ࡱ> 13./0q`ȢbjbjqPqP.::X;JJJJ\K ltLLLLLLM,M,M,)l+l+l+l+l+l+l`mho+l96+|M,66+lLLdlRRR6PLL)lR6)lRR U jUULhL X{JJ.!Umhzl0l)U,xpNfxpUUUUxpUM,/R12M,M,M,+l+luRM,M,M,l6666-=$ = Fermentation rate in White Riesling (Vitis vinifera L.) grape juice as affected by vineyard nitrogen fertilization Marko Karoglan1, Luna Maslov1, Ana Jeromel1, Mirela Osre ak1, Bernard Kozina1, Josip Marijanovi2 1Agronomski fakultet, Sveu iliate u Zagrebu, Zavod za vinogradarstvo i vinarstvo, Svetoimunska cesta 25, Zagreb (mkaroglan@agr.hr) 2student Agronomskog fakulteta u Zagrebu Summary Nitrogen (N) fertilizer (urea) was applied by hand to vineyard of White Riesling grapes at rates of 0, 23, 70 or 117 kg N/ha/year from 2006 to 2009. Fermentation rate increased with increasing N fertilization, although only one of four juices from 0 kg N/ha completed fermentation, while only two of four 23 kg N/ha completed fermentation. All juices from 70 and 117 kg N/ha completed fermentation, but fermentation rate was unecceptable slow. Long fermentation time was strongly correlated with low free amino nitrogen levels in all juices, varying from 13 to 40 mg/L. Nitrogen fertilization rate affected higher titratable acidity. Key words: fermentation rate, fertilization, grape juice, nitrogen Introduction It is well known that nitrogen (N) is required by yeast for growth and completion of alcoholic fermentation in grape juice (Bisson, 1991). Sluggish or stuck fermentations may result from nitrogen deficiencies of grape juice (Bell et al., 1979; Ingledew and Kunkee, 1985). Many producers utilize to fertilize the vineyard to avoid this problems, although this does not always assure sufficient content of nitrogen compounds in the grape (Bell and Henschke, 2005). Agenbach (1977) reported that concentrations greater than 140 mg/L of assimilable nitrogen were required for sufficient yeast growth to complete fermentation. Free amino nitrogen (FAN) content has been used as an indicator of total avaliable nitrogen in juice (Amerine and Ough, 1980), although chemical determinations of FAN do not discriminate between those free amino nitrogen containing compounds that can be utilized by Saccharomyces cerevisae for thier nitrogen content and those that cannot. Concentration of nitrogenous compounds in grape juice is influenced by viticultural practice such as vineyard nitrogen fertilization (Agenbach, 1977; Bell et al., 1979), cultivar (Huang and Ough, 1989), rootstock (Huang and Ough, 1989) and growing season (Spayd et al., 1994). Many studies examined the impact of vineyard nitrogen fertilization on the fermentation kinetics (Agenbach, 1977; Bell et al., 1979). Overall fermentation rate is lineary related to must total nitrogen. Application of N fertilizer to Thompson Seedless grapevines increased fermentation rate of juices which was found to be related to must total N concentration (Bell et al., 1979). Pre-harvest application of N fertilizer increased juice total N concentration, and fermentation time to dryness decreased lineary as the total N concentration in juice increased. However, this viticultural practice could create some problems as the high vineyard nitrogen application can result in reduced grape colour and total soluble solids concentration. Difficult fermentations of Riesling musts in north-western region of Croatia are fairly frequent. Spayd et al. (1994) reported that N concentration in Riesling juice are increased by even moderate (56 kg N/ha) applications of N fertilizer. The purpose of this study was to investigate the impact of the N fertilizer applications on fermentation rate in Riesling must. Materials and methods Research was laid out during four growing seasons (2006-2009). Grape harvest as well as vinification process happened in the experimental field of Department of enology and viticulture, Faculty of Agriculture in Zagreb, which is placed on the hills near Zagreb. The White Riesling grapevines were grafted on Vitis Berlandieri x Vitis Riparia SO4 rootstock. Nitrogen application rates were 0, 23, 70 or 117 kg N/ha and were replicated three times in the filed using a randomized complete block design. Each treatment comprised 36 grapevines, so there were total 124 White Riesling grapevines included in the experiment. Vines were spaced 1.2 m apart within rows and 2.1 m apart between rows. Nitrogen in the form of urea was applied by hand within 30-cm band on each side of each experimental row. Fertilizer application were made in spring, immediately after bloom. The fruits were harvested according to usual technological ripeness, and before harvest the vines were berry-sampled several times to determine maturity. Each replication was destemed and crushed separately, and immediately pressed. The juice was collected in 25-L glass containers, SO2 added and the juice settled per 24 hours. The samples of must were taken and analyzed before fermentation started. The musts were analyzed for total acidity and sugar content according to the official O.I.V. methods (2001). Free amino nitrogen (FAN) content was determined according to the NOPA procedure (Butzke and Dukes, 1998) The clear juice was racked from the lees, yeast inoculum of Saccharomyces cervisiae 228B was added, and the must was fermented at 16C. Fermentation progress was determined by measuring changes in sugar content. Fermentation rate were determined as days for the must to ferment to <1 g/L of residual sugar. Data were analyzed by ANOVA, with nitrogen fertilization rate as variability source. Results and discussion Must composition at harvest: Nitrogen fertilization didn't delayed fruit maturation regarding soluble solids content (Table 1). Moreover, the sugar content increased when nitrogen fertilizer was applied. In 2006 and 2009 the sugar content was the lowest in control musts, without nitrogen fertilization. Fertilization with 23 kg N/ha influenced the highest must sugar content in three of four experimental years, but statistical significance showed up only in 2006 and 2009 years. Peacock et al. (1991) stated that nitrogen fertilization generally influenced lower sugar content in grape must. Christensen et al. (1994) also found that fertilization with 112 kg N/ha reduced sugar content in grape juice. But there are also authors finding no differences in grape sugar content due to rate of nitrogen fertilization (Bell et al., 1979). Nitrogen fertilization rate affected higher titratable acidity (TA). The lowest content of titratable acidity was found in control musts in all growing seasons, but only in 2006 with statistical significancy. Bell et al. (1979) reported that total acidity increased significantly with increasing nitrogen fertilization. The explanation is the probable increase in vine vigor and leaf surface area due to increasing nitrogen fertilization. So, there is an increased production of acids in leaves and subsequent translocation to the grapes. The increased acidity may be due also to the denser leaf canopy that accompanied nitrogen fertilization, resulting in greater shading of fruit and lower fruit temperature than on unfertilized vines. Christensen et al. (1994) investigated nitrogen fertilization among 4 cultivars, and reported higher total acidity content but only in Grenache and Chenin blanc juices. According to Bavaresco et al. (2001) nitrogen fertilization affected higher acidity content, but without significant statistical differences. Table 1. Effect of nitrogen fertilization rate on sugar content of White Riesling grape juice, 2006-2009. kgN/ha2006200720082009095.788.399.786.392.523104.790.3100.092.096.870100.787.798.789.094.0117103.388.3100.787.795.0 Table 2. Effect of nitrogen fertilization rate on total acidity of White Riesling grape juice, 2006-2009. kgN/ha2006200720082009010.158.667.178.978.742313.638.927.318.989.717013.409.437.359.369.8911711.179.577.209.269.30 Free amino nitrogen (FAN): In all four years, when compared with no N fertilization, juice FAN concentrations were higher when N fertilizer was applied, esspecially rates of 70 and 117 kg N/ha (Table 2). Statistical significant differences were observed in all years except 2007. Linear increase in FAN concentration with increasing N fertilization was detected only in 2009 year. In the other three growing seasons fertilization with 117 kg N/ha had no significant effect compared with 70 kg N/ha. However, Spayd et al. (1994) found that juice FAN concentration at least doubled when 56 kg N/ha were aplied to the vines, and increased lineary till maximum fertilization rate of 224 kg N/ha. It is also worth to note that the FAN concentrations in this investigation are significantly lower than those recommended for completion of alcoholic fermentation. Several authors (Agenbach, 1977; Ingledew and Kunkee, 1985) found that the level of 140 mg N/L (meaning yeast assimilable nitrogen (YAN), that can be assimilated by wine yeasts) is necessary for completion of alcoholic fermentation. Spayd et al. (1996) examined 142 must samples of White Riesling from Washington state, and found an average of 156 mg/L of FAN. The same authors stated that in 96% samples of White Riesling was found less than 400 mg/L of FAN, and in 77% samples even less than 200 mg/l, claiming that White Riesling among all white cultivars had the lowest FAN concentration in must. When comparing with our results it is obvious that the FAN content in White Riesling musts is significantly lower, and far away from recommended and necessary concentrations for good alcoholic fermentation. Table 3. Effect of rate of nitrogen fertilization on free amino nitrogen in must (mg/L) of White Riesling, 2006-2009. kg N/ha2006200720082009013.2224,0527,2314,5119,752323.9225,5830,1118,9524,647029,0234,2840,5219,0330,7111721,7930,9237,2522,5728,13 Fermentation rates: The effect of nitrogen fertilization is shown in Figures 1-4. It is obvious that only in 2007 all must completed fermentation to dryness, with 70 and 117 kg N/ha in 29 days, and 0 and 23 kg N/ha in 37 days. That was the only year in which control treatment (without N fertilization) completed fermentation. Must treated with 23 kg N/ha completed fermentation only in 2006 and 2007 years, while 70 and 117 kg N/ha completed fermentations in all experimental years. Average number of days needed for completing fermentation for 70 and 117 kg N/ha treatments was 32 and 34 days respectively, which can be considered as a quite long term fermentation. Other two treatments showed no capability for completing fermentation, which was prolonged for even 52 or 56 days, and stuck with 6-8 g/L of residual sugar. The length of fermentation time is not strongly related to the FAN level in the juice, especially when looking year by year FAN levels. The explanation is in fact that FAN as a chemical definition do not discriminate between those free amino nitrogen containing compounds that can be utilized by Saccharomyces cerevisae for their nitrogen content and those that cannot.  SHAPE \* MERGEFORMAT   SHAPE \* MERGEFORMAT   SHAPE \* MERGEFORMAT   SHAPE \* MERGEFORMAT  Conclusions Nitrogen fertilization delayed fruit maturity as indicated by total acidity concentrations. The impact of nitrogen fertilization on FAN concentration varied from year to year, but was higher in fertilization treatments. In fact, in all treatments the FAN concentrations were extremely low, that can not assure adequate conditions for completing alcoholic fermentations in the appropriate time. Application of sufficient N fertilizer in the soil immediately after bloom, and in form of urea to assure complete fermentation of White Riesling juice produced from Zagreb vineyard hills is not possible, especially when considering very high partitioning of clay in the soil. It is necessary to examine possibility of leaf (foliar) application of N fertilizers, as well as other timings of nitrogen fertilizers application. Literature Agenbach, W. A. A. (1977). A study of must nitrogen content in relation to incomplete fermentations, yeast production and fermentation activity. In Proceedings of South African Society for Enology and Viticulture, Cape Town Amerine, M. A., Ough C. S. (1980). Methods for analysis of musts and wines. John Wiley and Sons, New York, 794p Bavaresco, L., Pezzutto S., Ragga A., Ferrari F., Trevisan M. (2001). Effect of nitrogen supply on trans-resveratrol concentration in berries of Vitis vinifera L. cv. Cabernet Sauvignon. Research note. Vitis. 40 (4): 229-230 Bell, A. A., Ough C. S., Kliewer W. M. (1979). Effects on must and wine composition, rates of fermentation and wine quality of nitrogen fertilization Vitis vinifera var. Thompson Seedless grapevines. Americain Journal of Enology and Viticulture. 30 (2):124-129 Bell, S.-J., Henschke, P. A. (2005). Implications of nitrogen nutrition for grapes, fermentation and wine. Australian Journal of Grape and Wine Research. 11(3): 242-295 Bisson, L. F. (1991). Influence of nitrogen on Yeasts and fermentations of grapes. International Symposium on Nitrogen in Grapes and Wine, J. M. Rantz (Ed.). pp 78-89. Am. Soc. Enol. Vitic., Davis, CA Butzke C. E., Dukes B. C. (1998): NOPA Procedure, Concentration of Primary Amino Acids in Grape Juice, UC Davis Cooperative Extension Christensen, L. P., Bianchi M. L., Peacock W. L., Hirschfelt D. J. (1994). Effect of nitrogen fertilizer timing and rate on inorganic nitrogen status, fruit composition, and yield of grapevines. Americain Journal of Enology and Viticulture. 45 (4): 377-387 Huang, Z., Ough C. S. (1989). Effect of vineyard locations, varieties, and rootstocks on the juice amino acid composition of several cultivars. American Journal of Enology and Viticulture. 40(2): 135-139 Ingledew, W. M., Kunkee R. E. (1985). Factros influencing sluggish fermentations of grape juice. American Journal of Enology and Viticulture. 36: 65-76 O.I.V. International Code of Oenological Practices, edition 2001, Paris Peacock, W. L., Christensen L. P., Hirschfelt D. J. (1991). Influence of timing of nitrogen fertilizer application on grapevines in the San Joaquin Valley. American Journal of Enology and Viticulture. 42(4): 322-326 Spayd, S. E., Wample R. L., Evans R. G., Stevens R. G., Seymour B. J., Nagel C. W. (1994). Nitrogen fertilization of White Riesling grapes in Washington. Must and wine composition. American Journal of Enology and Viticulture. 45(1): 34-42 Utjecaj duai ne gnojidbe na brzinu i tijek fermentacije moata Rizlinga rajnskog (Vitis vinifera L.) Sa~etak Duai no gnojivo (urea) aplicirano je ru no u vinograd Rizlinga rajnskog u koli inama od 0, 23, 70 ili 117 kg N/ha u periodu od 2006 do 2009 godine. Brzina fermentacija je rasla sa intenziviranjem duai ne gnojidbe, iako su samo jedan moat od 0 kg N/ha, odnosno dva moata od 23 kg N/ha uspjeano odfermentirala. Svi moatevi od 70 i 117 kg N/ha uspjeano su odfermentirali, ali je fermentacija trjala neprihvatljivo dugo. Produ~eno trajanje fermentacije u uskoj je vezi sa niskom razinom slobodnog amino duaika u moatu, koji je varirao od 13 do 40 mg/L. Duai na gnojidba utjecala je i na viau razinu ukupne kiselosti u moatu. 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Fermentation rate, White Riesling, 2006 0 50 100 150 200 250 300 28.09. 5.10. 11.10. 17.10. 23.10. 30.10. 2.11. 6.11. 10.11. 15.11. 19.11. 22.11. g/L 0 23 70 117 Graf 2. Fermentation rate, White Riesling, 2007. 0 50 100 150 200 250 12.09. 24.09. 3.10. 11.10. 16.10. 19.10. g/L 0 23 70 117 Graf 3. Fermentation rate, White Riesling, 2008. 0 50 100 150 200 250 16.10. 20.10. 26.10. 3.11. 10.11. 15.11. 21.11. 27.11. 2.12. 7.12. 12.12. g/L 0 23 70 117 Graf 4. Fermentation rate, White Riesling, 2009. 0 50 100 150 200 250 5.10. 9.10. 12.10. 21.10. 30.10. 4.11. 9.11. 13.11. g/L 0 23 70 117 cdhimnuv|} àʠˠàɠˠΠРѠӠՠנ٠۠ޠ!#&(+-35;=BDJLRTZ\_abdfhjloqġ)he5B*CJOJQJ\^JaJph)he5B*CJOJQJ\^JaJphhe#heB*CJOJQJ^JaJphKˠϠРҠӠ֠נڠ۠ߠ"#'(,-45<=CCDKLST[\`acdghklpqšơ͡Ρա֡ܡݡ ġơ̡Ρԡ֡ۡݡ !#&(XZ[]_adfiknpsuz|ĢƢǢhRL)he5B*CJOJQJ\^JaJph)he5B*CJOJQJ\^JaJph#heB*CJOJQJ^JaJphheH"#'(YZ\]`aefjkoptu{|ŢƢǢȢ$a$gduǢȢ h0PhX,1h. 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