Manipulating Soil Moisture and Nitrogen Availability Part 11.
Fermentable Nitrogen Content and Must and Wine Composition
Barney Watson, Kate Wall, Anna Specht, Hsiao-Ping Chen, and Mina
McDaniel
Department of Food Science and Technology, Oregon State University
Collaborators :
Carmo Vasconcelos, Jessica Howe, and Edward Hellman
Department of Horticulture, Oregon State University
INTRODUCTION
An approximation of the total yeast fermentable nitrogen content in juice or must is taken as
the sum of the nitrogen available from ammonia and the alpha-amino acids present (Bisson
1991 ; Dukes and Butzke 1998 ; Jiranek, Langridge, and Henshcke 1995). Recommended
levels of fermentable nitrogen needed by yeast for healthy fermentations are reported to vary
from as low as 140 mg (N)/L to as high as 500 mg (N)/L or more (Butzke, 1998 ; Spayd,
1998) . If assimilable nitrogen levels are too low, fermentations may be slow and may stick,
producing wines with undesirable levels of residual sugar. Problem fermentations are also
sometimes accompanied by production ofhydrogen sulfide and other reduced sulfur odors
(Kunkee 1991 ; Jiranek, Langridge, and Henshcke 1995). Other undesirable flavors have been
described in wines produced under drought and stressed conditions, including those related to
atypical aging (UTA) syndrome in white wines (Sponholz, 2000). Winemakers often add
supplements to juice and fermenting wines to balance perceived nutritional deficiencies
(Montiero and Bisson, 1992; Bisson, 1999). It is also thought that variations in climate, soil
type, and cultivation, soil moisture, and fertilizer practices may have an impact ofjuice and
must composition and nutrition (Butzke, 1998; Ingledew, 1985).
Slow and stuck fermentations are common in Oregon and winemakers often report
fermentation problems with fruit from specific vineyard blocks over several vintages. Petiole
analysis of Oregon vineyards over a three year study indicated that a high percentage of the
vines were consistently deficient in nitrogen, based upon bloom-time (California) and
veraison standards (Oregon) . Commercial juice samples (207) taken at harvest during the
three year period were analyzed for fermentable nitrogen content. The nitrogen available from
ammonia ranged from 0 to 145 mg (N)/L and the nitrogen available from the alpha amino
acids ranged from 35 to 380 mg (N)/L. The total estimated yeast assimilable nitrogen content
(YANG) ranged from 38 to 500 mg (N)/L . The percentage of commercial Chardonnay juice
samples with less than 140 mg (N)/L was 80% in 1997, 79% in 1998, and 17% in 1999. The
percentage ofcommercial Pinot noir juice samples with less than 140 mg (N)/L was 37% in
1997, 34% in 1998, and 8.6% in 1999. Petiole nitrate nitrogen levels at veraison were well
correlated with juice YANC in 1998 and 1999 with r=0.83 and r=0 .71, respectively (Watson,
Hellman, Specht, and Chen, 2000) .
The objectives of this study are to evaluate the effects of manipulating soil moisture and
nitrogen availability using different approaches, including supplemental irrigation, nitrogen
addition to the soil or to the leaves, and the elimination of competition for water and nutrients
between the ground cover and the grapevines by tilling. The effects ofthese viticultural
practices on juice and must composition, fermentable nitrogen content, fermentation behavior,
wine composition, aroma, flavor, and wine quality are being conducted over several vintages
at a mature commercial Pinot noir vineyard in the southern Willamette Valley of Oregon.
MATERIALS AND METHODS
Vineyard Experimental Design
The experimental design of this study is a factorial of irrigation, nitrogen soil and foliar
applications, and soil cultivation in a randomized block design . Each of 12 treatments was
replicated five times in groups of eleven vines each. The main factors and treatment
combinations are described in Manipulating Soil Moisture and Nitrogen Availability Part 1 :
Vine physiological performance, yield components, ripening dynamics, andfruit compostion
(Jessica Howe and Carmo Vasconcelos) .
Yeast assimilable nitrogen content
Cluster samples were taken at two-week intervals from veraison to harvest and were crushed,
pressed and the juice analyzed for the yeast fermentable nitrogen content. Berry weights were
determined from the weight of 100 berries sampled from each field replication. Ammonia
content was determined with a Sigma enzymatic diagnostic kit and the alpha amino acid
content was determined using the NOPA spectrophotometric assay (Dukes and Butzke, 1998).
The yeast assimilable nitrogen content (YANG) is expressed as mg (N)/L as the sum of the
assimilable nitrogen from ammonia plus the assimilable nitrogen from alpha amino acids.
Wineproduction
In 1999 and 2000 Pinot noir grapes were harvested from Benton Lane Vineyards from the 12
treatments (3 field replications each for a total of 36 lots) . The grapes were crushed,
destemmed, and 50 mg/L sulfur dioxide was added. Musts were inoculated with 1 g/L of
Lalvin RC 212 Bourgorouge yeast and the fermenting wines were punched down twice daily.
Fermentation temperatures reached a maximum of 32 °C for 48 hours and the wines were
pressed from the skins at dryness after seven days. Fermentation rates were monitored by
hydrometer readings taken at crushing and every 48 hours until pressing . The new wines
were settled, racked from the primary yeast lees, and inoculated with 0.025g/gallon with OSU
1-Step (Lalvin) freeze-dried malolactic bacteria . After completion of malolactic fermentation
the wines were cold stabilized at 4 °C, racked and bottled after addition of25 mg/L of sulfur
dioxide .
Must and wine analysis
Must samples taken after crushing and destemming were analyzed for degrees Brix, titratable
acidity (TA), pH, and malic acid content. New wines were analyzed after completion of
malolactic fermentation for alcohol content (%), TA, pH, residual fermentable sugar (RS),
volatile acidity (VA), color intensity, and for total anthocyanin and phenolic content. Color
intensity was measured as the absorbance at 520 + 420 nm at wine pH (1 mm cuvette with no
dilution). Total anthocyanin content was measured by absorbance at 520 nm at a pH<1 using
a 1% extinction coefficient of 380 (Singleton, 1982) . Total phenolic content was measured by
the Folin ciocalteau spectrophotometric assay and expressed as gallic acid equivalents (GAE)
(Ough and Amerine, 1988). Sulfides analysis will be done spring of 2001 by GC-MS and will
include hydrogen sulfide, ethyl and methyl mercaptan, ethyl and methyl polymercaptan, and
diethyl and dimethyl disulfide.
Sensory evaluation
The 1999 Benton Lane Vineyards Pinot noir wines (36 wines consisting of 3 replications each
from 12 field treatments) underwent sensory evaluation by an industry panel for differences in
color, aroma and flavor in the Sensory Sciences Laboratory of the Department of Food
Science and Technology (Manipulating soil moisture and nitrogen availability. Part III.
Effects on wine color, aroma andfavor, Heather Hjorth and Mina McDaniel). The 2000 Pinot
noir wines are currently undergoing malolactic fermentation (MLF). After completion of
MLF the wines will be analyzed for wine compositional differences and will undergo sensory
evaluation .
RESULTS AND DISCUSSION
Yeast assimilable nitrogen content andfermentation behavior
1999 Vintage
In 1999 the yeast assimilable nitrogen content (YANG) at harvest ranged from 143 to 208 mg
(N)/L with an average of 183 mg (N)/L (averaging 37 mg (N)/L from ammonia and 146 mg
(N)/L from alpha amino acids) (Table 1) . There were minimal differences in YANC among
treatments at harvest, although the Dry treatments tended to have slightly higher YANC (by
about 5 .6%) than the Irrigated treatments possibly due to a concentration effect due to smaller
berry size . Berry weights from the Dry treatments averaged about 5% less than berries from
the Irrigated treatments. From veraison to harvest the ammonia content in the berries
decreased by about 50% and the alpha amino acid content increased by about 250% for all
treatments . This resulted in a rapid net increase in YANC in the later stages of ripening
coinciding with a period of warm, dry weather and significant berry dehydration (berry
weights decreased by approximately 15-20% for all treatments) . At harvest all treatments had
a YANC greater than 140 mg (N)/L, the minimal recommended level of fermentable nitrogen,
and differences observed in the fermentation rates in 1999 were minimal . Had harvest been 7
to 10 days earlier, however, the YANC of all the treatments would have averaged about 140
mg (N)/L or less (Watson, McDaniel, Specht, Wall, and Chen, 2000).
2000 Vintage
In 2000 the yeast assimilable nitrogen content (YANC) at harvest ranged from as low as 59 to
156 mg (N)/L with an average of 117 mg (N)/L (averaging 21 mg (N)/L from ammonia and
113 mg (N)/L from alpha amino acids) (Table 2). All the Tilled treatments had greater YANC
at harvest than the Non Tilled treatments averaging 136 and 98 mg (N)/L, respectively . All of
the No Till treatments were lower in YANC than the minimal recommended level of 140 mg
(N)/L. The berry weights for all treatments averaged 1 .12 grams and no differences were
observed between the Dry treatments and the Irrigated treatments in 2000 .
From veraison to harvest the ammonia content decreased by about 26% and the alpha amino
acid content increased by about 70% . At harvest the YANC of all treatments averaged 36%
less than in 1999 . Many of the treatments in 2000 had less than the minimal recommended
fermentable nitrogen level of 140 mg (N)/L and differences were observed in fermentation
rates. Overall, the Till treatments with higher YANC tended to ferment more rapidly than the
Non Tilled treatments . For example, the Dry Till ON treatments averaged 138 mg (N)/L
YANC at harvest and fermented the most rapidly while the Irrigated NT SN averaged 89 mg
(N)/L at harvest and fermented the slowest (Figure 1). Observed vintage differences in YANC
may be due to differences in overall maturity as well as to differences in weather during
ripening and harvest (Watson, Hellman, Specht, and Chen, 2000).
Must and Wine Analysis
1999 must analysis
Significant differences in must analysis at harvest were observed in 1999. Till treatments had
higher Brix than No Till treatments, and Foliar Nitrogen (FN) and the Soil Nitrogen (SN)
treatments had lower titratable acidity (TA) and higher pH s than Zero Nitrogen (ON)
treatments. Malate levels were also lower for both FN and SN treatments compared to ON
treatments (Watson, M. McDaniel, A. Specht, K. Wall, and H.P. Chen, 2000).
2000 must analysis
Significant differences were also observed in must analysis at harvest in 2000 . Irrigated
treatments had higher Brix than Dry treatments and Till treatments had higher Brix than No
Till treatments (as occurred in 1999). Till treatments also had higher pH s than No Till
treatments . FN and SN treatments had lower TA s, lower malate levels, and tended to have
higher pH s at harvest than the ON treatments (also similar to the 1999 results) . These
differences suggest that the Irrigated, Soil N, and the Till treatments had slightly advanced
overall grape maturity at harvest compared to Dry, ON, and No Till treatments.
1999 wine analysis
In 1999 the wine color intensity (520+420 rim.) and the anthocyanin content were greater in
the Dry treatments than in the Irrigated treatments, possibly due to differences in berry
weights (Dry vs Irrigated treatments averaged 1 .07 vs 0.98g, respectively) . The total phenolic
content was greater in the SN treatment wines than either the FN or the ON treatment wines
(no differences were observed in average berry weights). The color intensity of the SN
treatment wines was also greater than either the ON or the FN treatments and was comparable
to the color intensity levels of the Dry treatment wines (Table 4). Preliminary evaluation of
wine color intensity was done at 9 months of age by a sensory panel using a 16-point intensity
scale (Helms, 2000). Wines produced from Dry treatments had higher average color intensity
ratings than wines produced from Irrigated treatments and wines produced from SN
treatments had higher average color intensity ratings than wines produced from FN
treatments. Wines produced from the ON treatments had the lowest average color intensity
ratings . Few differences were observed in color intensity ratings among wines produced from
Till compared to No Till treatments. The wines with the greatest color intensity had higher
anthocyanin content, higher polymeric anthocyanin content, and higher polymeric phenolic
content as analyzed by high performance liquid chromatography (ETS Laboratories, St.
Helena, CA) . For example, the Dry Till SN treatment wines had the highest average color
intensity rating of 12 .3 and had an average anthocyanin content of 330 mg/L, a polymeric
anthocyanin content of20 mg/L, and a polymeric phenolic content of403 mg/L. By
comparison, the Dry Till ON wines had a much lower average sensory color intensity rating of
7.2 and had an average anthocyanin content of 295 mg/L, a polymeric anthocyanin content of
13 mg/L, and a polymeric phenolic content of 257 mg/L (Figure 2). Polymeric anthocyanins
are essentially 100% colored while monomeric anthocyanins may be as little as 10% colored
at wine pH. The polymeric anthocyanin fraction, although present in relatively low
concentrations compared to the total anthocyanin content, may have a pronounced effect on
total color intensity.
2000 wine analysis
In 2000 the wine color intensity (520+420nm), the anthocyanin content, and the total phenolic
content were also greater in Dry treatment wines than in the Irrigated treatment wines, similar
to 1999. Again, these differences may reflect the effect of smaller average berry weights at
harvest in the Dry compared to the Irrigated treatments (1 .06 vs 1 .13g, respectively) .
Although little difference was observed in anthocyanin content in the ON, FN, and SN
treatments, the SN treatment wines tended to have greater wine color intensity and higher
total phenols than either ON or FN, again similar to the results in 1999. The SN average berry
weights were also slightly smaller than ON and FN in 2000 (Tables 5 and 6).
Overall, the differences in degrees Brix, TA, pH, and malate levels observed at harvest and
the differences in color intensity, anthocyanin and polymeric anthocyanin content, total
phenols and polymeric phenolic content observed in wines produced from the different
treatments suggests that there may be significant physiological differences occurring during
ripening with respect to the soil management and irrigation treatments used in this trial.
REFERENCES CITED
Bisson, L.F. 1991 .Influence of nitrogen on yeast and fermentation of grapes . In: Proceedings
of the International Symposium on Nitrogen in Grapes and Wines . Am. Soc. Enol. Vitic.,
Davis, CA., J.M. Rantz (Ed.), pp78-89.
Butzke, C.E. 1998 . Survey ofyeast assimilable nitrogen status in musts from California,
Oregon and Washington . American Journal of Enology and Viticulture. 49(2):220-224 .
Dukes, Bruce C., and Christian Butzke. 1998. Rapid determination of primary amino acids in
grape juice using an o-phthaldialdehyde/N-acetyl-L-cyseine spectophotometric assay. Am. J.
Enol. Vitic. 49:125-134 .
Helms, K. 2000 . The effects of nitrogen, tillage and irrigation on the color of Willamette
Valley Pinot noir wine . Undergraduate Honors Thesis, May 2000 . Oregon State University .
Ingledew, W.M . and R.E. Kunkee . 1985 . Factor s influencing sluggish fermentations of
grape juice. American Journal of Enology and Viticulture . 36(1):65-76 .
Jiranek, V., Langridge, P., and P.A. Henschke .1995. Regulation of hydrogen sulfide liberation
in wine producing Saccharomyces cerevisiae strains by assimilable nitrogen. J . Appl .
Microbiol . 61 :461467.
Kunkee, R.E.1991 . Relationship between nitrogen content of must and sluggish fermentation .
In: Proceedings of the International Symposium on Nitrogen in Grapes and Wines. Am. Soc.
Enol. Vitic., Davis, CA., J.M. Rantz (Ed.). pp148-55 .
Montiero, F.F. and L.F. Bisson . 1992. Nitrogen supplementation of grape juice. I . Effect
on amino acid utilization during fermentation. American Journal of Enology and Viticulture.
43(1) :1-10.
Ough C.S. and M.A. Amerine.1988 . Methods for analysis of musts and wines. 2nd Edition.
John Wiley & Sons .
Singleton, V.L. 1982. Grape and wine phenolics:Background and prospects . In:University
of California Davis Grape and Wine Centennial Symp. Proc. 1880-1980 . A.D. Webb, Ed.
pp 219-227. Department of Viticulture and Enology, University of California, Davis, CA
95616-8749 .
Spayd, S. E. January 1977. Nitrogen components and their importance in grape juice
fermentations : A Primer .WSU Wine & Grape Research Newsletter, Supplement No. l,
Irrigated Agricultural Research and Extension Center, Washington State University.
Sponholz, W.R., W. Grossman, and T.Huhn . 2000. The untypical ageing (UTA) in white
wines-its origins and prevention. 5ch International Symposium on Cool Climate Viticulture
and Oenology, January 16-20, 2000, Melbourne, Australia (In print).
Watson, B.T ., E . Hellman, A. Specht, and H.P. Chen. 2000 . Evaluation of nitrogen
deficiencies in Oregon grapevines and musts. Oregon State University Agricultural Research
Station Winegrape Research Progress Reports, 1999-2000, pp. 3-8.
Watson, B.T., M. McDaniel, A. Specht, K. Wall, and H .P. Chen. 2000 Manipulating soil
moisture and nitrogen availability to improve fermentation behavior and wine quality . Part II.
Effect of nitrogen, irrigation, and soil management strategy on yeast assimilable nitrogen,
juice composition at harvest, fermentation behavior, and wine composition and wine quality .
Proceedings of the 3`d International Burgundy-California-Oregon Wine Symposium . Terroir
and Quality. November 13-15, 2000, University of Burgundy, Dijon, France .
Table 1 . 1999 BL Pinot noir Yeast Assimilable Nitrogen at Harvest
Treatment
Berry wt.
grams
NH3
mg/L
NH3
mg N/L
NOPA(ile) YANC
mg N/L
mg N/L
Dry NT ON
Dry Till ON
1 .04
1 .14
44
61
36
50
151
154
187
204
Dry NT FN
Dry Till FN
1 .03
1 .13
47
57
39
47
157
161
196
208
Dry NT SN
D Till SN
1 .06
0.95
43
19
35
16
154
127
189
143
Irr NT ON
Irr Till ON
1 .18
1 .12
46
46
38
38
133
139
171
177
Irr NT FN
Irr Till FN
1 .16
1 .27
50
55
41
45
144
149
185
194
Irr NT SN
Irr Till SN
Average
1 .02
1 .18
1 .11
36
40
45
30
33
37
141
140
146
171
173
183
Table 2. 2000 BL Pinot noir Yeast Assimilable Nitrogen at Harvest
Treatment Berry wt.
rams
NH3
m /L
NH3
m N/L
NOPA(ile) YANC
m N/L
m g N/L
Dry NT ON
Dry Till ON
1 .13
1 .22
26
40
21
33
81
105
102
138
Dry NT FN
Dr Till FN
1 .18
1 .13
12
30
10
25
96
105
106
130
Dry NT SN
D Till SN
1 .12
0.92
18
19
15
16
97
127
112
143
Irr NT ON
Irr Till ON
1 .16
1 .13
9
33
7
27
52
90
59
117
Irr NT FN
Irr Till FN
1 .2
1.16
30
61
27
50
90
106
117
156
Irr NT SN
Irr Till SN
Average
0.97
1.08
1.12
10
21
26
8
17
21
81
113
95
89
130
117
Figure l . 2000 BL Pinot noir Fermentation Behavior
23
21
1
22 .8
22
21 .9
19
20.3
17
\16 .8
--0 -Dry NT ON
15
- -"- - Dry NT FN
13
- Dry NT SN
13.4
--0 -Dry T ON
--/- Dry T FN
r-- Dry T SN
7.5
f. - Irr NT ON
- -
- Irr NT FN
--~---Irr NT SN
--~---Irr T ON
"
Irr T FN
-- AL -Irr T SN
44
r
Days on skins
Table 3 . 2000 BL Pinot noir Must Analysis
Irrigation
Nitrogen
Soluble Solids
Brix
TA
g/L
pH
Malate
g/L
Irrigated
Dry
22.9
22.2
**
6.1
6.2
ns
3.18
3.15
ns
2.1
1.9
us
Zero N
Foliar N
Soil N
22.5
22.5
22.7
ns
6.5
6.2
5.9
*
3 .14
3.17
3.18
ns
2.2
1 .9
1 .8
23 .1
22
***
6.1
6.2
ns
3 .19
3 .14
*
2
2
ns
Cultivation
Till
No Till
Significant Treatment Interactions
none
ns, *, **, and *** indicate not significant and statistically significant at the 0 .05, 0.01,
and 0.001 levels of probability, respectively.
Table 4. 1999 BL Pinot noir Color and Phenols
Treatment
Berry
wt.
grams
Anthocyanin
_
mg/L
Color
Intensity
520+420nm
Hue
420/520nm
Total
Phenols
mg /L
Dry NT ON
Dry Till ON
1 .04
1 .14
352
343
7.3
6.8
0.72
0.70
1746
1689
Dry NT FN
Dr Till FN
1 .03
1 .13
368
341
7.8
7.0
0.72
0.79
1783
1786
Dry NT SN
D Till SN
1 .06
0.95
371
373
7.2
8.8
0.74
0.74
1776
2041
Irr NT ON
Irr Till ON
1 .18
1 .12
342
346
7.3
6.6
0.70
0.74
1677
1549
Irr NT FN
Irr Till FN
1 .16
1 .12
316
299
6.1
6.4
0.74
0 .77
1597
1713
Irr NT SN
Irr Till SN
1 .02
1 .18
323
331
7.4
7.0
0.77
0.79
1982
1842
Figure 2. 1999 BL Pinot noir Color Intensity and Phenolic Profiles
450
425
T-
~Anthocyanin mg/L
®Color Intensity Sensory Panel
-/-Poly Anth mg/L
--f--Polyphenols mg/L
520
400 -~375-t
350 1'I
325
016
016
016
016
015
300
214
0p414
13
275 -~250
225 --
9.53
9.17
200
7 .44
175 _j150
T
125 100 75 -,-
50 -~
,_
0`'
Treatments
0+
0+
20
/12
'13
~
12
Table 5. BL Pinot noir New Wine Analysis 2000
Anthocyanin Color Int.
mg/L
520+420nm
Irrigation
Total Phenol
mg/L
Irrigated
Dry
326
358
6 .8
7.5
0.68
0.71
1727
1803
Zero N
Foliar N
Soil N
346
331
7
6.8
0.66
0.7
1665
1720
350
7.6
0.73
1910
Till
No Till
339
345
7.1
7.2
0.7
0 .69
1770
1760
Nitrogen
Cultivation
Hue
420/520
Table 6. 2000 BL Pinot noir Color and Phenols
Treatment
Berry
wt.
grams
Anthocyanin Color
Intensity
mg/L
520+420nm
Hue
420/520nm
Total
Phenols
mg /L
Dry NT ON
Dry Till ON
1 .13
1 .22
191
163
5.1
4.2
0.65
0.69
2025
1875
Dry NT FN
D Till FN
1 .18
1 .13
187
175
4.7
4.1
0.75
0.74
1971
1882
Dry NT SN
D Till SN
1 .12
0.92
157
245
4.0
5.9
0.71
0.72
1932
1986
Irr NT ON
Irr Till ON
1 .16
1 .13
182
201
4.8
4.8
0.64
0.66
1702
1725
Irr NT FN
Irr Till FN
1 .20
1 .16
157
188
4.2
4.4
0.64
0.66
1922
1860
Irr NT SN
Irr Till SN
0.97
1 .08
214
187
5.5
4.4
0.74
0.76
1920
1871