YAN testing

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YAN is the amount of nitrogen from the combination of ammonium plus Free Amino Nitrogen (FAN) in the form of amino acids. We can measure YAN via titration using a few reagents and a pH meter:

Estimate of FAN by formol titration

This is rapid method for estimation of the fermentable nitrogen in juice, wine, or vinegar adapted from Zoecklein et al., 1999 and Gump, Zoecklein and Fugelsang, 2002 The method measures alpha-amino acids and ammonia.

Equipment

• pH meter to 0.05 pH unit
• Electrode (Orion, epoxy body, sure flow, semi-micro or equivalent)
• 5μm syringe filters, or equivalent
• 2-mL and 10-mL pipets, or equivalent
• 25-mL volumetric flasks
• 10-mL buret (± 0.05 mL)
• 30-mL beaker
• mini stirbars

Reagents

• Calibration buffers for the pH meter
• Sodium hydroxide (NaOH) 1 N
• Sodium hydroxide (NaOH) 0.05 N standardized against potassium hydrogen phthalate or equivalent
• Formaldehyde, reagent grade 37% (vol/vol or 40% wt/vol), neutralized to pH 8.0 with 1N NaOH

Products

MoreBeer

Reagents for the Vinmetrica. "Measures down to 20 mg/L of available nitrogen with accuracy of 10 mg/L of available nitrogen." Includes: YAN reagent (37% formaldehyde/15% methanol, formaldehyde neutralizer powder (45g sulfite), 3 plastic transfer pipettes, and a 15mL conical tube. 1 year shelf life.

Procedure

1. Check pH of formaldehyde and if not at pH 8.0 neutralize with 1N NaOH
2. Clarify the wine sample with a 5μm syringe filter or equivalent.
3. Transfer 10.0 mL of the clarified sample into a 25-mL volumetric flask. Bring to volume with deionized water, and mix well.
4. Transfer a 10.0 mL sample of the clarified and diluted wine into a 30-mL beaker, place calibrated pH/reference electrodes and a stirbar into the solution, mix, and adjust the pH of the sample to 8.0 with 1 N sodium hydroxide.
5. Add 2.0 mL of the previously neutralized formaldehyde (pH 8.0) to the aliquot, mix, and titrate back to pH 8.0 with the 0.05 N NaOH.
6. The concentration of fermentable nitrogen is given as follows:

   Fermentable Nitrogen (mg N/L) = (mL of 0.05 NaOH titrated) x 175

Notes

• Formaldehyde is carcinogenic and a bronchial irritant, so handle with proper ventilation.
• The pH of formaldehyde gradually shifts downward. Its pH should be checked and readjusted to 8.0 prior to each use.
• The Formol titration only titrates one nitrogen of arginine, it also titrates approximately 14% of the proline present. These two errors are, at least, partially offsetting.

The Vinmetrica YAN (Yeast Assimilable Nitrogen) Test Kit provides a simple and accurate way to determine YAN levels in wine, must and other samples.

Materials provided in the kit:

• YAN Reagent (37% Formaldehyde/15% methanol)
• YAN Formaldehyde Neutralizer Powder (Sulfite)
• Three Plastic Transfer Pipettes
• 15 mL conical tube

Things you will need:

• pH meter (0.02 pH resolution required).
• Solutions for pH calibration.
• TA Titrant and accessories.
• 25 mL burette recommended, or syringe for TA titration
• Distilled water (DI water), which can be found at most grocery stores.
• It's handy to have a wash bottle for rinsing.

Why Test for YAN? Yeast assimilable nitrogen is an important nutrient in fermentation of wines beers, and other juices. If levels are too low, fermentation may stop prematurely, and/or off odors can develop (mostly from generation of hydrogen sulfide — "rotten egg" smell). In this case you may want to add yeast nutrients before and/or during fermentation. Many yeast and nutrient manufacturers follow a study by the UC Davis Department of Viticulture and Enology relating optimal nitrogen levels (in milligrams of nitrogen per liter, mg N/L) to wine’s brix level at harvest: 21°Bx = 200 mg N/L 25°Bx = 300 mg N/L 23°Bx = 250 mg N/L 27°Bx = 350 mg N/L

How it works: A sample is titrated to pH 8.2 with TA Titrant (this gives the TA value as a bonus). Then the YAN reagent (formaldehyde) is added. This causes any amino groups or ammonia to become formylated, releasing one proton per amine/ammonia and lowering the pH. The sample is then titrated back to 8.2 as before. The YAN value is calculated from the volume of TA Titrant used in the second titration.

CAUTION! The YAN reagent (formaldehyde and methanol components) is flammable. Keep away from sparks and open flame. The YAN reagent is TOXIC and a possible CARCINOGEN! Do not inhale fumes or ingest any solutions containing it. Do not pipet by mouth! Perform all steps with formaldehyde in a fume hood or similar well-vented environment. Wear laboratory gloves and safety glasses at all times when handling formaldehyde. If contact with skin or clothes occurs, flush with plenty of water and apply a solution of the formaldehyde neutralizer powder (1 tsp/50 mL water).

Procedure:

1. Clarify wine, must or other sample if needed by decanting, filtration or centrifugation. De-gas any excess dissolved CO2 by taking about 15 mL of sample into a suitable container and shaking until outgassing ceases.
2. Calibrate your pH electrode in the usual manner.
3. Check and adjust pH of the YAN Reagent:
   • Pour off enough YAN Reagent into the 15 mL conical tube for your immediate needs. You need 1 mL per sample. Take a minimum of 3 mL and maximum of 5 mL at a time.
   • Rinse your pH electrode, remove excess water, then place in the 15 mL conical with formaldehyde and swirl. If the pH is below 7, use a plastic transfer pipette or eyedropper to add 1 drop TA Titrant, mix well and check again. Repeat this process carefully until the pH gets up to between 7.5 and 8.5 (2-4 drops typically is needed - If you're having trouble hitting the target, you can dilute the TA Titrant: add 1 big drop (about 0.05 mL) to about 0.5 mL water, mix, and use this dropwise once you get above pH 6.). If the pH goes above 8.7 you will need to bring it back down. In this case you can add a little more of the formaldehyde solution, or dropwise 0.1 M HCL solution (can make this by adding 1 mL of your SO2 Acid Solution to 20 mL water)
4. Optional: If you suspect your sample has high free SO2 levels (>150 ppm), you may need to precipitate it with barium chloride¹. Otherwise proceed to the next step.
5. Take a 10.0 mL degassed sample into a 100 mL beaker or similar vessel; add 10 mL water and do a TA titration (check your SC-200 or SC-300 manual), but note that since you are using a 10 mL sample rather than the usual 5 mL, it will probably require more than 5 mL of TA Titrant to reach the endpoint of pH 8.2 – 8.3. If the end point goes above pH 8.5, repeat the step. The TA value can now be calculated if desired², but is not needed to complete the test. We recommend using a 25 mL burette for this titration.
6. Do not pipette by mouth! Using another of your plastic transfer pipettes, add 1 mL of YAN Reagent that has been pH-adjusted as in step (3.). Mix well for about 1 minute.
7. The pH of the wine will drop back to around 6 to 7. Titrate back to 8.2 with the TA Titrant (be sure to note starting and ending volumes on your burette or syringe!).
8. Measure the volume in mL, ‘V’, of the TA Titrant used in step 7.

   YAN, mg N/L = V * 0.133 * 1400 [Eq. 1.]

Note: Typical values for YAN are 50 to 500 mg/L in must

¹ Add 1 mL 1 N BaCl2 to a 10 mL wine sample, mix and let stand 15 minutes. Bring to 20 mL with water. Filter through Whatman no. #1 paper. Titrate as in step 5.
² In this case, g/L Tartaric acid = mL TA Titrant used, i.e., do not multiply by 2, because you took twice as much sample.

Finishing Up:

1. Clean-up: To neutralize any residue of formaldehyde, add ¼ teaspoon (about 1.5 g) of the YAN Formaldehyde Neutralizer Powder for every 1 mL formaldehyde used. Mix well and allow to /stand for at least 1 hour. Dispose in accordance with local regulations with plenty of water.
2. Turn off your pH meter and rinse and store the electrode properly per its manual's instructions.
3. Reagents should be stored at room temperature out of direct sunlight and away from children.
4. Rinse all plastic ware with DI water.

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• https://vtechworks.lib.vt.edu/handle/10919/49450
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The Formol titration has the advantage of measuring both ammonia and FAN amino acids. However, the method over-titrates proline (which yeast cannot use) and under-titrates arginine, which the yeast do use. Our research suggested that this generally balances out [in wine].