Enzyme Inactivation and Quality Preservation of Sake by High-Pressure Carbonation at a

Moderate Temperature

Sake is traditional alcoholic beverages that reveal its best when it is fresh. However, non-
pasteurized sake contains lactic acid producing bacteria and enzymes caused sake cannot
be store for long. Pasteurization overcomes this but it will lose its fresh flavor. Carbon
dioxide (CO2) used in food processing because it is nontoxic, nonflammable, inexpensive
and can be readily removed from food products. High-pressure CO2 used to inactive
enzymes and microorganism in recent years. Non-pasteurized sake was brewed with 50%
polished rice and was stored at -20oC. Continuous treatment of sake then carried out. The
temperature of CO2 dissolving vessel was maintained at 5 ± 0.5oC. The sample and CO2
were heated to a given temperature by a heating coil, before being retained for a given
time in a residence coil. A cooling unit (-20oC) was attached after a pressure control valve
to prevent the volatile component from vaporizing. Three treatment conditions were set.
The average residence time was calculated from the flow rate of the sake and CO2. Heat
treatment performed by heating non-pasteurized sake in 90oC water bath and cooled
immediately in ice bath when its temperature reached 65oC. Each sample was stored at
3oC and 20oC for 6 month. After the storage it was frozen at -70 oC until needed for
analyses and sensory evaluation. The activity of enzymes which is α-glucosidase,
glucoamylase, α-amylase and acid carboxypeptidase was measured with their own kind
enzyme assay kit. The activity of isoamyl alcohol oxidase was measure by special
method. The residual activities of these enzymes are defined as residual percentage of
activity after the treatment. Sake meter and other general components were also
measured. Sugars were analyses using HPLC and its components measured by refractive
index detector. Organic acids analyzed by capillary electrophoresis. Flavor components
were measured using head-space method except for isovaleraldehyde which is the off-
flavor component in sake produce by isoamyl alcohol oxidase, analyzed by gel filtration
chromatography further with capillary GC chromatography. Sensory evaluation was
conducted by a panel of 5 experts, evaluating sake flavor, taste and total quality
according to five levels. After the treatment the results shows that the activity of five
enzymes at 40oC with residence time of 33 minutes had decrease to less than 6% of
activity in untreated sake. Residual activity of α-glucosidase in treated sake is 29%
which was most stable enzyme against high-pressure carbonation. Presumed that enzyme
inactivation in sake might due to irreversible denaturation of the enzyme protein. The
sake meter and alcohol content of carbonated sake were slightly lower than pasteurized
and fresh sake samples. These results suggest that such volatile compound such as
ethanol could have been removed along with CO2 gas in depressurization process. Acidity
and amino acidity were unchanged for both treatments. The composition of sugars,
organic acids and free amino acids were unchanged after the treatment. The flavor
components were decreased in carbonated sake than pasteurized sake which unchanged
compare to untreated sake. This result is influenced with sake meter results and alcohol
level measurements. The changes of sake during storage at 3oC and 20oC were also
measured. The amino acidity of heat-treated and carbonated sake remained stable for both
temperatures. Untreated sake amino acidity increased which was negative result because
it will caused undesirable tastes. Measurements of sake meter, alcohol level and acidity
were stable during storage at those temperatures except for acidity that slightly decreased
at 20oC for every sample. The sugars component concentration also stable during storage
for both treated samples. The untreated sake glucose content was increased due to
difference of enzyme activity even during storage at 3oC, but it was negative effect which
cause undesired taste. The content of free amino acids, aspartic acid, glutamine and
ammonia in sake during storage at 3oC and 20oC also remained almost unchanged for
both carbonated and heat-treated sake samples. The total free amino acids content in
untreated sake at 20oC were increased, while content of aspartic acid and ammonia were
increased at both temperatures. Formation of the main flavor component in sake,
isovaleraldehyde is also measured during storage. It shows that level of isovaleraldehyde
did not increase in both treated samples. Untreated sake sample shows increasing level of
isovaleraldehyde formation during storage which caused undesired taste. The sensory
evaluation shows that taste of carbonated sake flavor and total quality before storage is
slightly worse compare to heat-treatment and untreated sake. After 6 month storage
carbonated and heat-treatment sake were both retained good than untreated sake.
Conclude that high-pressure carbonation avoided unacceptable changes in sake taste and
flavor during storage and were effective to preserve sake at normal temperatures.
 Bioprocess Engineering Assignment: Journal Summary

Enzyme Inactivation and Quality Preservation of Sake by High-Pressure

Carbonation at a Moderate Temperature

Lecturer: Mr. Goh Kian Mau

KHAIRUL FIKRIAH BINTI HAMDAN

AS060117
870405025628
2SSB