Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
NITROGEN STATUS OF GRAPE JUICE AS AN INDICATOR OF WINE QUALITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0200531
Grant No.
(N/A)
Project No.
VA-135724
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2004
Project End Date
Jun 30, 2009
Grant Year
(N/A)
Project Director
Zoecklein, B. W.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
FOOD SCIENCE AND TECHNOLOGY
Non Technical Summary
Winemakers must seek to maximize production and quality, especially as international marketing of wine and grape products becomes more competitive. It is important for these winemakers to have valid analytical procedures, as well as knowledge of how and when to apply these procedures to better monitor the production of their wines. Develop an analysis procedure for the improvement of wine quality via a rapid method for juice and plant nitrogen.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50211311000100%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
1.Evaluate the relationship between proline/arginine ratios and the results of the standard titration assays. 2.Evaluate the relationship between fermentable nitrogen concentration of the juice and the production of wine volatiles. 3.Develop a simple titration test of arginine that growers and vintners could use to measure nitrogen status of the vine. 4.Evaluate the relationship between the nitrogen status of the vine and the juice analysis of fermentable nitrogen by Formol titration.
Project Methods
Procedure: Five cultivars (Cabernet Sauvignon, Merlot, Cabernet franc, Chardonnay, and Viognier), from vineyards in Virginia and California, will be used. Virginia fruit will be from vines at the AHS Agricultural Research and Extension Center at Winchester. Cabernet franc and Viognier vines grown under three different training systems (Geneva Double Curtain, Smart Dyson, and Vertical Shoot Position) will be compared consisting of six three-vine panels. Three vineyards in California will be used. These will produce Cabernet franc, Cabernet Sauvignon, Merlot, and Chardonnay grown on Vertical Shoot Positioned vines in three regions of the state: Fresno, Paso Robles-Santa Maria, and Napa Valley. Leaf petiole samples will be collected and analyzed by standard procedure each season. Vineyard sampling will be arranged in a randomized complete block design that consists of six replicates of three vine plots. A minimum of 50 berries will be randomly collected from each side of the vineyard canopy from each cultivar, training system, and region at commercial maturity, and immediately frozen and stored at minus 20 C until evaluated. At harvest, shoots per meter row, clusters per shoot, clusters per vine, and vine fruit and berry weight will be determined. Objective 1. Four analytical methods will be utilized: Formol titration (Gump et al., 1999), two spectrophotometric methods (NOPA and ARGOPA), as described by Austin and Butzke (1999) and high performance liquid chromatography (HPLC) for specific amino acids, as described by Zoecklein et al. (1995). Objective 2. In a separate series of fermentations, California Chardonnay grapes (750 pounds) will be harvested and processed. Free-run and press juice will be combined, cold settled (1 C for 24 hours) and racked. Juice will be treated with 250 mg/L of (dimethyldicarbonate) and divided into 16 carboys (11.5L), with 7.3L of juice per container. Juice nitrogen analysis will be as described above. Yeasts will be grown from 5 grams of dry active yeast, rehydrated in sterile water at 45 C for 15 minutes, transferred to sterilized juice, and developed for 10 hours at 30 C prior to inoculation. Four carboys will be randomly selected for each treatment and inoculated with a 3 percent (v/v) actively-growing culture containing 100,000 cell/mL of a single strain of Saccharomyces cerevisiae. Sensory analysis will be performed on each wine lot using triangle comparison sensory analysis, as described by Zoecklein et al. (1995), using a minimum of 40 judges under standard conditions. Objective 3 and 4. A standard addition method with be used to determine quantitative recovery of arginine via Formol analysis, first in model buffered solution, then in grape juices. Methods for the isolation of arginine from juice and petioles will be evaluated using cation exchange. Statistical analysis: One-way analysis of variance and least significant difference (LSD) comparison tests of SAS (SAS Institute, Cary, NC) will be used to statistically interpret differences in means, if any. Significance of the triangle test will be determined from published sources.

Progress 07/01/04 to 06/30/09

Outputs
OUTPUTS: Activities included multiple year analyses of fruit from a number of varieties, grown under different viticultural conditions, at different stages of grape maturity. Over 900 assays have been completed on juices for yeast assimiable nitrogen, free amino nitrogen, ammonia, and arginine. Seven short course programs were conducted involving discussions of these results and the practical impact of this research to the grape and wine industry. Three presentations were given at scientific meetings outlining the findings of this effort. Six Enology Notes, electronic technical briefs, we written and sent outlining the results of this research and the practical impacts. Analysis of the nitrogen components evaluated in this study were correlated to fruit maturity and evaluated using electronic nose technology. Such correlations would allow for a rapid, non-distructive determinations. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We determined YAN, ammonium nitrogen, free alpha amino acid fractions using a simply modified titration method we developed. We compared results with conventional assays. This has allowed the industry to use data derived from different analysis methods and compare results. We determined the range in fruit (from 30 to more than 550 mg/L)and the relative ratio of NH4+ concentration to free FAN. This information has allowed the industry to use less nitrogen supplementation and to use specific supplements for specific needs. The minimum levels of YAN required for successful completion of alcoholic fermentation as evaluated. We found that the minimum is lower than the reported values of 140 mg/L. This knowledge has allowed producers in our region to use fewer supplements under optimum conditions. We confirmed that both timing of nitrogen supplementation and the form of supplement may play a crucial role in successful completion of alcoholic fermentation. Our data demonstrates, at least in the mid-Atlantic region, that cultivar, site, season, vineyard management, and fruit maturity can have a profound influence on the fermentable N and N fractions. This knowledge has caused producers to evaluate their fermentable N status each season. We also domonstrated the impact of vineyard management and wine processing methods on the concentration of yeast assimible N, free alpha amino acids, ammonia, and arginine. This has highlighted the importance of monitoring N when stylictic processing changes are implemented. We evaluated fermentation anomalies arising from from several sourses including: deficiencies in the fruit and winemaking practices. The ratio of arginine to proline is much greater in the skins than the pulp. In other words, pulp juice taken off in bleeding for rose wine productions, for example, has a relatively high concentration of proline (approximately 55 percent) which cannot be used by the yeast, and a small concentration of the more potent amino acid arginine and others needed to carry out a healthy fermentation.

Publications

  • Zoecklein, B.W. 2009. Enology Notes, electronic technical briefs. Virginia Coop. Extension.
  • Yamuma S. Devarajan, B. W. Zoecklein and K. Mallikarjuan. 2009. Electronic nose evalution of the impact of clusters per shoot on Merlot and Cabernet franc (Vitis vinifera L.) grapes. Under review.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We conducted an extensive analytical survey of 235 juice samples during reporting period. The evaluation included total yeast assimible nitrogen (YAN), ammonia, and arginine concentrations. The concentration of YAN found in all red and white varieties at harvest, expressed as a percentage below 140 mg/L YAN as significant. The minimum level considered to be optimum for fermentation management if no other limiting factors are present is about 140 mg/L. It should be noted that the 2007 season was warmer and dryer than most and that these data represent only one year. A number of viticultural and environmental factors can impact fruit YAN. These may include: cultivar, soil, soil moisture, rot incidence, crop load, and maturity. From veraison onward, the following changes occur in the fruit were noted: Ammonia increases, then declines. FAN amino acids increase, then declined, with the rate of decline different among FAN components. With extended maturity, YAN declined. Results of traditional analyses were compared to a modified Formol procedure previously reported. Results confirm the Formal titration as a viable means of determining fermentable nitrogen within the precision and accuracy limitations required by industry. PARTICIPANTS: This study is done in conjunction with CSUFresno. The results of this effort have been convayed to members of the grape and wine industry. Industry has used this information to help improve wine quality. TARGET AUDIENCES: Grape growers and winemakers now understand the relationships between yeast fermentable nitrogen and aroma/flavor. The vast majority of the industry now measures nitrogen and makes adjustments, when needed. PROJECT MODIFICATIONS: The industry now understands the importance of measuring fermentable nitrogen.

Impacts
This study is designed to impact grower and winemaker economic profitability by increasing wine quality. Optimizing the concentration of fermentable nitrogen produced in the fruit allows for the production of wines with increased aroma intensity. A simple, in-house test, which could easily measure juice nitrogen, allows a greater link between vineyard management practices, juice nitrogen, and ultimate wine quality. Vineyard management practices impacting fruit nitrogen must be understood and evaluated based on their effect on ultimate wine quality. Grape growers and winemakers must seek to maximize wine quality, especially as international marketing becomes increasing more competitive.

Publications

  • Martin, A., Zoecklein, B., and K. Mallikarjunan. 2008. Discrimination of wines produced from Cabernet Sauvignon grapes treated with aqueous ethanol post-bloom using electronic nose Internation Journal of Food Engineering. Vol: 4, No. 2
  • Zoecklein, B. W., Wolf, T., Pelanne, L., Miller, K., and S. Birkenmaier. 2008. Evaluation of Viognier grapes and wines using VSP, Smart-Dyson or Geneva Double Curtain training systems. Am. J. Enology and Viticulture. 59: No. 1


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The goals and objectives include 1. evaluation of the relationship between proline/arginine ratios, 2. relationships between juice nitrogen and grape and wine volatiles,3. development of a simple assay that growers could use to test nitrogen status of their grapes and, 4. evaluate the relationships between N status of the vine and juice nitrogen. Items 1, 2 and 3 were previously reported. Over 300 assays were conducted durning the current reporting period to evaluate the N status of juice. This included nitrogen componet fractions such as arginine, total FAN (free alpha amino nitrogen) and ammonia. Each contributes to fermentable nitrogen, the nitrogen required by yeasts to properly conduct alcholic fermentations. The results of these assays have been shared with the wine industry throught three Wine Industry Roundtable meetings, at the annual meeting of the Virginia Vineyards Association, throught my electronic newsbrief, Enology Notes and posting on our web site at www.vtwines.info. PARTICIPANTS: Pofessor Ken Fugelsang, California State University, Fresno Dr. Barry Gump, California State University, Fresno Dr. Kumar Mallikarjunan, Virginia Tech Dr. Keith Patterson, California State University, CalPoly Mr. Ahmad Athamneh, Virginia Tech TARGET AUDIENCES: The wine industry. Efforts to target this audience have included scientific publications, trade journal publications, my electronic email technical briefs and short courses and seminars.

Impacts
The results of the analysis of fermentable nitrogen have allowed us to determine some variety and regional differences. This has provided the industry with an understanding of potential fermentation problems prior to the grape harvest season. Results have also aided in the understanding of seaonal differences with regard to total fermentable nitrogen and component parts. The strong positive correlation between fermentable nitrogen and sulfur-like off odor production by yeasts has long been established. Our analysis has allowed us help prevent this important fermentation problem. This has allowed for the production of wines that do not produce sulfur-like off odor taint. As such this effort has a positive economic impact as a result of increased wine quality.

Publications

  • Athameneh, A., B. Zoecklein, and K. Mallikarjunan. 2008. Electronic Nose Evaluation of Cabernet Sauvignon Fruit Maturity. J. Wine Research. In press.
  • Zoecklein, B. 2007. Use of a Hand Held Electronic Nose for Fruit Maturity Evaluation. Wine Business Monthy, December. In press.
  • Patterson, T. 2007. New Technology-Electronic Nose. Wines and Vines, December.
  • Zoecklein, B. 2007. Enology Notes. Electronic Newsbrief, Virginia Cooperative Extension.


Progress 10/01/05 to 09/30/06

Outputs
Cabernet Franc, Viognier and Traminette fruit grown on different training systems (Geneva Double Curtain, Smart-Dyson and Vertical Shoot Position) and rootstocks (own rooted or 3309) were evaluated for amino acid composition and total fermentable nitrogen status. Fruit samples were processed by whole berry pressing or complete maceration, and the amino acid profiles determined by HPLC. Processing method significantly impacted the fermentable N content, notably the ratio of proline to arginine. Amino acid composition differed by cultivar, rootstock, and training system. The accumulation of proline occurred in the later stages of fruit maturation, while arginine generally plateaued, then declined with increased fruit maturity. HPLC profiles were compared to results of fermentable N via a modified Formol titration. An average of 17 percent of the non-fermentable proline was titrated in the Formol analysis, while the fermentable arginine N was under-titrated. Results confirm the Formal titration as a viable means of determining fermentable nitrogen within the precision and accuracy limitations required by industry.

Impacts
This study is designed to impact grower and winemaker economic profitability by increasing wine quality. Optimizing the concentration of fermentable nitrogen produced in the fruit allows for the production of wines with increased aroma intensity. A simple, in-house test, which could easily measure juice nitrogen, will allow for a greater link between vineyard management practices, juice nitrogen, and ultimate wine quality. Vineyard management practices impacting fruit nitrogen must be understood and evaluated based on their effect on ultimate wine quality. Grape growers and winemakers must seek to maximize wine quality, especially as international marketing becomes increasing more competitive.

Publications

  • Zoecklein, B. W., Wolf, T., Pelanne, L., Miller, K., and S. Birkenmaier. 2006. Evaluation of Viognier grapes and wines using VSP, Smart-Dyson or Geneva Double Curtain training systems. Proceedings of the Sixth International Symposium for Viticulture and Enology.
  • Graves, H., Patterson, K., and B. Zoecklein. 2006. Effects of irrigation practices and rootstock on glycosyl glucose in Pinot noir winegrapes. Am.J. Enol. Vitic. Vol: 57:3
  • Graves, H., MS Thesis. CalPoly. 2006. Effects of irrigation practices and rootstock on Pinot noir winegrape composition.
  • Athamneh, A., Zoecklein, B., and K. Mallikarjunan. 2006. Electronic nose evaluation of Cabernet Sauvignon fruit maturity. Am. J. Enol. Vitic. Vol: 57:4


Progress 10/01/04 to 09/30/05

Outputs
A simple, in-house test, which could easily measure both plant and juice arginine, would allow for a closer link among plant nitrogen nutrition, juice nitrogen, and ultimate wine quality. The addition of nitrogen to the vineyard should be done with an understanding of the impact on wine quality, not simply on plant growth. Winemakers must seek to maximize production and quality, especially as international wine marketing becomes more competitive. Over 350 analyses using five methods for evaluating the nitrogen status, NOPA, Arginine NOPA, enzymatic NH3, Formol and HPLC were compared using Cabernet Sauvignon juice and tissue samples. Parallel recovery studies using model solutions of various amino acids and ammonia, present singly and in combination, were also conducted. Adjustment of formaldehyde pH prior to analysis was found to be critical to consistency of the Formol method. Low formaldehyde pH resulted in significant over-titration and falsely-high values of fermentable N. Average amino acid recoveries for the Formol titration ranged from 82 to 99 percent. Average recovery of the titrated proline was 16.9 +/- 0.4 percent. Ammonium nitrogen was also found to be titrated in the Formol procedure, at 84 +/- 3 percent. The Formol results trended significantly with NOPA. The correlation between Formol and NOPA+NH3 was 0.87 with the Formol values generally being higher. The average deviation between the Formol and HPLC plus NH4 was 8.5 percent, while that between the NOPA and HPLC as -8.4 percent.

Impacts
The Formol procedure has been modified to be able to be conducted by every winery creating minimal waste products. The recovery of Arginine as a function of this test, a ARGOFORMOL method for plant material is under study.

Publications

  • Zoecklein, B. W., B.H. Gump and K.C. Fuglesang. 2002. Nutritional Status of Grape Juice. In: Methods in Biotechnology, p. 413-423.
  • Gump, B. H., B. W. Zoecklein, K.C. Fugelsang. 2002. Prediction of Prefermentation Nutritional Status of Grape Juice. In: Methods in Biotechnology, p. 283-298.
  • Gump, B.H., B. W. Zoecklein, K. C. Fugelsang. 2002. Comparison of Analytical Methods for Prediction of Prefermentation Nutritional Status of Grape Juice. Am. J. Enol. Vitic. 53:325-328.


Progress 10/01/03 to 09/30/04

Outputs
Five methods for evaluating nitrogen status, Nitrogen by o-phthalaldehyde (NOPA), Arginine NOPA, enzymatic ammonia, Formol, and HPLC were compared using 70 Cabernet Sauvignon juice samples. Parallel recovery studies using model solutions of various amino acids and ammonia, present singly and in combination, were also conducted. The results from two fruit processing methods were compared using immature and mature berries. NOPA measurements were significantly higher in mature, pressed whole berry-derived samples, compared with homogenized juice. Adjustment of formaldehyde pH prior to analysis was found to be critical to consistency of the Formol method. Average amino acid recoveries for the Formol titration ranged from 82 to 99 percent. Average recovery for proline was 16.9 plus or minus 0.4 percent. Ammonium nitrogen was also recovered (84 plus or minus 3 percent) in the Formol procedure. Formol results trended significantly with NOPA. The correlation coefficient between Formol and NOPA plus Ammonium was 0.87, with the Formol values being higher. The average deviation between the Formol and HPLC plus Ammonium was 7.3 percent, while that between the NOPA plus Ammonium and HPLC plus Ammonium was minus 7.3 percent.

Impacts
This research evaluated methods for assimilable N and modified the Formol procedure that is simple and can provide a useful index of the nutritional status of a juice or must. We are currently evaluating the impact of total N and amino acid concentration on wine volatiles to provide specific recommendations to growers regarding N fertilization, and to winemakers regarding nitrogen supplementation of their fermenting juices.

Publications

  • Fugelsang, K.C. and B. W. Zoecklein. 2004. Population dynamics and effects of Brettanomyces bruxellensis strains on Pinot noir Vitis vinifera L. wines. Am. J. Enol. Vitic. 54:294-298.
  • Mansfield, A.K., and B.W. Zoecklein. 2003. Effects of fermentation, post-fermentation heat treatment and post-bottling heat treatment on Cabernet Sauvignon Vitis vinifera L. glycosides and glycoside fractions. Am J. Enol. Vitic.54: 99-104.
  • Zoecklein, B.W., B. H. Gump and K.C. Fugelsang. 2005. Analysis techniques in wine and brandy. In: Handbook of Enology. V.L. Joshi Ed. Haworth Press, New York, NY. In press.