|Abstract or Summary
- The presence of diacetyl in sufficient concentrations to cause
off-flavor in beer is a serious economic problem affecting the brewing
industry. Research was carried out in an attempt to provide an
industrially acceptable method for reducing diacetyl to safe levels
in beer, with additional applications to frozen concentrated orange
juice, and other distilled products.
A modified method of the Owades and Jakovac diacetyl determination
was employed to evaluate the diacetyl content of beer and
orange juice samples. The beer was found to contain a residual
concentration of 0.2 ppm while none of the orange juice samples
showed any detectable diacetyl. All samples were spiked with an
additional 0.5 ppm before being treated for diacetyl removal.
Diacetyl reductase, a reduced nicotinamide adenine dinucleotide
(NADH) requiring enzyme,was used for diacetyl removal studies; it was extracted from cells of Aerobacter aerogenes 8724 and subjected
to a series of characterizations. On the basis of activity in the presence
of NADH, diacetyl reductase was effectively purified with ammonium
sulfate fractionation and Sephadex gel separation, but was
rendered inactive when lyopholized in either purified form. As an
unpurified dialysate, or lyopholized preparation, the enzyme remained
stable for at least four months when stored at -20°C. It
was also determined that a pH less than 5.5 and an alcohol content
comparable to that found in beer reduced the activity of an unprotected
enzyme preparation by approximately 50%.
Any effective activity of diacetyl reductase in beer necessitated
a means of protecting the enzyme from inactivation by acids during incubation.
For this purpose a system using Swift's superclear gel
and a Fleischmann's yeast cell suspension, as an NADH substitute
or regenerative system, were incorporated with the enzyme and
prepared as a thin film dried at room temperature. The gel-yeast-enzyme
system was found to work efficiently in beer while allowing
none of its components to enter the beer. The best activity was
obtained when the gel flakes were freely suspended; if necessary
the flakes could be reused two or more times and still allow diacetyl
removal. Using parameters based on industrial requirements of
reduction to 0.2 ppm diacetyl in 48 hours and minimizing the time
required for reduction to 0.1 ppm, several combinations of gel, yeast, and enzyme were evaluated. The yeast alone was found to
be effective in removing diacetyl, but required the presence of at
least 0.05 percent enzyme to meet the time limits. A purified
enzyme preparation provided no additional activity in the system.
A detailed analysis of all optimum concentrations, with economic
considerations, is being prepared through a statistical regression
analysis for inclusion in a later publication.
Removal of diacetyl from orange juice required approximately
double the amount of gel-yeast-enzyme of that used in beer. Other
distillers' products with a proof comparable to that of whiskey could
not be analyzed.