Estimation Of Yeast Assimilable Nitrogen Using The Formol Titration Technique
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3Estimation of Yeast Assimilable Nitrogen using the Formol Titration Technique
Successful fermentation of wine requires grapes with adequate level of yeast nutrients. The primary
nutrient requirement of yeast is limited by the availability of fermentable nitrogen sources distributed in
the form of ammonium ions (NH
) and certain alpha-amino acids. Yeasts that are deprived of adequate
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nutrients show signs of stress including the production of hydrogen sulfide and other undesirable
byproducts of fermentation. A shortfall of yeast nutrients could be handled by dumping lots of
commercially available yeast nutrients (DAP, Fermaid, etc.) into all batches of juice or must. But such a
solution is not optimal because (1) overfeed yeast populations can grow too quickly during the
reproductive stage of fermentation, and (2) unused yeast nutrients can foster the growth of spoilage
microorganisms following fermentation. The formol titration technique is a relatively simple procedure
that can be used to provide a rough (but useful) estimate of the nutritional status of grape juices and
musts expressed in terms of mg/L of yeast assimilable nitrogen (YAN). When formaldehyde is added to
a properly conditioned sample of grape juice or must it reacts with the amino acids that provide the
+
major sources of nitrogen used in yeast metabolism. These reactions free-up hydrogen ions (H
) in
proportion to the available nitrogen and, as a result, yield a systematic decrease in the pH of the test
+
sample. The amount of NaOH needed to neutralize the H
ions freed by the formaldehyde addition
provides an estimate of the nitrogen content provided by amino acids; and, as a bonus, also reflects the
nitrogen available as ammonia in the juice or must. Given the results of the formol titration and a
determination of the target nutritional requirements of the grapes based upon varietal, climate and
viticultural practices, the wine maker can add optimal levels of yeast nutrients.
Procedure
1.
Filter a small, representative sample of juice/must using a 5-μm syringe filter or filter paper.
2.
Pipette precisely 10 ml of filtered sample to a 25 ml volumetric flask and top-off with
distilled water.
3.
Pipette 10 ml of the diluted/filtered sample to a small beaker, insert a magnetic stir bar and
pH probe. Add enough distilled water to ensure that the pH probe is sufficiently submerged.
4.
Excessive levels of SO
(previously added to the juice/must) can introduce error into the YAN
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determination. If SO
> 150 PPM, add 1 ml of 3% hydrogen peroxide to neutralize the SO
.
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5.
Turn on the magnetic stirrer and titrate the sample with 0.05 N NaOH until neutralized (pH=8.0).
6.
Add 2 ml (nominal) of previously neutralized formaldehyde solution to the sample using a small
graduated cylinder. The formaldehyde will react with the YAN sources yielding a proportionate
release of H+ ions (thus, lowering pH).
7.
Record the current level of 0.05 N NaOH in your burette.
8.
Titrate the sample with 0.05 N NaOH until pH = 8.0 to neutralize the H+ ions released in Step 6.
9.
Record the new level of 0.05 N NaOH in your burette and calculate the amount needed to
neutralize the sample following the addition of the formaldehyde solution.
10.
Estimate yeast assimilable nitrogen (YAN) level as follows:
YAN (mg N/L) = (ml of 0.05 N NaOH titrant) x 175
[Eq. 1]
Version 1.2 – December 7, 2012
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