Changes in the content of water-soluble vitamins in Actinidia chinensis during cold storage J. Serb. Chem. Soc. 81 (6) 623–632 (2016) UDC 582.688.4:577.164.1/.2+54–145.2+ JSCS–4872 621.565.92 Original scientific paper 623 Changes in the content of water-soluble vitamins in Actinidia chinensis during cold storage XIAN-BO ZHU1,2, LIANG PAN2, WU WEI2, JIA-QING PEN2, YIN-WEI QI3 and XIAO-LIN REN1* 1College of Horticulture, Northwest A & F University, Yangling, 712100, Shaanxi, China, 2Institute of Economic Crop Research, Shiyan Academy of Agricultural Sciences, Shiyan, 442714, Hubei, China and 3School of Agriculture Ningxia University, Yinchuan, 750021, Ningxia, China (Received 28 October, revised 18 December 2015, accepted 29 January 2016) Abstract: The effects of cold storage on nine water-soluble vitamins in seven cultivars of Actinidia chinensis (kiwifruit) were assessed using high-perform- ance liquid chromatography. Samples were collected at three time points during cold storage: one day, 30 days, and when edible. It was found that the vitamin C content in most cultivars increased with cold storage time, but there was no consistent increasing or decreasing trend for the other water-soluble vitamins across the cultivars during storage. After one day of cold storage, vitamins B1 and B2 were the most prevalent vitamins in the control (wild) fruit, while vitamins B5 and B6 were most prevalent in the Hongyang and Qihong cultivars. However, B12 was the most prevalent vitamin in the Qihong cultivar after 30 days of cold storage. Vitamins B3, B7, B9, and C were detected at the edible time point in the Huayou, Hongyang and Jingnong-2 cultivars and in the control fruit. The vitamin contents varied significantly among kiwifruit culti- vars following different durations of cold storage. Out of the three durations tested, 30 days in cold storage was the most suitable for the absorption of water-soluble vitamins by A. chinensis. Keywords: vitamin B; kiwifruit; cultivar; storage; vitamin C. INTRODUCTION Water-soluble vitamins are found in very small amounts in edible foods, but they play an essential role in the metabolism of the human body. Their deficiency leads to a variety of clinical abnormalities that range from anemia to growth retardation and neurological disorders.1 Vegetables and fruits are the major diet- ary sources for water-soluble vitamins in humans, and much research has been conducted on the vitamin content of various vegetables and fruits, such as six * Corresponding author. E-mail: caihou2014@163.com doi: 10.2298/JSC151028012Z _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 624 ZHU et al. date palm cultivars, green leafy vegetables, Rhodiola imbricata root, black garlic, and okra.2–6 Changes to the water-soluble vitamin content of black garlic and six date palm cultivars were registered following thermal processing or vegetable storage.2,3,5 Kiwifruits have a unique flavor and are rich in vitamin C, dietary fiber, and a variety of mineral nutrients, making them popular with consumers around the world. The most commonly cultivated types are Actinidia chinensis and A. deli- ciosa. A. chinensis kiwifruits are endemic to China, and the Chinese public generally prefers A. chinensis to A. deliciosa. Although it was found that kiwifruit might contain up to nine water-soluble vitamins, little research has been performed to determine which factors influence the vitamin content and, in particular, what the ideal cold-storage period is for vitamin uptake.7 Water-soluble vitamins can be measured with either liquid chromatography– –mass spectrometry (LC–MS) or high-performance liquid chromatography (HPLC), though HPLC tends to be the more cost-effective method.2,5,7–16 Thus, in this study, the effects of cold storage on the water-soluble vitamin content of 7 A. chinensis cultivars were assessed using HPLC. It is hoped that this research may provide a guide for how to maximize the uptake of water-soluble vitamins by kiwifruit. EXPERIMENTAL Plant materials Seven kiwifruit cultivars were selected for this study: A. chinensis wild kiwifruit (here after referred to as control), Wudang-1, Hongyang, Qihong, Huayou, Jingnong-2, and Cuiyu. The control (wild) kiwifruit were picked on the Qinling Mountains in Shaanxi province. Wudang-1 kiwifruit were obtained from the Institute of Economic Crop Research Center in Shiyan City (32°65′N, 110°79′E), Hubei, China. Hongyang and Qihong kiwifruit were retri- eved from two well-managed orchards in Meixian (34°29′N, 107°76′E), Shaanxi, China. Hua- you, Jingnong-2 and Cuiyu were acquired from the Kiwifruit Experiment Station of the Northwest Agricultural & Forestry University in Shaanxi province. All plant material were picked when the total soluble solids were 6.5–9.5 % of the harvest term and immediately transported to the laboratory, where they were rapidly pre-cooled before being stored under refrigerated conditions.17 As some cultivars took over two months in cold storage (1±0.5 °C) to soften, samples were collected from all cultivars at the picking period (Day 1), after 30 days of storage (Day 30), and then when they reached the edible period (EP) for assessment of the content of water-soluble vitamins. The storage times were as follows: control, 56 days; Wudang-1, 61 days; Qihong, 61 days; Hongyang, 62 days; Cuiyu, 81 days; Huayou, 71 days and Jingnong-2, 61 days. Reagents All chemicals were of analytical reagent grade, and Milli-Q water was distilled and filtered through a 0.22-μm membrane filter. Thiamine hydrochloride (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine hydrochloride (vitamin B6), D-biotin (vitamin B7), folic acid (vitamin B9), cyanocobalamin (vitamin B12), and ascorbic acid (vitamin C) were purchased from Sigma–Aldrich (St. Louis, MO, USA). _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ WATER-SOLUBLE VITAMINS IN KIWIFRUITS 625 Sodium hexanesulfonate and phosphoric acid were purchased from Kermel Chemical Reagent Co., Ltd. (Tianjing, China). HPLC-grade methanol was purchased from ThermoFisher Company (Waltham, USA). Sample preparation Samples (10 g) were prepared from the pulp of five kiwifruits for each cultivar and were then homogenized. Sample extracts were prepared with ultrasonic assistance in 30 mL of HCl (0.005 M) for 30 min at 30 °C. All extracts were centrifuged at 10,000×g for 5 min at 4 °C and then filtered through a 0.22-µm nylon membrane. During all these steps, the samples were protected from light. Three independent replicates were prepared for each sample. Determination of firmness and total soluble solids (TSS) Firmness was measured in five kiwifruits, and the measurements were repeated three times. A TA-XT2i texture analyzer (Stable Microsystems, Godalming, UK) was used to deter- mine firmness using 8 mm diameter samples. The fruits were peeled at opposite side and then the firmness was determined for the peeled position.18 The TSS was assessed by juicing five kiwifruits and then using a PAL-1 refractometer (Atago, Japan). HPLC analysis A Shimadzu SCL-10AVP HPLC system was used. The mobile phase consisted of (A) methanol and (C) 0.005 M sodium hexanesulfonate (phosphoric acid was used to adjust the pH to 3.0). A C18 (YMC-Pack ODS-A, 4.6×250 mm, 5 μm) column was used for the deter- mination of water-soluble vitamins. In all cases, the flow rate was 0.7 mL min-1 using isocra- tic elution (A:C = 3:7). The column temperature was set at 25 °C, and injection volume was 10 μL. Dual wavelength recording was used detect the vitamins simultaneously under the same conditions: B5 and B7 were detected at 210 nm, while the other seven water-soluble vitamins were detected at 270nm.19 HPLC chromatograms are shown in Supplementary material to this paper (Fig. S-1) Statistical analysis IBM SPSS Statistics software v.20 and SigmaPlot 12.0 were employed for data elabor- ation and statistical analysis. All analyses were run in three duplicates with five kiwifruits of each cultivar used in each duplicate and the results are expressed as means ± standard devi- ation (SD). One-way analysis of variance (ANOVA) was employed to assess the differences in the vitamin contents for different storage times. Differences (Duncan) tests were considered statistically significant at p < 0.05. RESULTS AND DISCUSSION Determination of firmness and total soluble solids Firmness is usually used as a proxy for the degree of maturity of kiwifruit, while the total soluble solids (TSS) is an important indicator of kiwifruit quality.17 When kiwifruit picked period follow 6.5≤TSS≤7.5, and the firmness ≤1 N mm–1, there was no need to store. The first period followed 6.5≤TSS≤7.5, the second was stored 30 days, and the last period was when the firmness was ≤1 N mm–1 (Table I). When the sampled kiwifruit were separated from the vine, their firmness decreased, while TSS increased in all cultivars with the duration of storage (Table I). _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 626 ZHU et al. TABLE I. Changes in firmness and total soluble solids (TSS) during cold storage of kiwifruit; each value represents the mean ± standard deviation. Different letters (a–c) within a row indicate significant differences at p < 0.05 over the three storage time points for each cultivar Cultivar Firmness, N mm-1 Total soluble solids, % Day 1 Day 30 Edible period Day 1 Day 30 Edible period Control 6.47±0.07a 1.58±0.06b 0.36±0.02c 6.46±0.05c 12.9±0.13a 12.0±0.11b Wudang-1 6.29±0.06a 3.96±0.05b 0.89±0.02c 6.52±0.06c 11.8±0.08b 13.6±0.15a Huayou 6.23±0.05a 5.30±0.07b 0.96±0.07c 6.66±0.07c 9.7±0.12b 12.5±0.12a Cuiyu 7.37±0.08a 7.00±0.05a 0.71±0.03b 7.46±0.15c 11.2±0.08b 16.5±0.12a Jingnong-2 4.77±0.04a 4.38±0.04b 0.44±0.01c 9.40±0.11c 14.1±0.11b 17.2±0.10a Hongyang 5.33±0.06a 5.28±0.07a 0.75±0.02b 6.86±0.16c 11.0±0.10b 17.2±0.10a Qihong 6.59±0.08a 4.37±0.09b 0.97±0.02c 7.12±0.15c 15.3±0.13b 20.0±0.12a Changes in B1, B2, B3, and B5 in cold storage For each of the nine vitamins assessed, the water-soluble vitamin content ranged from undetectable to more than 2500 µg g–1 fresh weight (FW). Out of the nine water-soluble vitamins previously reported in kiwifruit, only B9, B12 and vitamin C were detected in all cultivars across all three periods. In most cultivars, these three vitamins became more prevalent in the fruit over time. Vitamin B1 was not detected on Day 1 in the Wudang-1 samples, while it was only detected on Day 30 in the Cuiyu samples and only on Day 1 in the Qihong fruit (Fig. 1A). In the Jingnong-2 cultivar, the vitamin B1 content rem- ained constant over the three time points, indicating that cold storage had no effect on the B1 content in this cultivar. Previous studies found that the B1 con- tent increased over time, a phenomenon that was observed in the present study for the Wudang-1 and Hongyang cultivars. In contrast, it was found that the B1 content in the control and Huayou fruit decreased with storage.3 Vitamin B2 can be destroyed by light and heat; therefore, rapid extraction in a dark room was used to avoid decomposition. The vitamin B2 content in all cultivars across the three points was found to be no higher than 0.1 µg g–1 FW (Fig.1B), except for in the Control cultivar on Day 1. The Vitamin B2 content increased in Huayou fruit over time, perhaps due to microbial growth.20 The B2 content decreased in the control and Hongyang cultivars, while the others exhi- bited no significant differences across the three time points despite a low vitamin B2 content. This may be because B2 participates in physiological metabolism in storage.21 The control, Wudang-1 and Qihong cultivars had the highest vitamin B3 content on Day 30, while the Huayou, Cuiyu, and Jingnong-2 cultivars experi- enced an increase in B3 content across the three time points (Fig.1 C). The max- imum B3 content (over 0.2 µg g–1 FW) was found in the Huayou fruit at the edible period. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ WATER-SOLUBLE VITAMINS IN KIWIFRUITS 627 Fig. 1. Changes in the B1 (A), B2 (B), B3 (C) and B5 (D) contents of A. chinensis during cold storage. Interestingly, the vitamin B5 content increased in three cultivars over time and decreased in three cultivars over time. Jingnong-2 was found to have the highest B5 content with more than 8 µg g–1 FW on Day 30 (Fig. 1D). Changes of B6, B7, B9, B12 and vitamin C in cold storage Of the seven cultivars, the vitamin B6 content was not detectable in only two cultivars: Huayou on Day 1 and Cuiyu on Day 1 and at the EP (Fig. 2A). The B6 _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 628 ZHU et al. Fig. 2. Changes in the B6 (A), B7 (B), B9 (C), B12 (D) and vitamin C (E) contents of A. chinensis during cold storage. Each value represents the mean ± standard deviation. Different letters (a–c) within a row indicate significant differences at the p < 0.05 level over the three storage time points for each cultivar. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ WATER-SOLUBLE VITAMINS IN KIWIFRUITS 629 content in some cultivars was not detected, which could be attributed to the hyd- roxylation of B6 in the presence of vitamin C. In general, except for Huayou, the B6 contents of the other six cultivars were lower at the edible time point than at Day 30. Similar results were previously found with the storage of green leafy vegetables.3 Biotin (vitamin B7) is involved in amino acid catabolism, gluconeogenesis, and fatty acid synthesis. The vitamin B7 content of four cultivars increased with the duration of cold storage. Jingnong-2, on the other hand, had the highest B7 content on Day 30, while the other cultivars had their lowest B7 contents at that time (Fig. 2B). The highest B7 content registered in this study appeared in the Hongyang fruit and was nearly 20 µg g–1 FW. Rapid increases in the vitamin B9 content were found from Day 30 to the edible time point in the control, Wudang-1 and Huayou kiwifruit (Fig. 2C). In the same period, a rapid decrease in the Qihong cultivar was found. The highest B9 content (nearly 0.5 µg g–1 FW) was found in the control fruit at the EP. For most cultivars, the vitamin B12 content increased with increasing dur- ation of cold storage. The exceptions were Wudang-1, where B12 decreased over time, and Huayou and Qihong, where Day 30 exhibited the highest B12 content (Fig. 2D). Vitamin C was found to be abundant in kiwifruit, although no regular pattern was found across the seven cultivars with cold storage. For example, for the con- trol, Wudang-1 and Qihong cultivars, the lowest vitamin C content was detected on Day 30, while the content decreased over time for the Huayou fruit (Fig. 2E). For the other cultivars, the vitamin C content increased with increasing duration of storage. For all the studied A. chinensis cultivars, Day 30 was found to be the time point most suitable for absorbing water-soluble vitamins, as most of them could be detected in the seven cultivars at this time (Figs. 1 and 2). Although all water- soluble vitamins were detected at all three time points in the control (wild) kiwi- fruit, these fruit had the lowest TSS content (Table I), making the fruit less palat- able for human consumption. Previous studies found a significant depletion of vitamin C in fruit juice with cold storage.22 In the present study, however, the vitamin C content increased for many cultivars. This phenomenon was also found in pea leaves, perhaps due to the bound form of vitamin C being released at dif- ferent times.3 Similarly, the effects of cold storage on the other water-soluble vit- amins were not similar to those seen in fruit juice; this perhaps be due to water- soluble vitamins in living plant materials having some activity.3,22 The Hongyang cultivar is one of the most popular cultivars and of high eco- nomic importance in northwest China, and it was found to be rich in all water- soluble vitamins (Figs. 1 and 2). It is therefore the most suitable cultivar of the seven cultivars for absorbing water-soluble vitamins. Water-soluble vitamins not _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 630 ZHU et al. detectable in some cultivars at some time points, which corresponded to LC–MS results.10 The B1, B2, B5 and B6 contents in some cultivars increased over time. Similar results were obtained during vegetables storage, indicating that the levels of these three vitamins reflect not only degradation but also synthesis.3 Some previous studies used LC–MS to detect water-soluble vitamins in green and yellow kiwifruit.7 In the present study, the contents of B6 and B9 found were lower and that of B7 were higher than the values found in previous studies, perhaps due to differences in varieties studied or cultivation environments.7 Interestingly, the B7 contents found in the two red kiwifruit (Hongyang and Qihong cultivars) were higher than those of green kiwi, red kiwifruit may have more efficient synthetic pathways in regards to this vitamin.7 In green leafy vegetables, the contents of several water-soluble vitamins change during storage, but B7 was not one of these. In fact, sometimes B7 could not be detected at all for it is often in the bound form and not often in the free form.3 Vitamin B9 was detected in all kiwifruit cultivars across the three time points. This is in contrast to stored vegetables, which do not have abundant vit- amin B9.3 Similarly, stored vegetables have very low vitamin B12 contents, while A. chinensis cultivars contain considerable levels of B12.3 CONCLUSIONS The contents of nine water-soluble vitamins changed over time in Actinidia chinensis. Different cultivars exhibited different trends for different vitamins. For example, vitamin C in most cultivars increased with storage, while the contents of the other vitamins showed no similar change trends. Some vitamins were undetectable in some cultivars at one or two time points. In general, more water- soluble vitamins could be detected following storage for 30 days, indicating this is the optimal duration of cold storage for the uptake of water-soluble vitamins. SUPPLEMENTARY MATERIAL HPLC chromatograms of a standard mixture for the studied water-soluble vitamins are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. This work was supported by the earmarked fund for Modern Agro- industry Technology Research System of China. We would like to thank Dr. Jiangtao Suo for material assistance. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ WATER-SOLUBLE VITAMINS IN KIWIFRUITS 631 И З В О Д ПРОМЕНА САДРЖАЈА ВИТАМИНА РАСТВОРНИХ У ВОДИ У БИЉЦИ Actinidia chinensis ТОКОМ ЧУВАЊА НА ХЛАДНОМ XIAN-BO ZHU1,2, LIANG PAN2, WU WEI2, JIA-QING PEN2, YIN-WEI QI3 и XIAO-LIN REN1 1College of Horticulture, Northwest A & F University, Yangling, 712100, Shaanxi, China, 2Institute of Economic Crop Research, Shiyan Academy of Agricultural Sciences, Shiyan, 442714, Hubei, China и 3School of Agriculture Ningxia University, Yinchuan, 750021, Ningxia, China Испитана је промена у садржају девет витамина растворних у води седам сорти Actinidia chinensis (киви) током чувања на хладном, применом методе HPLC. Сакупљена су три узорка током чувања: првог дана, после 30 дана и у тренутку сазревања за јело. Садржај витамина Ц је порастао у већини сорти током чувања, али за друге витамине није нађена правилност у промени. Након једног дана чувања на хладном, витамини Б1 и Б2 су били доминантни у контролном (дивљем) плоду, док су витамини Б5 и Б6 били доминантни у сортама Hongyang и Qihong. После 30 дана чувања, витамин Б12 је постао главни у сорти Qihong. Витамини Б3, Б7, Б9 и Ц су детектовани тек у зрелим плодовима сорти Huayou, Hongyang, Jingnong-2 и у контролној сорти. Садржај витамина је јако варирао између сорти кивија у различитим периодима чувања. Упоређивањем три вре- менска периода чувања, закључено је да је, након 30 дана чувања, најповољнија комби- нација витамина A. chinensis за апсорпцију. (Примљено 28. октобра, ревидирано 18. децембра 2015, прихваћено 29. јануара 2016) REFERENCES 1. B. Klejdus, J. Petrlová, D. Potěšil, V. Adam, R. Mikelová, J. Vacek, R. Kizek, V. Kubáň, Anal. Chim. Acta 520 (2004) 57 2. J. Aslam, S. H. Khan, S. A. Khan, J. Saudi Chem. Soc. 17 (2013) 9 3. J. Santos, J. A. Mendiola, M. B. P. P. Oliveira, E. Ibanez, M. Herrero, J. Chromatogr. A 1261 (2012) 179 4. A. B. 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Available on line at www.shd.org.rs/JSCS/ << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice