Graduate Thesis Or Dissertation
 

An enzymatic method for the removal of diacetyl from fermented beverages

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/j3860b39p

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  • 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.
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