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Yeast Assimilable Nitrogen (YAN)


Steve Monson

on 9 July 2010

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Transcript of Yeast Assimilable Nitrogen (YAN)

Spectrophotometric Determination
of Yeast Assimilable Nitrogen Purpose Organic Nitrogen vs. Molecular Nitrogen Principle Materials and Methods Yeast needs nitrogen for fermentation Simple kits available for testing ammonia Ammonia and YAN concentrations are not directly correlated Levels of nitrogen below 140 mg N/L can lead to stuck fermentation and production of off flavors Yeast Assimilable Nitrogen (YAN) Ammonia N2 NH3 - Free ions in Juice 78% of our atmosphere, can't be used by plants unless reduced Alpha-Amino Acids Amino acids react with ortho-phthalaldehyde (OPA) Possible to measure reaction based on excitation of OPA with a spectrophotometer Analytical Balance
Graduated Cylinder
1-5 mL pipettor with tips
200-1000 ul pipettor with tips
20-100 ul pipettor with tips
100 mL volumetric flask
1000 mL volumetric flask
Spectrophotometer 95% Ethanol
DI water
N-acetyl-L-cysteine (NAC)
Boric Acid
Sodium Hydroxide Standard - 28, 56, 84, 112, 140 mg N/L Reagent Preparation Reagent A:
Weigh .671 grams of OPA, transfer to 100 mL volumetric flask, bring to volume with EtOH
Weigh 3.837 g NaOH, 8.468 g boric acid, and .816 g NAC into a 1000 mL volumetric flask. Add 100 mL of OPA solution, bring to volume with DI water

Reagent B:
Identical solution except without OPA
To be used with blank sample Standard Preparation:
Pipette following volumes into 6 cuvettes
Blank - 50 ul DI water
10 ul isoleucine + 40 ul DI water
20 ul isoleucine + 30 ul DI water
30 ul isoleucine + 20 ul DI water
40 ul isoleucine + 10 ul DI water
50 ul isoleucine Sample Preparation
Pipette as follows for each sample
Blank - 50 ul juice
2 cuvettes of 50 ul juice
2 cuvettes of 25 ul juice and 25 ul DI water
Reagent Addition and Sample Reading
Pipet 3 mL of Reagent B into blank
Pipet 3 mL of Reagent A into standards and samples
Allow reaction for 10 minutes
Record absorbance at 335 nm on the spectrophotometer By: Steve Monson Calculating YAN Subtract water blank absorbance from all leucine standards
Subtract juice blank absorbance from all samples
Insert data into Excel and determine R-squared for the standard
Insert sample absorbance into this equation Use for the winemaker Can encourage better fermentation with the addition of nitrogen
Diammonium Phosphate (DAP)
Too much nitrogen can stimulate yeast to synthesize off flavors or even ethyl carbamate, a carcinogen.
YAN levels combined with ammonia levels should be:
200 mg N/L at 21 Brix
250 mg N/L at 23 Brix
300 mg N/L at 25 Brix
350 mg N/L at 27 Brix Questions? Sampling For white wines, a homogenous sample of juice after pressing For red wines, a winemaker must collect and centrifuge a sample from fresh must Must be done before yeast addition Limitations:
Up to 30% more nitrogen unnaccounted
Seeds, skins, pulp etc...
Some "wiggle room" in optimum N content
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