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FERMENTABLE NITROGEN

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  1. 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)

  2. 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.

  3. 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

  4. 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

  5. INFLUENCES • GRAPE GROWING PARAMETERS –rootstock, climate, soil, fertilization, irrigation, fruit maturity, cultivar • WINEMAKING PARAMETERS – distribution between skins, seeds, & pulp, juice clarification, fining practices

  6. 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

  7. ANALYTICAL METHODS • NOPA – FAN • HPLC – SPECIFIC AMINO ACIDS • NH3 – by enzymes or electrode • ARGOPA – selective for Arginine • FORMOL TITRATION -- FAN

  8. 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

  9. 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

  10. 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

  11. Comparisons of reduced volume Formol titrations

  12. 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

  13. 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)

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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.

  23. 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

  24. Suggestions Use 0.01 NNaOH for YAN titration Build database for cultivar, vineyard, other harvest conditions vs. YAN values