- Improved sensitive techniques were developed for the
determination of soluble proteins in grape, juice and wine.
These techniques which permitted direct sample analysis
without prior dialysis or concentration, were used for: 1)
characterization of heat unstable proteins, 2) evaluating
the effect of bentonite fining and ultrafiltration (UF) on
the soluble proteins and heat unstable proteins in
Gewurztraminer and Riesling juices and wines. Protein
molecular weights (MW), isoelectric points (pI) and
glycoproteins were determined by using LDS-polyacrylamide
gel electrophoresis and 2-dimensional isoelectric focusing
(IF)-LDS electrophoretic techniques with silver staining as
well as protein blotting for glycoprotein detection.
Relative concentrations of proteins in stained gels were
determined by laser scanning densitometry. Bentonite fining
tends to remove higher pi (5.8-8.0), intermediate MW
(32,000-45,000) protein fractions first. In general, it is
necessary to remove the lower pI (4.1-5.8), lower MW
(12,600 and 20,000-30,000) fractions and glycoproteins to "protein stabilize" wines to heat testing. Unstable
proteins precipitated by heat test were recovered and
analyzed. These proteins were mainly of low MW (<30,000)
and primarily glycoproteins. Protein fractions with MW of
greater than 14,000 were more heat sensitive than lower MW
fractions. Wines and juice were ultrafiltered with Romicon
and Millipore systems operated with membranes of "nominal
MW cut-off (MWCO)" of 10,000-100,000 daltons. A progressive
increase in membrane retention of soluble protein was
observed with decreasing MWCO, up to 99% of wine protein
being retained with membranes of 10,000 dalton MWCO.
However, certain high MW protein fractions also remained in
permeates even with 10,000 MWCO membranes. In the order of
3-2 0 mg protein/L frequently remained in UF wine permeates,
this correlated with the periodic detection of heat
instability and of a low bentonite requirement for "protein
stability" as determined by sensitive heat testing.
"Protein stability" could be obtained with MWCO of 10,000
and 3 0,000; however, when not obtained, reductions in the
order of 80-95% in "bentonite demand" were achieved.
Protein stabilization of wines by UF is similar to that by
bentonite fining in that it is necessary to remove the
lower MW (12,600 and 20,000-30,000), lower pI (4.1-5.8)
fractions, and glycoproteins to stabilize wines to heat
testing. UF membranes were more effective at retaining wine
proteins than juice proteins. UF juices were more
susceptible to heat induced haze formation than UF wines.