For the specific and rapid assay of L-arginine, urea and ammonia in grape juice/must and wine.
INTRODUCTION:
The addition of nutrient supplements to grape juice prior to/during fermentation can now be managed accurately in terms of Yeast Available Nitrogen (YAN). YAN is especially important to quantitate accurately, as too little available nitrogen can result in sluggish or “stuck” fermentation, and the generation of hydrogen sulphide (H2S) gas, while too much nitrogen, such as in the form of diammonium phosphate (DAP), can lead to the formation of the carcinogenic compound ethyl carbamate, especially where starting levels of
L-arginine in the juice are high.
Total YAN (YANT) is comprised of three components:
(a) free ammonium ions1,
(b) primary amino nitrogen (PAN, from free amino acids)2, and
(c) the contribution from the side chain of L-arginine (after
hydrolysis by yeast arginase that creates ornithine and urea)1.
All three components must therefore be measured accurately before
any informed decision can be made regarding the addition of extra
YAN in the form of DAP or yeast extract (permitted nutrient
supplements). The YAN from ammonia and L-arginine (YANAG)
(a plus c) can be conveniently and rapidly determined using the
Megazyme L-Arginine/Urea/Ammonia kit (K-LARGE); the PAN
component of YAN (b) is measured using the Megazyme
Primary Amino Nitrogen kit (K-PANOPA). Total YAN is
determined using both the K-PANOPA and K-LARGE kits, but
because both measure the primary amino group of L-arginine, this
has to be allowed for in the calculations (see page 7-8 of this
booklet).
The rapid assay procedure (K-LARGE) described in this booklet enables the determination of both ammonium ions and L-arginine in a simple sequential format. Urea can also be quantified by this procedure, and the levels of this compound should be minimised to prevent significant production of ethyl carbamate.
KITS:
Kits suitable for performing 50 assays are available from Megazyme. The kits contain the full assay method plus:
Bottle 1: Buffer (18 mL, pH 8.0) plus sodium azide
(0.02% w/v) as
a preservative.
Stable for
> 2 years at 4°C.
Bottle 2: NADPH. Lyophilised powder.
Stable for > 5 years at -20°C.
Bottle 3: Glutamate dehydrogenase suspension (1.1 mL).
Stable for
> 2 years at 4°C.
Bottle 4: Urease solution (2.7 mL).
Stable for
> 2 years at -20°C.
Bottle 5: Arginase suspension (1.1 mL).
Stable for
> 2 years at 4°C.
Bottle 6: Ammonia (0.04 mg/mL) standard solution.
Stable for
> 2 years at 4°C.
Bottle 7: L-Arginine powder (2 g) standard.
Stable for
> 2 years stored dry at 4°C.