was placed in tubes and mixed with 4 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in water bath at 95 °C for 90 min. After the samples were cooled down to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. For blank, 0.4 mL of methanol was used in place of sample. The antioxidant activity was determined using a standard curve with ascorbic acid solutions as the standard and the reducing capacity of the extracts was expressed as mg of ascorbic acid equivalents (AAE)/g extract. 2.4. DPPH antiradical activity The antiradical activity of pomace extracts was determined using a, a-diphenyl-b-picrylhydrazyl (DPPH) method of Lee et al. (1998). The capacity of ethanolic extracts to scavenge the lipid soluble DPPH radical, which results in the bleaching of the purple colour exhibited by the stable DPPH radical, was monitored at 517 nm. 50 lL aliquots of the proper ethanolic extract dilution at a concentration range of 0.1–2 mg/mL was mixed with 450 lL Tris–HCl buffer (pH = 7.4) and 1 mL of the ethanolic DPPH solution (0.1 mM). The mixtures were kept in the dark at 25 °C for 30 min. The absorbance was measured using a spectrophotometer (Shimadzu UV–visible 1700, Tokyo-Japan) at 517 nm after 30 min, versus methanol as blank. IC50 (concentration causing 50% inhibition) value of each extract was determined graphically. Inhibition of free radical DPPH (I%) was expressed as percentage inhibition of DPPH radical and calculated using following equation:
Absorbance of sample 100 Percentage inhibitionðI %Þ ¼ 1 Absorbance of control
2.5. Quantification of phenolic compounds by RP-HPLC Phenolic compounds were evaluated by reversed-phase highperformance liquid chromatography (RP-HPLC, Shimadzu Scientific Instruments, Tokyo, Japan). Detection and quantification were carried out with a LC-10ADvp pump, a Diode Array Detector, a CTO-10Avp column heater, SCL-10Avp system controller, DGU14A degasser and SIL-10ADvp auto sampler (Shimadzu Scientific Instruments, Columbia, MD). Separations were conducted at 30 °C on AgilentÒ Eclipse XDB C-18 reversed-phase column (250 mm 4.6 mm length, 5 lm particle size). The mobile phases were A: 2.0% acetic acid in distilled water and B: methanol. Flow rate was 0.8 mL/min. For analysis, 25 mg of dry pomace extract was dissolved in 1 mL of methanol and injection volume of the sample solution was 10 lL. A gradient programme was used for separation of phenolic compounds (Kammerer, Claus, Schieber, & Carle, 2005). Phenolic compositions of the extracts were determined by a modified method of Schulz, Steuer, Kruger, Junghanns, and Weinreich (2001). Gallic acid, protocatechuic acid, (+)-catechin, ()-epicatechin, caffeic acid, chlorogenic acid, vanillin, p-coumaric acid, ferulic acid, vitexin, o-coumaric acid, rutin, trans-resveratrol, hesperidin, eriodictyol, trans-cinnamic acid, quercetin, luteolin, genistein, kaempferol and apigenin were used as standard. Identification and quantitative analysis were done by comparison with standards. The amount of each phenolic compound was expressed as lg per gram of extract.
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2.6. Analytical quality assurance In general, all analysis was conducted in triplicate with three replications. Triplicate values varied by approximately ±5%. For RP-HPLC analysis, the choice of appropriate detection mode is critical to ensure that all the components can be detected. With DAD, this challenge could be overcome using a multiple wavelength scanning programme that can monitor several wavelengths simultaneously. This also provides assurance that all UV–vis absorbing components are detected, if these are found in adequate amount. Chromatograms of the compounds are recorded by using DAD used to carry out simultaneous record. Therefore, in this study, DAD was used to optimise determination wavelength in the work. The UV– vis spectrums of the phenolic standards dissolved in the mobile phase were obtained by DAD. UV–vis absorbing chromatograms of 21 flavanoids are shown in Fig. 2a. The HPLC showed linearity in the range used. 2.7. Antifungal activity 2.7.1. Yeast cultures Two osmophilic yeast cultures; Z. rouxii DSM 70540 and Z. bailii DSM 70492 were obtained from German Collection of Microorganisms and Cell Cultures (Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH, DSMZ, Germany). 2.7.2. Agar-well diffusion assay The agar-well diffusion method was used to determine the antifungal activities of pomace extracts. The yeast strains were suspended and activated (104–105 cfu/mL) in Malt Extract Broth (MEB, Merck, Germany), after incubation at 25 °C for 24 h. Then,
1.0% concentration of each yeast isolate was added into a flask containing 25 mL sterile Malt Extract Agar (Merck, Germany) at 45 °C and poured into petri dishes (9 cm in diameter) and the agars were allowed to solidify at 4 °C for 1 h. Four equidistant wells (4 mm in diameter) were cut from the agars using a sterile corkborrer. 1.0%, 2.0%, 5.0% and 10.0% concentrations of the pomaces or extracts were diluted in pure ethanol and 50 lL of pomace and extract solutions were applied to the wells. Pure ethanol without pomace or extract was used as a control and inhibition zone was observed in the control wells. Then, the yeast plates were incubated at 25 °C for 24–48 h in an inverted position (Aureli, Costantini, & Zolea, 1992). At the end of the incubation period, all plates were examined for any zones of growth inhibition and the diameters of the formed zones were measured in millimetres using a digital calliper.
2.7.3. Antifungal activity in apple and orange juices In situ antifungal activity assay was performed in apple and orange juices from apples (Starking) and oranges (Washington) procured from a local market in Kayseri, Turkey. To obtain fruit juices, these fruits were pressed in a laboratory hydraulic press (Tefal, Type 8314, China). The juices were then filtered through a mesh filter and transferred into the tubes with 15 mL of capacity. Then, aliquots of these juices were sterilized at 121 °C for 15 min. To determine antifungal activity of the pomaces and extract groups, the stock cultures of Z. rouxii (DSM 70540) and Z. bailii (DSM 70492) were firstly activated in Malt Extract Broth (Merck) at 25 °C for 24 h, followed by the second activation in the Malt Extract Broth at 25 °C for 24 h. Then, these tubes were allocated into five groups, and each of them was separately and aseptically added with the previously described pomace powders and extracts at
Table 1 Total extract yield, phenolic content, antioxidant activity, antiradical activity and the amounts of phenolics in pomace extracts of five Vitis vinifera L. cultivars. Analysis results
Chemical analysisb Yield (%) TPC (mg GAE/g extract) TAA (mg AAE/g extract) DPPH (IC50, lg/g) Phenolic Compounds (lg/g) Phenolic acids Chlorogenic acid Gallic acid Protocatechuic acid Caffeic acid p-Coumaric acid o-Coumaric acid Ferulic acid trans-Cinnamic acid Vanillin Flavonoids (+)-Catechin ()-Epicatechin Kaempferol Quercetin Rutin Eriodictyol Hesperidin Apigenin Vitexin Luteolin Genistein Stilbens trans-Resveratrol Total a b
Pomace extracts of grape cultivarsa Emir
Gamay
Kalecik karasi
Narince
Okuzgozu
Average
4.58a 75.5e 64.6e 428.1a
2.09d 255.4b 126.6b 151.8d
2.49c 205.7c 107.6c 189.6c
1.14e 138.1d 87.9d 245.6b
2.63b 281.4a 139.1a 109.8e
2.59 191.2 225.0 105.2
3778.1b 674.8d –d 15.7c 29.7b 178.7c –d –c –c
525.6e 989.9b 87.3c 11.7c 22.5c 21.0e –d –c 105.4a
925.8d 1524.5a 166.9a 45.5b –e 87.6d 17.7c 11.9b –c
5326.6a 1510.4a 173.4a 55.4b 65.0a 377.0a 33.9b 19.7a 33.3b
1340.1c 750.9c 117.6b 100.2a 20.0d 282.0b 42.9a –c –c
2379.2 1090.1 109.0 45.7 27.4 189.3 18.9 6.3 27.7
3645.9cd 334.1b 59.9c 105.5d 1013.3c 235.4b 119.4b –c – – –
3365.7d 79.3c 432.1b 799.0c 109.5e 26.0d 125.5b 30.25b – – –
5493.3b 21.5d 713.4a 1144.2a 191.6d 114.2c 156.0b 39.10a – – –
8973.3a 504.1a 434.0b 1097.2b 1127.2b 270.1a 122.7b –c – – –
3936.9c 25.0d 94.7c 105.1d 1662.0a 242.0b 367.5a –c – – –
5083.0 192. 8 346.8 650.2 998.5 177.5 178.2 13.8 – – –
–c 10190.5
12.2b 20123.3
14.6a 10653.1
–c 6633.3
–c 9086.8
3.1
In each row, difference (p < 0.01) between means (a–e) for grape varieties (–: not detectable). TPC (total phenolic content) expressed as gallic acid equivalent (GAE), TAA (total antioxidant activity) expressed as ascorbic acid equivalent (AAE).
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2.8. Statistical analysis Data were analysed using one way analysis of variance (ANOVA) through the general linear models (GLM) procedure of the statistical analysis software (SPSS 10.01). Significant differences between the means were further analysed using the Tukey Test. Bivariate correlations between variables were analysed by Pearson’s test using Minitab 14.0 software.
extracts of grape cultivars had strong TAA (Table 1). Significant differences (p < 0.05) were found in the TAAs of the grape cultivars. Emir and Narince were determined to have the lowest level of TAA, while the extracts of red cultivars; Gamay, Kalecik karasi and Okuzgozu had the highest, with an average value of 225.0 mg AAE/g extract.
160
128
(mg AAE/g)
four different concentrations (0.0%, 2.0%, 5.0% and 10.0%, based on 15 mL of fruit juice). Then, each group was inoculated with the previously activated and reproduced osmophilic yeasts; Z. rouxii and Z. bailii (106–107 cfu/mL) at 1.0% concentration and incubated in refrigerator at 7 ± 1 °C (the storage temperature of juice) for 2, 6, 18, 24, 48, 72 and 120 h. The yeasts were enumerated in the 2, 6, 18, 24, 48, 72 and 120 h, in pour plates of Malt Extract Agar (Merck, Germany), after pipetting of 0.1 mL of prepared serial decimal solutions (0.85% NaCl) onto surface of the agar plates and incubation at 25 °C for 24–48 h.
Total antioxidant activity
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96 y = 0.351x + 37.94
64
R² = 0.995
32 3. Results and discussion
0
3.1. Total extract yield, phenolic content, antioxidant activity and antiradical activity
100
150
200
250
300
Total phenolic content (mg GAE/g) 450
360
y = -1.401x + 493.0
,µ g/g) 50
(IC
DPPH
R² = 0.915
270
180
90
0 50
100
150
200
250
300
Total phenolic content (mg GAE/g) 450
DPPH
360
(IC 50 µ g/g)
Total extract yield, total phenolic content, antiradical and antioxidant activities of the ethanolic pomace extracts of commercial Turkish grape cultivars were determined and the results were presented in Table 1. The extract yields ranged from 1.14% to 4.58%. Amongst the grape cultivars; Emir extract had the highest extract yields, whereas Narince extract had the lowest one. The research showed differences in the mass amounts of total phenolic compounds amongst the selected grape cultivars, as expected. The total phenolic contents (TPC) of the extracts were determined to range from 75.5 to 281.4 mg GAE/g dry extract with an average value of 191.2 mg GAE/dry extract. Extracts of white grape cultivars; Emir and Narince were determined to have the lower level of total phenolics, while the extracts of red cultivars; Gamay, Kalecik karasi and Okuzgozu had the higher. Therefore, highly pigmented red cultivars; Gamay, Kalecik karasi and Okuzgozu can be emphasised as cultivars with grape extracts rich in phenolic compounds. These results were in agreement with the findings of Katalinic et al. (2010) who studied the total phenolic compounds of different grape cultivars. Generally, they determined the red grape cultivars to contain higher amounts of total phenolics than white cultivars, especially in the grape skin extracts. On the other hand, the TPC results for the extracts of grape cultivars in this study were different from those reported in literature. Gokturk-Baydar, Ozkan, and Sagdic (2004) reported that the total phenolic contents of solvent (ethyl acetate:methanol:water) extract of Narince was 627.9 mg GAE/g. Ozkan, Sagdic, Gokturk-Baydar and Karamahmutoglu (2004) determined the total phenolic contents of Emir and Kalecik karasi pomace extracts to be 68.8 and 96.3 mg GAE/g. These differences in total phenolic contents might be due to the difference between the extraction methods; in fact, phenolic content of grape pomace extracts are expected to strongly depend on extraction conditions as well as the solvent used (Ozcan, 2006). Therefore, comparison of results with literature data was difficult. The total antioxidant activity (TAA) of the ethanolic extracts of grape cultivars tested was determined through the phosphomolybdenum assay. This method is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and the formation of green Mo (V) complexes with a maximal absorption at 695 nm (Prieto, Pineda, & Aguilar, 1999). The results indicated that the ethanolic
50
y = -3.979x + 643.4 R² = 0.915
270
180
90
0 50
70
90
1 10
130
150
Total antioxidant activity (mg AAE/g) Fig. 1. Bivariate correlations between total phenolic content (TPC), total antioxidant activity (TAA) and DPPH antiradical activity.
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Pearson’s test was used to analyse bivariate correlations between the values of TPC and TAA. The TPC exhibited a significantly linear correlation with TAA (R = 0.998; p < 0.01) for extracts of grape cultivars. The correlation (R2 = 0.995) indicated that 95.5% of the phenolic content in the extracts were accounted for total antioxidant activity (Fig. 1). The results were consistent with previous reports because some authors have shown that high total phenol content increases the antioxidant activity (Kumaran & Karunakaran, 2007). The free radical-scavenging of ethanolic extracts of grape cultivars tested were determined using the DPPH method, which is a useful method to investigate the free radical-scavenging activities of the phenolic compounds. Free radical-scavenging assays may provide information on how capable an antioxidant is in preventing reactive radical species from reaching lipoproteins, polyunsaturated fatty acids, DNA, amino acids, proteins and sugars in biological and food systems (Katalinic et al., 2010). Amongst the extracts of grape cultivars, those of the white grape cultivars; Emir and Narince were determined to have the highest level of DPPH, on
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a lg analyte basis (IC50 = 428.1 and 245.6 lg/g). It was reported by Katalinic et al. (2010) that antioxidants can deactivate or scavenge stable free DPPH radical by two major mechanisms: by reduction via electron transfer or by hydrogen atom transfer that may occur also in parallel and steric accessibility is one of the major determinants of the reaction. The ethanolic grape pomace extracts of selected, both red and white grape cultivars could interact with the stable free DPPH radicals efficiently and quickly, with an average IC50 of 105.2 lg/g. Differences for DPPH IC50 between the extracts of grape cultivars could be probably due to differences in polyphenolic content of analysed extracts. Fig. 1 indicates a significant negative correlations between TPC and IC50 (p < 0.01; R = 0.957) and between TAA and IC50 (p < 0.01; R = 0.956) values were observed for pomace extracts of grape cultivars, indicating direct contribution of phenolics to this activity. This result was consistent with the observations in the literature (Meda, Lamien, Romito, Millogo, & Nacoulma, 2005). Several factors might have contributed to our results. First, the DPPH radical-scavenging assay determined free antioxidants in
Fig. 2. A chromatogram of phenolic standards and grape pomace extracts at 278 nm in HPLC.
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the extracts, whereas the assay of total phenols with Folin–Ciocalteau reagents determined both free phenolics and bound phenolics in the extracts (Singleton, Orthofer, & Lamuela-Raventos, 1999). It is also known that only flavonoids of a certain molecular structure, particularly those with a certain hydroxyl position, will determine the antioxidant properties present. In general, these properties depend on the ability to donate hydrogen and electrons to a free radical (Meda et al., 2005). Therefore, the negative correlation of IC50 with TPC and TAA were partly due to the presence of other phenolics in the extracts of grape cultivars, which might have contributed to the antioxidant activity. Second, the reactions of antioxidants to the DPPH free radicals were different from their reactions to the Folin–Ciocalteau reagent in the total-phenol assay. The Folin–Ciocalteau reagent is sensitive to a broad range of substrates, which are easily oxidised, but the DPPH free radicals exhibit different sensitivity to various antioxidants, which indicate fast, intermediate, or slow kinetic reactions to the DPPH free radicals (Yang, Paulino, Janke-Stedronsky, & Abawi, 2007). Third, some phenolic antioxidants reacting strongly to the Folin–Ciocalteau reagent may not react to the DPPH free radicals (Yang et al., 2007). 3.2. Identification of phenolic compounds by RP-HPLC It is obvious that the total phenolic content measured by the Folin–Ciocalteau procedure does not give a full picture of the qualification or quantification of the phenolic constituents in the grape pomace extracts (Wojdylo, Oszmianski, & Czemerys, 2007). Therefore, the phenolic acids, flavonoids and stilbenes in the extracts of grape cultivars were determined by the HPLC method and the HPLC results are presented in Table 1. Eighteen phenolic compounds; Chlorogenic acid, gallic acid, protocatechuic acid, caffeic acid, p-coumaric acid, o-coumaric acid, ferulic acid, trans-cinnamic acid, vanillin, (+)-catechin, ()-epicatechin, kaempferol, quercetin, rutin, eriodictyol, hesperidin, apigenin and trans-resveratrol were identified by comparison with the retention times and UV spectra of authentic standards analysed under identical conditions, while the quantitative data was calculated from their respective calibration curves. Chromatograms of the phenolic standards and the phenolic constituents of the extracts are shown in Fig. 2. As a general categorisation proposed by Shi et al. (2003) phenolic compounds in grape can be divided into two groups: phenolic
acids (precursors of flavonoids) and flavonoids. The most common phenolic acids encountered in grape are cinnamic acids (coumeric, caffeic, ferulic, chlorogenic and neochlorogenic acids) and benzoic acids (p-hydroxybenzoic, protocatechuic, vanillic and gallic acids) (Ozcan, 2006). Although these results were obtained for grapes, similar results could also be obtained for grape pomace extracts in this study. Of phenolic acids, the major compound present in the extracts was identified to be chlorogenic acid with an average value of 2379.2 lg/g, followed by gallic acid (average 1090.1 lg/g). Amongst flavonoids, (+)-catechin was the major compound identified as an average value of 5083 lg/g. The least abundant compound present in the extracts was trans-resveratrol (3.05 lg/g). On the other hand, protocatechuic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, vanillin, apigenin and trans-resveratrol could not be identified in the all extracts tested. Furthermore, none of the extract variety was found to contain vitexin, luteolin and genistein. Gamay and Kalecik karasi extracts were the only grape varieties extracts of which was determined to contain trans-resveratrol. This result should be important because of the great interest that has been devoted to resveratrol and its derivatives (Katalinic et al., 2010). Although there are many reports dealing with phenolic constituents of some grape varieties, data about these compounds in pomace extracts of grape cultivars, especially belonging to Turkish flora have appeared to be very limited. Therefore, the phenolic compounds in the extracts of grape cultivars studied could not be directly compared with those in the literature both qualitatively and quantitatively. Tukey test analysis results revealed that a significant (p < 0.01) difference was found between the extracts of grape cultivars in respect of their phenolic compounds (Table 1). In general, amongst the grape cultivars was Narince extract of which had the highest levels of phenolic compounds, with an average value of 20123.3 lg/g extract. Chlorogenic acid, gallic acid, p-coumaric acid and o-coumaric acid were the most abundant phenolic acids, while (+)-catechin, (-)-epicatechin and eriodictyol were the most abundant flavonoids in Narince extract. Kalecik karasi extract had the highest gallic acid, protocatechuic acid, kaempferol and quercetin than did the other extracts. On the other hand, caffeic acid and ferulic acid were determined to be the most abundant phenolic acids; rutin and hesperidin to be the most abundant flavonoids in Okuzgozu, red grape cultivar. These results indicate that
Table 2 In vitro antifungal activity values (means ± SD) indicated by inhibition zones (mm) formed by grape pomaces and extracts at different concentrations of 1%, 2%, 5% and 10%. Grape variety
Emir
Gamay
Kalecik Karası
Narince
Okuzgozu
a
Concentration (%)
1 2 5 10 1 2 5 10 1 2 5 10 1 2 5 10 1 2 5 10
Pomaces
Extracts
Z. rouxii
Z. bailii
Z. rouxii
Z. bailii
–a – – 11 ± 1.0 – – – 10.5 ± 0.5 – – – 14 ± 1.0 – – – – – – – –
– – – – – – – 7.5 ± 0.5 – – – 9 ± 1.0 – – – – – – – –
– – – 13.5 ± 1.5 – – 10.5 ± 0.5 16 ± 1.0 – – 12.5 ± 0.5 14.5 ± 0.5 – – 13 ± 1.0 15.5 ± 0.5 – – 15 ± 1.0 17.5 ± 0.5
– – – – – – – 17 ± 1.0 – – 11 ± 1.0 15 ± 1.0 – – 14 ± 1.0 18 ± 1.0 – – 9 ± 1.0 12.5 ± 0.5
Not detectable. The detection limit was P12 lg/ml, the minimum amount of pomaces and extracts required to show an inhibition zone in the agar-well diffusion assay.
Table 3 Effect of grape pomace or extracts on Z. rouxii and Z. bailii counts (log cfu/mL) in apple juice at different concentrations aand storage periodsb. Treatments
Control Pomaces Emir
Gamay
Narince
Okuzgozu
Extracts Emir
Gamay
Kalecik Karasi
Narince
Okuzgozu
a b
Z. rouxii
Z .bailii
2h
6h
18 h
24 h
48 h
72 h
120 h
2h
6h
18 h
24 h
48 h
72 h
120 h
0
4.69Ad
5.05Aab
4.91Abc
5.18Aa
4.86Ac
4.42Ae
4.11Af
5.69Ab
6.25Aa
6.11Aa
5.26Ac
4.91Ad
4.93Ad
5.01Ad
2 5 10 2 5 10 2 5 10 2 5 10 2 5 10
4.08Ba 3.82Ca 3.37Da 3.73Ba 3.69Ba 3.24Ca 3.64Ba 3.55Ba 3.18Ca 3.52Ba 3.15Ca 3.10Ca 3.28Ba 3.23Ba 3.21Ba
3.94Ba 3.66Cab 2.69Db 3.69Ba 2.66Cb 2.05Db 3.40Bba 2.93Cb 2.08Db 3.40Ba 3.00Cb 2.69Db 3.21Ba 3.19Ba 3.08Ba
3.62Bb 3.48Bbc 2.25Cc 3.49Bba 1.97Cc –Dc 3.15Bbc 2.09Cc –Dc 3.27Ba 2.81Cc 2.29Dc 3.05Bba 2.69Cb 2.19Db
3.51Bb 3.29Cc 1.98Dd 3.23Bbc –Cd –Cc 2.98Bdc –Cd –Cc 3.18Bba 2.69Cc 1.91Dd 2.88Bbc 2.39Cc 1.92Dc
3.24Bc 2.92Cd 1.69De 3.01Bdc –Cd –Cc 2.87Bd –Cd –Cc 2.86Bbc 2.46Cd –De 2.65Bc 2.03Cd –Dd
3.12Bcd 2.81Cd –Df 2.88Bde –Cd –Cc 2.76Bd –Cd –Cc 2.51Bdc 2.09Ce –De 2.22Bd –Ce –Cd
2.98Bd 2.65Cd –Df 2.69Be –Cd –Cc –Be –Bd –Bc 2.22Bd –Cf –Ce 2.04Bd –Ce –Cd
5.14Ba 5.04Ba 5.03Ba 5.10Ba 5.11Ba 4.69Ca 5.36Ba 5.23CBa 5.11Ca 5.11Ba 4.82Ca 4.82Ca 5.11Ba 5.02Ba 5.00Ba
4.97Bb 4.94CBba 4.78Cb 5.08Bba 4.72Cb 4.62Ca 5.17Bba 5.08Ba 4.69Cb 5.10Ba 4.84CBa 4.67Cba 5.06Bba 4.82Cba 4.72Cb
4.95Bcb 4.79Cbc 4.52Dc 4.98Bba 4.57Cb 4.46Db 5.11Bbac 5.05Ba 4.49Cc 5.01Bba 4.69Ca 4.50Cbc 4.95Bba 4.69Cba 4.44Dc
4.91Bcbd 4.72Cc 4.41Ddc 4.84Bbc 4.39Cc 4.28Cc 4.94Bbdc 4.53Cb 4.29Cd 4.79Bba 4.55Cba 4.37Cc 4.79Bbc 4.33Cbc 4.16Cd
4.85Acbd 4.50Bd 4.23Cde 4.64Bdc 4.24Cdc 4.08Dd 4.82Aedc 4.44Bb 4.02Ce 4.67Abc 4.31Bbc 4.22Bdc 4.58Bdc 4.29Cc 3.93De
4.80Acd 4.40Bed 4.17Cfe 4.51Bd 4.15Cd 4.01Ced 4.66Bed 4.30Cb 3.96De 4.42Bdc 4.26Bbc 3.95Cde 4.57Bdc 4.12Cdc 3.82De
4.75Bd 4.26Ce 4.02Df 4.40Bd 3.95Ce 3.87Ce 4.57Be 4.22Cb 3.82Df 4.25Bd 4.07Bc 3.75Ce 4.35Bd 3.90Cd 3.45Df
2 5 10 2 5 10 2 5 10 2 5 10 2 5 10
3.57Ba 3.51Ba 2.69Ca 3.66Ba 3.56Ba 2.69Ca 3.66Ba 3.66Ba 2.52Ca 3.61Ba 3.46CBa 3.32Ca 3.18Ba 3.02Ca 3.03Ca
3.48Ba 3.17Cb 2.03Db 3.48Bb 2.54Cb 1.96Db 3.28Bb 2.26Cb 1.98Db 3.08Bb 2.96Bba 2.14Cb 3.01Bba 2.77Cb 2.09Db
3.39Bba 3.08Cb –Dc 3.30Bc –Cc –Cc 3.10Bb –Cc –Cc 2.84Bc 2.60Cbc –Dc 2.88Bb 2.47Cc –Dc
3.25Bbc 2.69Cc –Dc 3.13Bc –Cc –Cc 2.86Bc –Cc –Cc 2.67Bd 2.48Cbc –Dc 2.58Bc 2.19Cd –Dc
3.06Bdc –Cd –Cc 2.88Bd –Cc –Cc 2.48Bd –Cc –Cc 2.44Be 2.11Cc –Dc 2.32Bd 1.94Ce –Dc
2.92Bde –Cd –Cc 2.58Be –Cc –Cc 2.07Be –Cc –Cc 2.13Bf 1.93Cd –D 2.04Be –Cf –Cc
2.83Be –Cd –Cc 2.14Bf –Cc –Cc –Bf –Cc –Cc 1.93Bg –Ce –Cc –Bf –Bf –Bc
5.15Ba 5.10Ba 5.08Ca 5.08Ba 4.98Ba 4.82Ca 5.10Ba 4.71Ca 4.49Da 5.25Ba 5.07Ba 4.82Ca 5.12Ba 5.02Ba 4.69Ca
5.08Bb 4.87Cb 4.69Db 5.01Bba 4.69Cb 4.39Db 5.08Ba 4.59Ca 4.25Db 4.77Bb 4.59Cb 4.19Db 4.71Bb 4.59Cb 4.19Db
4.90Bcb 4.71Ccb 4.45Dc 4.85Bbc 4.48Cc 4.21Dc 4.83Bb 4.37Cb 4.10Dc 4.54Bc 4.39Cc 4.02Dcb 4.52Bc 4.29Ccb 3.95Dc
4.84Bc 4.67Bc 4.19Cd 4.73Bc 4.20Cd 4.04Cc 4.60Bc 4.10Cc 3.85Dc 4.48Bdc 4.33Cdc 3.93Dcd 4.35Bd 4.16Cc 3.51Dd
4.51Bd 4.41Bd 4.15Cd 4.52Bd 4.11Cd 3.84Dd 4.43Bdc 4.08Cc 3.71Dd 4.37Bd 4.17Cde 3.78Dd 4.14Be 3.98Cc 3.30De
4.43Bed 4.33Bed 4.10Ced 4.47Bd 4.06Cd 3.78Dd 4.24Bde 3.89Cd 3.41Dd 4.21Be 4.01Ce 3.49De 4.08Be 3.53Cd 3.19Ce
4.31Be 4.20Be 3.91Ce 4.27Be 3.88Ce 3.54De 4.17Be 3.48Ce 2.94De 3.84Bf 3.62Cf 3.09Df 3.93Bf 3.12Ce 2.83Df
O. Sagdic et al. / Food Chemistry 126 (2011) 1749–1758
Kalecik Karasi
Con.(%)
In each column, difference (A–D) between the concentrations of pomace or extracts of grape cultivars (p < 0.01). In each row, difference (a–g) between the storage periods (p < 0.01). –: Not detectable (
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RP-HPLC–DAD analysis of phenolic compounds in pomace extracts from five grape cultivars: Evaluation of their antioxidant, antiradical and antifungal activities in orange and apple juices Osman Sagdic a,⇑, Ismet Ozturk a, Gulcan Ozkan b, Hasan Yetim a, Lutfiye Ekici a, Mustafa Tahsin Yilmaz c a b c
Erciyes University, Engineering Faculty, Food Engineering Department, 38039 Kayseri, Turkey Suleyman Demirel University, Engineering Faculty, Food Engineering Department, 32260 Isparta, Turkey Erciyes University, Safiye Cikrikcioglu Vocational College, Food Technology Department, 38039 Kayseri, Turkey
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 26 October 2010 Accepted 15 December 2010 Available online 19 December 2010 Keywords: Grape pomace extracts Phenolic compounds RP-HPLC Antioxidant activity Antifungal activity in vitro and in situ
a b s t r a c t Phenolic compounds, related to antioxidative and antifungal properties of ethanolic extracts from five commercial grape cultivars (three red and two white) grown in Turkey were determined. A reversedphase high performance liquid chromatography (RP-HPLC) procedure was developed, and a total 18 different phenolic compounds were identified. Total phenolic contents of the extracts were determined using Folin–Ciocalteau method. Antioxidant activities of the extracts were evaluated by using DPPH radical scavenging and phosphomolybdenum methods. All extracts exhibited strong antioxidant and antiradical activity. Phenolic compounds and antioxidant activities of the extracts were variety dependent. Antifungal activities of the pomaces and extracts were screened by both in vitro agar-well diffusion assay and antifungal activity in apple and orange juices in situ using Zygosaccharomyces rouxii and Z. bailii. Antifungal activities revealed that the pomaces and extracts of Gamay and Kalecik karasi could be more effective antifungal agents than those of Emir, Narince and Okuzgozu grape cultivars. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Winary by-products are of particular interest since grape (Vitis vinifera) is amongst the world’s largest fruit crop, with an annual production of more than 61 million tonnes according to the data of 2005 year (FAO, 2010). At present; however, only minimum amounts of these wastes are up-graded or recycled, which is particularly true in Europe, where vegetable wastes are generally dumped or used as animal feed and compost, without any pretreatment (Ruberto et al., 2007). This is also case for Turkey, where 4 million metric tons are processed per year, and 15–25% of the processed grapes is left over as pomace and not revalued anyway (Anonymous, 2008). Grape pomace is a waste product of grape juice and wine industry (Gokturk-Baydar, Sagdic, Ozkan, & Cetin, 2006; Ozkan, Sagdic, Gokturk-Baydar, & Kurumahmutoglu, 2004). These products may contain high phenolic compounds because of poor extraction during the winemaking processes; hence it makes their utilisation worthwhile and support sustainable agricultural production (Arvanitoyannis, Ladas, & Mavromatis, 2005). From the nutritional point of view, these compounds are polyphenols and the most
⇑ Corresponding author. Tel.: +90 352 4374937x32726; fax: +90 352 437 54 84. E-mail address: [email protected] (O. Sagdic). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.075
important constituents of grape pomace (Kammerer, Claus, Schieber, & Carle, 2005). In addition, many recent studies have highlighted the beneficial effects of dietary phenolic compounds in grape for human health (Ruberto et al., 2007). Grape seed procyanidins have been reported to be potent free radical scavengers and were found to exhibit novel cardio-protective properties (Bagchi et al., 2003). It has also been indicated that the growth of pathogen fungi such as Penicillium chrysogenum, Penicillium expansum, Aspergillus niger and Trichoderma viride could be inhibited by grape pomace extracts (Corrales, Butz, Tauscher, & Cabrejas, 2010). Therefore, pomace extracts can show their antifungal effects on some yeast too. Grape extracts contain large quantities of monomeric phenolic compounds such as (+)-catechins, ()-epicatechin and ()-epicatechin-3-o-gallate, and dimeric, trimeric and tetrameric procyanidins (Saito, Hosoyama, Ariga, Kataoka, & Yamaji, 1998). For instance, trans-resveratrol is produced from grape berries in response to fungal infection and UV irradiation. Some red grape varieties may be genetically richer in this compound than the others (Kallithraka, Arvanitoyannis, El-Zajouli, & Kefalas, 2001). In addition, quantitative and qualitative distribution of polyphenols in grape pomace may show significant differences, depending on other factors, such as varietal differences of the grapes, climate, location of the cultures and wine-making procedures (Ruberto
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et al. 2007). Therefore, several grape varieties should be analysed to differ them with respect to their antioxidative activities. The distribution of total extractable phenolics of fresh grape was reported to be approximately 10% in pulp, 60–70% in seeds, and 28–35% in skin (Shi, Yu, Pohorly, & Kakuda, 2003). The pomace consist of seeds, skins and stems; thus, separating and using grape seeds alone as a raw material would give lower results in total phenolics than grape pomace (Ozcan, 2006). Therefore, antioxidant and antimicrobial properties of pomace extract should also be studied. However, there are very limited reports on the pomace extract of V. vinifera species, especially belonging to Turkish flora (Ozcan, 2006) even though phenolic composition and antioxidant activity of V. vinifera species have been investigated in different countries. Furthermore, no study has appeared to report in vitro and in situ antifungal effect of these grape species on some yeast; for example osmophilic yeasts Z. rouxii and Z. bailii. Therefore, this work was aimed to determine the total phenolic content, phenolic composition, antioxidant and in vitro and in situ antifungal activities of five different Turkish grape pomace extracts. 2. Materials and methods 2.1. Grape cultivars and preparation of pomaces and extracts The pomace samples of wine grape ( V. vinifera L) cultivars grown in Turkey were collected from local wine processing plants. The grape pomace cultivars were Emir (white grape cultivar) grown in Cappadocia district of Nevsehir province, Gamay (red grape cultivar) grown in Sarkoy-Murefte district of Trakya Region, Kalecik karasi (red grape cultivar) grown in Ankara province, Narince (white grape cultivar) grown in Tokat province and Okuzgozu (red grape cultivar) grown in Elazig province. The grape pomaces were only used in the antifungal assays in this study. The grape pomaces were used as powder form; therefore, they were dried at 70 °C for 72 h, and ground to obtain fine powder using a laboratory type grinder (Faema MPN, Italy). To obtain the pomace extracts, the powdered pomace was extracted in a Soxhlet apparatus with petroleum ether (Merck, Darmstadt, Germany) at 60 °C for 6 h to remove fatty materials. The defatted pomace powder (100 g) was re-extracted in the Soxhlet extraction apparatus with 100 mL of ethanol:water (95:5) at 60 °C for 8 h. After filtered through Whatman filter paper (Whatman No:1), all extracts were concentrated by a rotary evaporator (Buchi, R 200, Switzerland) under vacuum conditions at 50 °C to get crude pomace extracts. The pomace extracts were freeze-dried using a lyophilizator (Labconco FreeZone 2.5, USA) and then stored in an ultra high deep freeze (Hettich HS4486, Germany) at 80 °C until analysis. 2.2. Determination of total phenolic content Total phenolics in grape pomace extracts were determined using the Folin–Ciocalteau reagent (Singleton & Rossi, 1965). The reaction mixture was prepared by mixing 40 lL of methanolic solution of extract, 2400 lL of distilled water, 200 lL of the Folin–Ciocalteau’s reagent and 600 lL sodium carbonate (20% Na2CO3). After the mixture was kept for 2 h at room temperature, the reduction of the Folin–Ciocalteau reagent by phenolic compounds under alkaline conditions, which resulted in the development of a blue colour, was recorded at 765 nm (spectrophotometer, Shimadzu UV–visible 1700, Tokyo-Japan). The absorbance was measured against blank that had been prepared in a similar manner, by replacing the extract with distilled water. The absorbance was compared to a gallic acid calibration curve. The total phenolic content was calculated as mg of gallic acid equivalents (GAE)/g extract.
2.3. Antioxidant activity determination by phosphomolybdenum method The antioxidant activities of extracts were evaluated using the phosphomolybdenum method as outlined (Prieto, Pineda, & Aguilar, 1999). An aliquot of 0.4 mL of the sample solution was placed in tubes and mixed with 4 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in water bath at 95 °C for 90 min. After the samples were cooled down to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. For blank, 0.4 mL of methanol was used in place of sample. The antioxidant activity was determined using a standard curve with ascorbic acid solutions as the standard and the reducing capacity of the extracts was expressed as mg of ascorbic acid equivalents (AAE)/g extract. 2.4. DPPH antiradical activity The antiradical activity of pomace extracts was determined using a, a-diphenyl-b-picrylhydrazyl (DPPH) method of Lee et al. (1998). The capacity of ethanolic extracts to scavenge the lipid soluble DPPH radical, which results in the bleaching of the purple colour exhibited by the stable DPPH radical, was monitored at 517 nm. 50 lL aliquots of the proper ethanolic extract dilution at a concentration range of 0.1–2 mg/mL was mixed with 450 lL Tris–HCl buffer (pH = 7.4) and 1 mL of the ethanolic DPPH solution (0.1 mM). The mixtures were kept in the dark at 25 °C for 30 min. The absorbance was measured using a spectrophotometer (Shimadzu UV–visible 1700, Tokyo-Japan) at 517 nm after 30 min, versus methanol as blank. IC50 (concentration causing 50% inhibition) value of each extract was determined graphically. Inhibition of free radical DPPH (I%) was expressed as percentage inhibition of DPPH radical and calculated using following equation:
Absorbance of sample 100 Percentage inhibitionðI %Þ ¼ 1 Absorbance of control
2.5. Quantification of phenolic compounds by RP-HPLC Phenolic compounds were evaluated by reversed-phase highperformance liquid chromatography (RP-HPLC, Shimadzu Scientific Instruments, Tokyo, Japan). Detection and quantification were carried out with a LC-10ADvp pump, a Diode Array Detector, a CTO-10Avp column heater, SCL-10Avp system controller, DGU14A degasser and SIL-10ADvp auto sampler (Shimadzu Scientific Instruments, Columbia, MD). Separations were conducted at 30 °C on AgilentÒ Eclipse XDB C-18 reversed-phase column (250 mm 4.6 mm length, 5 lm particle size). The mobile phases were A: 2.0% acetic acid in distilled water and B: methanol. Flow rate was 0.8 mL/min. For analysis, 25 mg of dry pomace extract was dissolved in 1 mL of methanol and injection volume of the sample solution was 10 lL. A gradient programme was used for separation of phenolic compounds (Kammerer, Claus, Schieber, & Carle, 2005). Phenolic compositions of the extracts were determined by a modified method of Schulz, Steuer, Kruger, Junghanns, and Weinreich (2001). Gallic acid, protocatechuic acid, (+)-catechin, ()-epicatechin, caffeic acid, chlorogenic acid, vanillin, p-coumaric acid, ferulic acid, vitexin, o-coumaric acid, rutin, trans-resveratrol, hesperidin, eriodictyol, trans-cinnamic acid, quercetin, luteolin, genistein, kaempferol and apigenin were used as standard. Identification and quantitative analysis were done by comparison with standards. The amount of each phenolic compound was expressed as lg per gram of extract.
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2.6. Analytical quality assurance In general, all analysis was conducted in triplicate with three replications. Triplicate values varied by approximately ±5%. For RP-HPLC analysis, the choice of appropriate detection mode is critical to ensure that all the components can be detected. With DAD, this challenge could be overcome using a multiple wavelength scanning programme that can monitor several wavelengths simultaneously. This also provides assurance that all UV–vis absorbing components are detected, if these are found in adequate amount. Chromatograms of the compounds are recorded by using DAD used to carry out simultaneous record. Therefore, in this study, DAD was used to optimise determination wavelength in the work. The UV– vis spectrums of the phenolic standards dissolved in the mobile phase were obtained by DAD. UV–vis absorbing chromatograms of 21 flavanoids are shown in Fig. 2a. The HPLC showed linearity in the range used. 2.7. Antifungal activity 2.7.1. Yeast cultures Two osmophilic yeast cultures; Z. rouxii DSM 70540 and Z. bailii DSM 70492 were obtained from German Collection of Microorganisms and Cell Cultures (Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH, DSMZ, Germany). 2.7.2. Agar-well diffusion assay The agar-well diffusion method was used to determine the antifungal activities of pomace extracts. The yeast strains were suspended and activated (104–105 cfu/mL) in Malt Extract Broth (MEB, Merck, Germany), after incubation at 25 °C for 24 h. Then,
1.0% concentration of each yeast isolate was added into a flask containing 25 mL sterile Malt Extract Agar (Merck, Germany) at 45 °C and poured into petri dishes (9 cm in diameter) and the agars were allowed to solidify at 4 °C for 1 h. Four equidistant wells (4 mm in diameter) were cut from the agars using a sterile corkborrer. 1.0%, 2.0%, 5.0% and 10.0% concentrations of the pomaces or extracts were diluted in pure ethanol and 50 lL of pomace and extract solutions were applied to the wells. Pure ethanol without pomace or extract was used as a control and inhibition zone was observed in the control wells. Then, the yeast plates were incubated at 25 °C for 24–48 h in an inverted position (Aureli, Costantini, & Zolea, 1992). At the end of the incubation period, all plates were examined for any zones of growth inhibition and the diameters of the formed zones were measured in millimetres using a digital calliper.
2.7.3. Antifungal activity in apple and orange juices In situ antifungal activity assay was performed in apple and orange juices from apples (Starking) and oranges (Washington) procured from a local market in Kayseri, Turkey. To obtain fruit juices, these fruits were pressed in a laboratory hydraulic press (Tefal, Type 8314, China). The juices were then filtered through a mesh filter and transferred into the tubes with 15 mL of capacity. Then, aliquots of these juices were sterilized at 121 °C for 15 min. To determine antifungal activity of the pomaces and extract groups, the stock cultures of Z. rouxii (DSM 70540) and Z. bailii (DSM 70492) were firstly activated in Malt Extract Broth (Merck) at 25 °C for 24 h, followed by the second activation in the Malt Extract Broth at 25 °C for 24 h. Then, these tubes were allocated into five groups, and each of them was separately and aseptically added with the previously described pomace powders and extracts at
Table 1 Total extract yield, phenolic content, antioxidant activity, antiradical activity and the amounts of phenolics in pomace extracts of five Vitis vinifera L. cultivars. Analysis results
Chemical analysisb Yield (%) TPC (mg GAE/g extract) TAA (mg AAE/g extract) DPPH (IC50, lg/g) Phenolic Compounds (lg/g) Phenolic acids Chlorogenic acid Gallic acid Protocatechuic acid Caffeic acid p-Coumaric acid o-Coumaric acid Ferulic acid trans-Cinnamic acid Vanillin Flavonoids (+)-Catechin ()-Epicatechin Kaempferol Quercetin Rutin Eriodictyol Hesperidin Apigenin Vitexin Luteolin Genistein Stilbens trans-Resveratrol Total a b
Pomace extracts of grape cultivarsa Emir
Gamay
Kalecik karasi
Narince
Okuzgozu
Average
4.58a 75.5e 64.6e 428.1a
2.09d 255.4b 126.6b 151.8d
2.49c 205.7c 107.6c 189.6c
1.14e 138.1d 87.9d 245.6b
2.63b 281.4a 139.1a 109.8e
2.59 191.2 225.0 105.2
3778.1b 674.8d –d 15.7c 29.7b 178.7c –d –c –c
525.6e 989.9b 87.3c 11.7c 22.5c 21.0e –d –c 105.4a
925.8d 1524.5a 166.9a 45.5b –e 87.6d 17.7c 11.9b –c
5326.6a 1510.4a 173.4a 55.4b 65.0a 377.0a 33.9b 19.7a 33.3b
1340.1c 750.9c 117.6b 100.2a 20.0d 282.0b 42.9a –c –c
2379.2 1090.1 109.0 45.7 27.4 189.3 18.9 6.3 27.7
3645.9cd 334.1b 59.9c 105.5d 1013.3c 235.4b 119.4b –c – – –
3365.7d 79.3c 432.1b 799.0c 109.5e 26.0d 125.5b 30.25b – – –
5493.3b 21.5d 713.4a 1144.2a 191.6d 114.2c 156.0b 39.10a – – –
8973.3a 504.1a 434.0b 1097.2b 1127.2b 270.1a 122.7b –c – – –
3936.9c 25.0d 94.7c 105.1d 1662.0a 242.0b 367.5a –c – – –
5083.0 192. 8 346.8 650.2 998.5 177.5 178.2 13.8 – – –
–c 10190.5
12.2b 20123.3
14.6a 10653.1
–c 6633.3
–c 9086.8
3.1
In each row, difference (p < 0.01) between means (a–e) for grape varieties (–: not detectable). TPC (total phenolic content) expressed as gallic acid equivalent (GAE), TAA (total antioxidant activity) expressed as ascorbic acid equivalent (AAE).
O. Sagdic et al. / Food Chemistry 126 (2011) 1749–1758
2.8. Statistical analysis Data were analysed using one way analysis of variance (ANOVA) through the general linear models (GLM) procedure of the statistical analysis software (SPSS 10.01). Significant differences between the means were further analysed using the Tukey Test. Bivariate correlations between variables were analysed by Pearson’s test using Minitab 14.0 software.
extracts of grape cultivars had strong TAA (Table 1). Significant differences (p < 0.05) were found in the TAAs of the grape cultivars. Emir and Narince were determined to have the lowest level of TAA, while the extracts of red cultivars; Gamay, Kalecik karasi and Okuzgozu had the highest, with an average value of 225.0 mg AAE/g extract.
160
128
(mg AAE/g)
four different concentrations (0.0%, 2.0%, 5.0% and 10.0%, based on 15 mL of fruit juice). Then, each group was inoculated with the previously activated and reproduced osmophilic yeasts; Z. rouxii and Z. bailii (106–107 cfu/mL) at 1.0% concentration and incubated in refrigerator at 7 ± 1 °C (the storage temperature of juice) for 2, 6, 18, 24, 48, 72 and 120 h. The yeasts were enumerated in the 2, 6, 18, 24, 48, 72 and 120 h, in pour plates of Malt Extract Agar (Merck, Germany), after pipetting of 0.1 mL of prepared serial decimal solutions (0.85% NaCl) onto surface of the agar plates and incubation at 25 °C for 24–48 h.
Total antioxidant activity
1752
96 y = 0.351x + 37.94
64
R² = 0.995
32 3. Results and discussion
0
3.1. Total extract yield, phenolic content, antioxidant activity and antiradical activity
100
150
200
250
300
Total phenolic content (mg GAE/g) 450
360
y = -1.401x + 493.0
,µ g/g) 50
(IC
DPPH
R² = 0.915
270
180
90
0 50
100
150
200
250
300
Total phenolic content (mg GAE/g) 450
DPPH
360
(IC 50 µ g/g)
Total extract yield, total phenolic content, antiradical and antioxidant activities of the ethanolic pomace extracts of commercial Turkish grape cultivars were determined and the results were presented in Table 1. The extract yields ranged from 1.14% to 4.58%. Amongst the grape cultivars; Emir extract had the highest extract yields, whereas Narince extract had the lowest one. The research showed differences in the mass amounts of total phenolic compounds amongst the selected grape cultivars, as expected. The total phenolic contents (TPC) of the extracts were determined to range from 75.5 to 281.4 mg GAE/g dry extract with an average value of 191.2 mg GAE/dry extract. Extracts of white grape cultivars; Emir and Narince were determined to have the lower level of total phenolics, while the extracts of red cultivars; Gamay, Kalecik karasi and Okuzgozu had the higher. Therefore, highly pigmented red cultivars; Gamay, Kalecik karasi and Okuzgozu can be emphasised as cultivars with grape extracts rich in phenolic compounds. These results were in agreement with the findings of Katalinic et al. (2010) who studied the total phenolic compounds of different grape cultivars. Generally, they determined the red grape cultivars to contain higher amounts of total phenolics than white cultivars, especially in the grape skin extracts. On the other hand, the TPC results for the extracts of grape cultivars in this study were different from those reported in literature. Gokturk-Baydar, Ozkan, and Sagdic (2004) reported that the total phenolic contents of solvent (ethyl acetate:methanol:water) extract of Narince was 627.9 mg GAE/g. Ozkan, Sagdic, Gokturk-Baydar and Karamahmutoglu (2004) determined the total phenolic contents of Emir and Kalecik karasi pomace extracts to be 68.8 and 96.3 mg GAE/g. These differences in total phenolic contents might be due to the difference between the extraction methods; in fact, phenolic content of grape pomace extracts are expected to strongly depend on extraction conditions as well as the solvent used (Ozcan, 2006). Therefore, comparison of results with literature data was difficult. The total antioxidant activity (TAA) of the ethanolic extracts of grape cultivars tested was determined through the phosphomolybdenum assay. This method is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and the formation of green Mo (V) complexes with a maximal absorption at 695 nm (Prieto, Pineda, & Aguilar, 1999). The results indicated that the ethanolic
50
y = -3.979x + 643.4 R² = 0.915
270
180
90
0 50
70
90
1 10
130
150
Total antioxidant activity (mg AAE/g) Fig. 1. Bivariate correlations between total phenolic content (TPC), total antioxidant activity (TAA) and DPPH antiradical activity.
O. Sagdic et al. / Food Chemistry 126 (2011) 1749–1758
Pearson’s test was used to analyse bivariate correlations between the values of TPC and TAA. The TPC exhibited a significantly linear correlation with TAA (R = 0.998; p < 0.01) for extracts of grape cultivars. The correlation (R2 = 0.995) indicated that 95.5% of the phenolic content in the extracts were accounted for total antioxidant activity (Fig. 1). The results were consistent with previous reports because some authors have shown that high total phenol content increases the antioxidant activity (Kumaran & Karunakaran, 2007). The free radical-scavenging of ethanolic extracts of grape cultivars tested were determined using the DPPH method, which is a useful method to investigate the free radical-scavenging activities of the phenolic compounds. Free radical-scavenging assays may provide information on how capable an antioxidant is in preventing reactive radical species from reaching lipoproteins, polyunsaturated fatty acids, DNA, amino acids, proteins and sugars in biological and food systems (Katalinic et al., 2010). Amongst the extracts of grape cultivars, those of the white grape cultivars; Emir and Narince were determined to have the highest level of DPPH, on
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a lg analyte basis (IC50 = 428.1 and 245.6 lg/g). It was reported by Katalinic et al. (2010) that antioxidants can deactivate or scavenge stable free DPPH radical by two major mechanisms: by reduction via electron transfer or by hydrogen atom transfer that may occur also in parallel and steric accessibility is one of the major determinants of the reaction. The ethanolic grape pomace extracts of selected, both red and white grape cultivars could interact with the stable free DPPH radicals efficiently and quickly, with an average IC50 of 105.2 lg/g. Differences for DPPH IC50 between the extracts of grape cultivars could be probably due to differences in polyphenolic content of analysed extracts. Fig. 1 indicates a significant negative correlations between TPC and IC50 (p < 0.01; R = 0.957) and between TAA and IC50 (p < 0.01; R = 0.956) values were observed for pomace extracts of grape cultivars, indicating direct contribution of phenolics to this activity. This result was consistent with the observations in the literature (Meda, Lamien, Romito, Millogo, & Nacoulma, 2005). Several factors might have contributed to our results. First, the DPPH radical-scavenging assay determined free antioxidants in
Fig. 2. A chromatogram of phenolic standards and grape pomace extracts at 278 nm in HPLC.
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the extracts, whereas the assay of total phenols with Folin–Ciocalteau reagents determined both free phenolics and bound phenolics in the extracts (Singleton, Orthofer, & Lamuela-Raventos, 1999). It is also known that only flavonoids of a certain molecular structure, particularly those with a certain hydroxyl position, will determine the antioxidant properties present. In general, these properties depend on the ability to donate hydrogen and electrons to a free radical (Meda et al., 2005). Therefore, the negative correlation of IC50 with TPC and TAA were partly due to the presence of other phenolics in the extracts of grape cultivars, which might have contributed to the antioxidant activity. Second, the reactions of antioxidants to the DPPH free radicals were different from their reactions to the Folin–Ciocalteau reagent in the total-phenol assay. The Folin–Ciocalteau reagent is sensitive to a broad range of substrates, which are easily oxidised, but the DPPH free radicals exhibit different sensitivity to various antioxidants, which indicate fast, intermediate, or slow kinetic reactions to the DPPH free radicals (Yang, Paulino, Janke-Stedronsky, & Abawi, 2007). Third, some phenolic antioxidants reacting strongly to the Folin–Ciocalteau reagent may not react to the DPPH free radicals (Yang et al., 2007). 3.2. Identification of phenolic compounds by RP-HPLC It is obvious that the total phenolic content measured by the Folin–Ciocalteau procedure does not give a full picture of the qualification or quantification of the phenolic constituents in the grape pomace extracts (Wojdylo, Oszmianski, & Czemerys, 2007). Therefore, the phenolic acids, flavonoids and stilbenes in the extracts of grape cultivars were determined by the HPLC method and the HPLC results are presented in Table 1. Eighteen phenolic compounds; Chlorogenic acid, gallic acid, protocatechuic acid, caffeic acid, p-coumaric acid, o-coumaric acid, ferulic acid, trans-cinnamic acid, vanillin, (+)-catechin, ()-epicatechin, kaempferol, quercetin, rutin, eriodictyol, hesperidin, apigenin and trans-resveratrol were identified by comparison with the retention times and UV spectra of authentic standards analysed under identical conditions, while the quantitative data was calculated from their respective calibration curves. Chromatograms of the phenolic standards and the phenolic constituents of the extracts are shown in Fig. 2. As a general categorisation proposed by Shi et al. (2003) phenolic compounds in grape can be divided into two groups: phenolic
acids (precursors of flavonoids) and flavonoids. The most common phenolic acids encountered in grape are cinnamic acids (coumeric, caffeic, ferulic, chlorogenic and neochlorogenic acids) and benzoic acids (p-hydroxybenzoic, protocatechuic, vanillic and gallic acids) (Ozcan, 2006). Although these results were obtained for grapes, similar results could also be obtained for grape pomace extracts in this study. Of phenolic acids, the major compound present in the extracts was identified to be chlorogenic acid with an average value of 2379.2 lg/g, followed by gallic acid (average 1090.1 lg/g). Amongst flavonoids, (+)-catechin was the major compound identified as an average value of 5083 lg/g. The least abundant compound present in the extracts was trans-resveratrol (3.05 lg/g). On the other hand, protocatechuic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, vanillin, apigenin and trans-resveratrol could not be identified in the all extracts tested. Furthermore, none of the extract variety was found to contain vitexin, luteolin and genistein. Gamay and Kalecik karasi extracts were the only grape varieties extracts of which was determined to contain trans-resveratrol. This result should be important because of the great interest that has been devoted to resveratrol and its derivatives (Katalinic et al., 2010). Although there are many reports dealing with phenolic constituents of some grape varieties, data about these compounds in pomace extracts of grape cultivars, especially belonging to Turkish flora have appeared to be very limited. Therefore, the phenolic compounds in the extracts of grape cultivars studied could not be directly compared with those in the literature both qualitatively and quantitatively. Tukey test analysis results revealed that a significant (p < 0.01) difference was found between the extracts of grape cultivars in respect of their phenolic compounds (Table 1). In general, amongst the grape cultivars was Narince extract of which had the highest levels of phenolic compounds, with an average value of 20123.3 lg/g extract. Chlorogenic acid, gallic acid, p-coumaric acid and o-coumaric acid were the most abundant phenolic acids, while (+)-catechin, (-)-epicatechin and eriodictyol were the most abundant flavonoids in Narince extract. Kalecik karasi extract had the highest gallic acid, protocatechuic acid, kaempferol and quercetin than did the other extracts. On the other hand, caffeic acid and ferulic acid were determined to be the most abundant phenolic acids; rutin and hesperidin to be the most abundant flavonoids in Okuzgozu, red grape cultivar. These results indicate that
Table 2 In vitro antifungal activity values (means ± SD) indicated by inhibition zones (mm) formed by grape pomaces and extracts at different concentrations of 1%, 2%, 5% and 10%. Grape variety
Emir
Gamay
Kalecik Karası
Narince
Okuzgozu
a
Concentration (%)
1 2 5 10 1 2 5 10 1 2 5 10 1 2 5 10 1 2 5 10
Pomaces
Extracts
Z. rouxii
Z. bailii
Z. rouxii
Z. bailii
–a – – 11 ± 1.0 – – – 10.5 ± 0.5 – – – 14 ± 1.0 – – – – – – – –
– – – – – – – 7.5 ± 0.5 – – – 9 ± 1.0 – – – – – – – –
– – – 13.5 ± 1.5 – – 10.5 ± 0.5 16 ± 1.0 – – 12.5 ± 0.5 14.5 ± 0.5 – – 13 ± 1.0 15.5 ± 0.5 – – 15 ± 1.0 17.5 ± 0.5
– – – – – – – 17 ± 1.0 – – 11 ± 1.0 15 ± 1.0 – – 14 ± 1.0 18 ± 1.0 – – 9 ± 1.0 12.5 ± 0.5
Not detectable. The detection limit was P12 lg/ml, the minimum amount of pomaces and extracts required to show an inhibition zone in the agar-well diffusion assay.
Table 3 Effect of grape pomace or extracts on Z. rouxii and Z. bailii counts (log cfu/mL) in apple juice at different concentrations aand storage periodsb. Treatments
Control Pomaces Emir
Gamay
Narince
Okuzgozu
Extracts Emir
Gamay
Kalecik Karasi
Narince
Okuzgozu
a b
Z. rouxii
Z .bailii
2h
6h
18 h
24 h
48 h
72 h
120 h
2h
6h
18 h
24 h
48 h
72 h
120 h
0
4.69Ad
5.05Aab
4.91Abc
5.18Aa
4.86Ac
4.42Ae
4.11Af
5.69Ab
6.25Aa
6.11Aa
5.26Ac
4.91Ad
4.93Ad
5.01Ad
2 5 10 2 5 10 2 5 10 2 5 10 2 5 10
4.08Ba 3.82Ca 3.37Da 3.73Ba 3.69Ba 3.24Ca 3.64Ba 3.55Ba 3.18Ca 3.52Ba 3.15Ca 3.10Ca 3.28Ba 3.23Ba 3.21Ba
3.94Ba 3.66Cab 2.69Db 3.69Ba 2.66Cb 2.05Db 3.40Bba 2.93Cb 2.08Db 3.40Ba 3.00Cb 2.69Db 3.21Ba 3.19Ba 3.08Ba
3.62Bb 3.48Bbc 2.25Cc 3.49Bba 1.97Cc –Dc 3.15Bbc 2.09Cc –Dc 3.27Ba 2.81Cc 2.29Dc 3.05Bba 2.69Cb 2.19Db
3.51Bb 3.29Cc 1.98Dd 3.23Bbc –Cd –Cc 2.98Bdc –Cd –Cc 3.18Bba 2.69Cc 1.91Dd 2.88Bbc 2.39Cc 1.92Dc
3.24Bc 2.92Cd 1.69De 3.01Bdc –Cd –Cc 2.87Bd –Cd –Cc 2.86Bbc 2.46Cd –De 2.65Bc 2.03Cd –Dd
3.12Bcd 2.81Cd –Df 2.88Bde –Cd –Cc 2.76Bd –Cd –Cc 2.51Bdc 2.09Ce –De 2.22Bd –Ce –Cd
2.98Bd 2.65Cd –Df 2.69Be –Cd –Cc –Be –Bd –Bc 2.22Bd –Cf –Ce 2.04Bd –Ce –Cd
5.14Ba 5.04Ba 5.03Ba 5.10Ba 5.11Ba 4.69Ca 5.36Ba 5.23CBa 5.11Ca 5.11Ba 4.82Ca 4.82Ca 5.11Ba 5.02Ba 5.00Ba
4.97Bb 4.94CBba 4.78Cb 5.08Bba 4.72Cb 4.62Ca 5.17Bba 5.08Ba 4.69Cb 5.10Ba 4.84CBa 4.67Cba 5.06Bba 4.82Cba 4.72Cb
4.95Bcb 4.79Cbc 4.52Dc 4.98Bba 4.57Cb 4.46Db 5.11Bbac 5.05Ba 4.49Cc 5.01Bba 4.69Ca 4.50Cbc 4.95Bba 4.69Cba 4.44Dc
4.91Bcbd 4.72Cc 4.41Ddc 4.84Bbc 4.39Cc 4.28Cc 4.94Bbdc 4.53Cb 4.29Cd 4.79Bba 4.55Cba 4.37Cc 4.79Bbc 4.33Cbc 4.16Cd
4.85Acbd 4.50Bd 4.23Cde 4.64Bdc 4.24Cdc 4.08Dd 4.82Aedc 4.44Bb 4.02Ce 4.67Abc 4.31Bbc 4.22Bdc 4.58Bdc 4.29Cc 3.93De
4.80Acd 4.40Bed 4.17Cfe 4.51Bd 4.15Cd 4.01Ced 4.66Bed 4.30Cb 3.96De 4.42Bdc 4.26Bbc 3.95Cde 4.57Bdc 4.12Cdc 3.82De
4.75Bd 4.26Ce 4.02Df 4.40Bd 3.95Ce 3.87Ce 4.57Be 4.22Cb 3.82Df 4.25Bd 4.07Bc 3.75Ce 4.35Bd 3.90Cd 3.45Df
2 5 10 2 5 10 2 5 10 2 5 10 2 5 10
3.57Ba 3.51Ba 2.69Ca 3.66Ba 3.56Ba 2.69Ca 3.66Ba 3.66Ba 2.52Ca 3.61Ba 3.46CBa 3.32Ca 3.18Ba 3.02Ca 3.03Ca
3.48Ba 3.17Cb 2.03Db 3.48Bb 2.54Cb 1.96Db 3.28Bb 2.26Cb 1.98Db 3.08Bb 2.96Bba 2.14Cb 3.01Bba 2.77Cb 2.09Db
3.39Bba 3.08Cb –Dc 3.30Bc –Cc –Cc 3.10Bb –Cc –Cc 2.84Bc 2.60Cbc –Dc 2.88Bb 2.47Cc –Dc
3.25Bbc 2.69Cc –Dc 3.13Bc –Cc –Cc 2.86Bc –Cc –Cc 2.67Bd 2.48Cbc –Dc 2.58Bc 2.19Cd –Dc
3.06Bdc –Cd –Cc 2.88Bd –Cc –Cc 2.48Bd –Cc –Cc 2.44Be 2.11Cc –Dc 2.32Bd 1.94Ce –Dc
2.92Bde –Cd –Cc 2.58Be –Cc –Cc 2.07Be –Cc –Cc 2.13Bf 1.93Cd –D 2.04Be –Cf –Cc
2.83Be –Cd –Cc 2.14Bf –Cc –Cc –Bf –Cc –Cc 1.93Bg –Ce –Cc –Bf –Bf –Bc
5.15Ba 5.10Ba 5.08Ca 5.08Ba 4.98Ba 4.82Ca 5.10Ba 4.71Ca 4.49Da 5.25Ba 5.07Ba 4.82Ca 5.12Ba 5.02Ba 4.69Ca
5.08Bb 4.87Cb 4.69Db 5.01Bba 4.69Cb 4.39Db 5.08Ba 4.59Ca 4.25Db 4.77Bb 4.59Cb 4.19Db 4.71Bb 4.59Cb 4.19Db
4.90Bcb 4.71Ccb 4.45Dc 4.85Bbc 4.48Cc 4.21Dc 4.83Bb 4.37Cb 4.10Dc 4.54Bc 4.39Cc 4.02Dcb 4.52Bc 4.29Ccb 3.95Dc
4.84Bc 4.67Bc 4.19Cd 4.73Bc 4.20Cd 4.04Cc 4.60Bc 4.10Cc 3.85Dc 4.48Bdc 4.33Cdc 3.93Dcd 4.35Bd 4.16Cc 3.51Dd
4.51Bd 4.41Bd 4.15Cd 4.52Bd 4.11Cd 3.84Dd 4.43Bdc 4.08Cc 3.71Dd 4.37Bd 4.17Cde 3.78Dd 4.14Be 3.98Cc 3.30De
4.43Bed 4.33Bed 4.10Ced 4.47Bd 4.06Cd 3.78Dd 4.24Bde 3.89Cd 3.41Dd 4.21Be 4.01Ce 3.49De 4.08Be 3.53Cd 3.19Ce
4.31Be 4.20Be 3.91Ce 4.27Be 3.88Ce 3.54De 4.17Be 3.48Ce 2.94De 3.84Bf 3.62Cf 3.09Df 3.93Bf 3.12Ce 2.83Df
O. Sagdic et al. / Food Chemistry 126 (2011) 1749–1758
Kalecik Karasi
Con.(%)
In each column, difference (A–D) between the concentrations of pomace or extracts of grape cultivars (p < 0.01). In each row, difference (a–g) between the storage periods (p < 0.01). –: Not detectable (
