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FERMENTABLE NITROGEN Key to successful fermentations! Barry H. Gump, Ph.D. Professor of Beverage Management Florida International University Co-Pi – VESTA (Viticulture & Enology Science & Technology Alliance) FERMENTABLE NITROGEN

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fermentable nitrogen
FERMENTABLE NITROGEN
  • Key to successful fermentations!

Barry H. Gump, Ph.D.

Professor of Beverage Management

Florida International University

Co-Pi – VESTA

(Viticulture & Enology Science & Technology Alliance)

fermentable nitrogen2

FERMENTABLE NITROGEN

Nitrogen compounds in grapes play important roles as nutrients for microorganisms involved in winemaking and wine spoilage and as aroma substances and precursors.

nitrogen
NITROGEN
  • GROWTH RATE – Saccharomyces cerevisiae – structural and functional proteins
  • SUGAR TO ALCOHOL CONVERSION – protracted or stuck fermentations
  • ODOR/FLAVOR-ACTIVE METABOLITES – H2S, higher alcohols, esters, organic acids
  • ETHYL CARBAMATE
nutritional status of grape juice

Nutritional Status of Grape Juice

The nitrogenous components of grapes and juice which are metabolically available to yeasts are present as ammonium salts (NH4+) and primary or “free alpha-amino acids” (FAN). FAN – alpha-amino acids – arginine, serine, threonine, lysine, -butyric, aspartic, glutamic acids

YANC – yeast assimilable nitrogen compounds – a-amino acids + NH3

Not proline

influences
INFLUENCES
  • GRAPE GROWING PARAMETERS –rootstock, climate, soil, fertilization, irrigation, fruit maturity, cultivar
  • WINEMAKING PARAMETERS – distribution between skins, seeds, & pulp, juice clarification, fining practices
fan yanc
FAN/YANC
  • FAN – alpha-amino acids – arginine, serine, threonine, lysine, -butyric, aspartic, glutamic acids
  • YANC – yeast assimilable nitrogen compounds – a-amino acids + NH3
  • Not proline
analytical methods
ANALYTICAL METHODS
  • NOPA – FAN
  • HPLC – SPECIFIC AMINO ACIDS
  • NH3 – by enzymes or electrode
  • ARGOPA – selective for Arginine
  • FORMOL TITRATION -- FAN
nitrogen by opa butzke and dukes procedure ajev 1998
Nitrogen by OPA(Butzke and Dukes Procedure)AJEV 1998
  • Ortho-Phthaldialdehyde (OPA)
  • isoleucine stock solution
  • N-acetyl-L-cysteine  (NAC)
  •  Record absorbance at 335 nm
  • measures primary amino acids (mg/L N)
  • Ammonia by ISE or enzyme kit
slide9
HPLC
  • OPA and FMOC chemistry (338nm and 262nm)
  • Sample prep using autosampler
  • Simple gradient
  • Reproducible and Sensitive
  • Results as mg/L N or specific amino acid
  • Assumes 2 fermentable nitrogens in Arginine
the formol titration
The Formol Titration
  • Sodium hydroxide solution, 0.10 N
  • Formaldehyde, 37% neutralized to pH 8.0 with sodium hydroxide – couples with amino acid – titratable acid function
  • pH meter sensitive to  0.05 pH
  • Titrate 4 mL sample to pH 8.0 with 0.1 N NaOH, add formaldehyde, back to pH 8.0 with 0.05 N NaOH (or more dilute)
  • mg N/L (NH4+ + amino nitrogen) = VNaOHxNNaOHx14x1000/Sample Volume
titratable acidity
Titratable Acidity
  • Measures organic acid content of juice or wine

(any organic compounds that react with NaOH up to pH 8.2)

  • Used as a harvest parameter of quality
  • Used to calculate acid additions or de-acidification requirements
titratable acidity15

Titratable Acidity

Reagents normally used for measurement of TA:

(A) 0.1N NaOH,

(B) phenolphthalein, or calibrated pH meter

Titrate 5 mL sample to pH 8.2 with 0.1 N NaOH (or more dilute) – various sample dilutions used

TA (g/L) = VNaOHxNNaOHx0.150x1000/2x Sample Volume (5 mL)

combined ta formol methods
Combined TA & Formol Methods
  • Reagents for TA
    • 0.100 N NaOH – for titrating acidity
    • 0.100 HCl – or KHP for standardizing NaOH
    • Phenolphthalein or pH meter
  • reagents for FORMOL
    • 37% formaldehyde neutralized to pH 8.2 (with 0.10 N NaOH)
    • 0.05 N NaOH for titrating nitrogen compounds
procedure continued
Procedure (continued)
  • Measure TA of juice sample (use 5 mL juice sample and measure volume of NaOH to pH 8.2 [or phenolphthalein])
    • TA (g/L as Tartaric) = VNaOH x NNaOH x 15
  • Measure YAN (continued with neutralized sample and add 2 mL formaldehyde. Measure volume of 0.05 N NaOH to pH 8.2)
    • YAN (mg N/L) = VNaOH x NNaOH x 4800
sample data juice
Sample Data - Juice

AOAC TA (200 mL - pH 8.2)

Classic YAN (5 mL, 0.1NNaOH

pH 8.0)

N = 4, Ave = 217 +/- 8mg N/L

YAN (200 mL dilution, pH 8.2)

N = 4, Ave = 56 +/- 1 mg N/L

  • N = 5, Ave = 2.7 +/- 0.1g/L
  • Combined AOAC TA 

N = 4, Ave = 2.6 +/- 0.1 g H2T/L

sample data new procedure
Sample Data – New Procedure

AOAC TA (0 mL - pH 8.2)

Classic YAN (5 mL, no dilution, 0.1NNaOH , pH 8.0)

N = 4, Ave = 280 +/- 7mg N/L

YAN (0 mL dilution, pH 8.2)

N = 4, Ave = 92 +/- 6 mg N/L

  • N = 4, Ave = 6.91 +/- 0.06 g H2T/L
  • Combined AOAC TA 

N = 4, Ave = 2.6 +/- 0.1 g H2T/L

white grape juice
White Grape Juice

TA with 5 mL sample, no dilution to pH 8.2

n = 5, Ave = 5.6 +/- 0.1 g H2T/L

Combined TA & YAN

n = 10, Ave = 5.6 +/- 0.1 g H2T/L

YAN with 5 mL sample, no dilution to pH 8.2, using 0.05 NNaOH

n = 6, Ave = 204 +/- 9 mg N/L

n = 10, Ave = 209 +/- 9 mg N/L

red grape juice
Red Grape Juice

TA with 5 mL sample, no dilution to pH 8.2

n = 5, Ave = 6.41 +/- 0.06 g H2T/L

Combined TA & YAN

n = 10, Ave = 6.48 +/- 0.06 g H2T/L

YAN with 5 mL sample, no dilution to pH 8.2, using 0.05 NNaOH

n = 5, Ave = 234 +/- 9 mg N/L

n = 10, Ave = 240 +/- 9 mg N/L

students in analysis class white grape juice
Students in Analysis Class – White Grape Juice

TA with 5 mL sample, no dilution to pH 8.2

n = 5, Ave = 6 +/- 1 g H2T/L

Comparison to TA & YAN

n = 10, Ave = 5.72 +/- 0.09 g H2T/L

YAN with 5 mL sample, no dilution to pH 8.2, using 0.05 NNaOH

n = 5, Ave = 214 +/- 13 mg N/L

values run by Brittany

n = 10, Ave = 209 +/- 6 mg N/L

cabernet sauvignon grapes
Cabernet Sauvignon Grapes

TA with 5 mL sample, 200 mL dilution to pH 8.2

n = 8, Ave = 2.93 +/- 0.09 g H2T/L

Combined TA & YAN 5 mL

n = 6, Ave = 2.97 +/- 0.03 g H2T/L

YAN with 5 mL sample, no dilution to pH 8.2, using 0.05 NNaOH

n = 6, Ave = 162 +/- 5 mg N/L

no dilution to pH 8.2

n = 6, Ave = 183 +/- 9 mg N/L

correcting nitrogen deficiency

Correcting Nitrogen Deficiency

Nitrogen deficiency in fermenting juice/must is often corrected by addition of assimilable nitrogen in the form of diammonium phosphates DAP and/or one of several commercially available nitrogen supplements.

correcting nitrogen deficiency25

Correcting Nitrogen Deficiency

Supplementation with nitrogen is best accomplished with incremental additions starting at 48 hr (for reds) and 72 hr (for whites) post-inoculation or a single addition midcourse during the fermentation

suggestions

Suggestions

Use 0.01 NNaOH for YAN titration

Build database for cultivar, vineyard, other harvest conditions vs. YAN values