Note: Descriptions are shown in the official language in which they were submitted.
~ ~ ~2 1~ ~
The present invention relates generally to a
method of preparing a beer. More particularly, it relates
to a method of preparing a low calorie beer which comprises
introducing an enzyme extracted from rice, a traditional
brewing source, into the brewing process. It also relates
to a method of extracting the enzyme from whole or polished
rice and a storage stable form of the enzyme.
In the production of beer, yeast is used to ferment
into ethyl alchohol a substrate made of a mixture of ferment-
able carbohydrates. The wort carbohydrates involved whichcan be fermented by brewer's yeast are primarily maltose,
glucose, maltotriose and traces of sucrose and fructose.
They are obtained by allowin~ malt enzymes (alpha and beta
amylase) to transform starch molecules from malt and other
adjuncts into the fermentable sugars outlined above. This
is done during the mashing operation. Following mashing the
soluble materials are extracted during lau-tering, leaving
behind the spent grain. A clear liquid (wort) is obtained
which is transferred to a brew kettle and boiled for a
period of time (kettle boil) to inactivate all malt enzymes.
Hops are usually added at kettle boil after which the wort
is cooled, aerated, pitched with yeast and allowed to fer-
ment. Wort compositions vary depending on the bill of
materials, mash cycle employed, etc. However, a typical
wort is made up of approximately 65 to 80% fermentable
carbohydrates of the type mentioned before and about 20 to
35% nonfermentable carbohydrates. After fermentation a
beverage is obtained which usually contains from 3 to 5~
alcohol with approximately equal amounts of residual dextrin
forming the bulk of dissolved solids, commonly referred to
as real extract. This residue remains because of the inabil-
ity of malt amylases -to hydrolyze the alpha 1-6 linkages of
,_i,
~Zl(14
the starch. When the wort described below is fermented a
product is obtained which contains approximately 110 calor-
ies per 12 oz. bottle when packaged at 3.3g/100 ethanol.
In the production of low calorie, superattenuated
beers, an attempt is made to obtain a higher proportion of
alcohol and a much lower amount of residual dextrin. This
results in a beer which has a lower specific gravity at end
fermentation than normally obtained. The first superatten-
uated products made were produced by a process which con-
sisted of adding an external enzyme in ~he fermenter. That
particular enzyme, a glucoamylase, has the capability of
hydrolyzing both alpha 1-4 and alpha 1-6 linkages of the
starch and is usually obtained from the mold Aspergillus
niger. The use of glucoamylase is not without certain
disadvantages. They are the following:
(a) The enzyme has some difficultv hydrolyzing the
alpha 1-6 linkages. It is much more efficient at
hydrolyzing alpha 1-4 linkages, and,
(b) The enzyme may be considered to be exogenous to
the brewing process. That is, it is not present
nor is it isolated from traditional brewing mater-
ials, e.g., malt, rice, corn, or yeast.
Another approach which has been suggested consists
of using an alpha 1-6 carbohydrase or pullulanase combined
with a beta amylase of microbiological origin.
There are three basic classes of starch debranching
enzymes. They are the glucoamylases, the isoamylases, and
the pullulanases. The distinctions between these classes
are well known to those skilled in the art. Basically,
pullulanases cleave the alpha 1,6 linkages of pullulan (an
alpha 1,6 polymer of maltotriose isolated from a mold cell
wall) to yield maltotriose. Pullulanases are specific for
~ 2~63~
alpha l,6 linkages and can debranch the wort limit dextrins
producing alpha 1,4 polysaccharides which can be converted
by various alpha 1,4 carbohydrases to sugars which are
fermentable ~y brewer's yeast.
Attempts have been made in the past to isolate a
debranching enzyme from sources germane to beer production,
such as malt. The so-called "R enzyme" has been reported in
the literature. It seems, however, that to date a good
efficient way of isolating the "R enzyme" has not been
found.
It is the general object of the present invention
to disclose the discovery that a well known traditional
brewing material can be used as a source for a debranching
enzyme to produce a superattenuated beer.
The present invention broadly comprises using rice
as the source of debranching enzyme in the preparation of a
low calorie beer. It also includes a method of isolating
the debranching enzyme from rice.
Rice has traditionally been used in the brewing
industry. Usually it is used as an adjunct, or an addi-
tional source of carbohydrates, like corn grits or corn
syrup. The rice used for this purpose is usually a food
grade rice, that is rice which has been put through the
conventional drying process and subsequently dry milled.
Brewers generally use the broken kernels from the polishing
operation. The traditional process consists of using rice
in the cereal cooker. Usually some malt is added, together
with enough water so that some conversion of rice starch is
obtained in the cooker. This mixture is cooked for a period
of time and added to the mash where malt enzymes convert the
starch from the malt and the rice into fermentable carbohy-
drates. The rice adjunct used in this fashion has no en~yme
3ll.~*Z1~4
activity, all of it having been inactivated in the cereal
cooker. The traditional process, therefore, does nok use
rice as a source of enzyme.
We have discovered that the use of a debranching
enzyme which naturally occurs in rice provides good results
when used in the brewing process to prepare a low calorie
beer.
The debranching enzyme from rice originates in a
traditional brewing ingredient, and it appears to be more
effective than glucoamylase in reducing the highly branched
high molecular weight dextrin fraction.
The method of the present invention is an improve-
ment in the method of producing a superattenuated beer by
fermenting brewers wort with yeast which comprises adding a
rice pullalanase in an amount effective to reduce the amount
of residual dextrins in the real extract by cleaving the
alpha 1,6 linkages of limit dextrins to form alpha 1,4 dex-
trins which are converted by 1,4 carbohydrases to ferment-
able sugars which are ~ermented by the yeast to alcohol.
The enzyme can be introduced at various stages. In a pre-
ferred embodiment, either rice or the debranching enzyme
extracted from the rice is added to the wort which contains
grain amylase from a suitable source, i.e., malt at the
fermentor. The debranching enzyme from the rice hydrolyzes
the residual 1-6 linkages of the limit dextrins and the
grain amylase cleaves the resulting linear alpha 1~4 poly-
saccharides into fermentable sugars which are then converted
to ethanol by the yeast.
In another embodiment of the method, the debranch-
ing enzyme extracted from the rice is added to the mash tohelp cleave the 1-6 linkages of the limit dextrins which
otherwise would be formed. The natural malt enzymes hydrol-
~14;~
yze the 1-4 linkages thus producing higher levels of fer-
mentable sugars.
Beer of palatable quality can be produced by each
of the above-described procedures. In each instance, the
end product has been found to contain a greater proportion
of alcohol to real extract and fewer calories per unit of
volume when packaged at constant alcohol than a control beer
produced with no enzyme addition.
The enzyme which has been found to be useful in
the preparation of a low calorie or superattenuated beer is
a starch debranching enzyme which naturally occurs in rice.
The enzyme of the present invention is classed as a pullul-
anase because it hydrolyzes the alpha 1,6 linkages of the
diagnostic substrate pullulan.
In the extraction method of the present invention
the enzyme is extracted from whole or commercially polished
rice with an aqueous buffer system having a pH of about 6 at
temperatures ranging from 0-60C. The preferred conditions
are to slurry polished rice in 0.1 M potassium phosphate
buffer--0.2 M NaCl, pH 6.0 at about 50C for about 3 hr.
The pullulanase-containing supernatant from the
extraction may be further purified by~ acidification of
the crude extract; and (2) precipitation of the rice enzyme
with (NH4)2S04. These procedures will be illustrated in the
examples below.
The enzyme may be stored in a liquid form, or as a
freeze-dried or spray dried powder. The freeze dried powder
is obtained by diafiltering the enzyme containing buffered
pH 6 extract against 0.lM ammonium bicarbonate solution.
Ammonium bicarbonate is a volatile salt which sublimes on
freeze drying to yield a salt free enzyme powder. Other
sublimable salts which do not interfere with the process or
adversely affect the enzymatic activity also may be used if
desired.
The amount of rice or extracted enzyme to be added
in the brewing process depends on many factors such as the
enzymatic content of the rice, the activity of the enzyme,
the stage of the brewing process at which it is added and
the brewing conditions, e.g., pH, temperature, and time.
Generally, the amount of rice or extract to be added will be
an amount effective to reduce the amount of residual limit
dextrins in the real extract by about 30 to about 80%.
Normally, for preconversion of the dextrins prior to fer-
mentation an enzyme source, either extract or rice, con-
taining from about 100 units to about 300 units of pullul-
anase activity per liter will be added to the wort or about
300 units to about 700 units per liter to the mash. Smaller
amounts containing about 2 units to about 75 units of pull-
ulanase activity per liter are effective when added to the
wort in the fermenter. Larger amounts than those normally
used can be used if desired or needed. Obviously, some
testing may be required to determine the precise amounts to
be used. However, such testing and determination are well
within the skill of those skilled in brewing art.
In one embodiment of the method, the debranching
enzyme is added to the fermentor with a carbohydrase, such
as a grain diastase. This combination is required because
the rice debranching enzyme cleaves the highly branched
alpha 1,6 limit dextrins, and the added carbohydrase cleaves
the resulting alpha 1,4 dextrins into sugars that can be
used by the yeast. The effective amounts of each enzyme to
be added will depend upon the content of limit dextrins nor-
mally present in the product of the fermentation and the
extent of caloric reduction desired. Normally, -the debranch-
Zl~
ing enæyme will be present in an amoun-t of about 2 units to
about 60 units of pullulanase activity per liter of wort and
the carbohydrase or grain diastase will be present in an
amount ranging from about 20 units per to about 140 units of
amylase activity per liter of wort.
In still another embodiment, the addition of the
rice debranching enzyme to the fermentor is accompanied by
the addition of a glucoamylase such as that derived from
Aspergillus niger which is active vs. both alpha 1,6 and
alpha 1,4 linkages. The introduction of the combination of
these two enzymes at the fermentation stage significantly
reduces the fermentation time normally required to prepare a
superattenuated beer, e.g., from 12 to 7 days. Although
both of the enzymes possess debranching activity the rice
enzyme is more potent than glucoamylase and as a result the
fermentation time is reduced. The concentration of the rice
enzyme in such a mixture may be lower than that normally
employed, e.g., 2-4 units of pullulanase and the gluco-
amylase will be present in about 2 units to about 10 units
of glucoamylase activity per liter.
The following analytical procedures were used in
the examples described below. Protein was determined by the
Lowry method as modified by Miller (l). Pullulanase activ-
ity was determined by hydrolysis of 0.5% w/v pullulan at pH
5.0 and 50C. Amylase activity was determined by the hydrol-
ysis of 0.5% w/v Linter soluble starch at pH 5.0 and 50C.
The appearance of reducing sugars was monitored by the
dinitrosalicylic acid method of Bernfield (2~. A unit of
activity in both assays was defined as the appearance of l
mg reducing sugar (as maltose)/minute. Specific activities
are expressed as units/mg protein.
~ lucoamylase activity was determined by a modi-
fication of the method of Pazur (3), using maltose as
1~9LZ~
substrate, at pH 5.0 and 25C. The appearance of glucose
was monitored using the coupled glucose oxidase-peroxidase
reaction with o-dansidine as the indicator dye (3). A unit
of activity was defined as the hydrolysis of 1 micromole
maltose/minute under these conditions.
Fermentations were monitored by the decrease in
specific gravity using the Mettler DMA-45 calculating den-
sitometer. When the beers were judged to be end-fermented,
refractive indices were obtained on a Zeiss immersion re-
fractometer. These measurements were used to calculate the
alcohol ~4,5,6) and real extract (5,6) of the beers. The
caloric content of a standard 12 oz. container was calcu-
lated at 3.3 g~100 ethanol, as described by Helbert (7).
Carbohydrate profiles were obtained by high-
pressure liquid chromatography on Bio Rad Q 15S resin as
described by the ASBC Subcommittee on brewery sugars and
syrups (8) and by Scobell, et al (9). Unless otherwise
stated, all diafiltrations were performed on an Amicon DC-2
apparatus equipped with an H-lP-10 cartridge (m.w. cutoff =
10,000) (Amicon Corporation, Lexington, Mass.).
Examples 1-5 will illustrate the isolation and
some properties of rice pullulanase.
Example 1. Isolation of pullulanase from
whole rice. Fi~e-hundred grams seed grade LaBelle rice were
blended in 0.1 M potassium phosphate buffer--0.2 M NaCl, pH
6.0, using a Waring blender. The blended grain was trans-
ferred to a vessel in a bath maintained at 50C and stirred
under 2 liters buffer for 3 hr. The spent grain was removed
by filtration through cheesecloth and the filtrate clarified
by centrifugation.
Further clarification may be achieved by reducing
the pH of the extract to 5~0. The resulting precipitate was
~421~4
removed by centrifugation, and the pH of the supernatant was
readjusted to 6Ø
The extract may be purified and concentrated by
(NH4)2S04 fractionation. This salt was added to the pH
adjusted supernatant at the rate of 40 g solid (NH4)2SO4 per
100 ml solution. The suspension was stirred for 1 hr. at
room temperature, and the precipitate was removed by centri-
fugation. The precipitate was dissolved in and diafiltered
vs. the extraction buffer. Table 1 summarizes the extrac-
tion of whole rice detailed above.
Example 2. Localization of pullulanase withinthe rice kernel. LaBelle rice from Example 1 was pearled
and the following fractions isolated: (13 husks; (2) brown
or dehusked rice; (3) rice bran; and (4) polished white
rice. Each fraction was extracted and clarified as de-
scribed in Example 1 for whole rice. Analysis of these
extracts, summarized in Table 2, revealed that the great
majority of pullulanase activity was localized in the endo-
sperm (polished rice). In addition, this preparation had a
much greater specific activity than preparations obtained
from either whole or brown rice. Thus, polished rice is the
preferred enzyme source.
Example 3. Extraction of pullulanase from
pollshed rice. Two kilograms of commercially polished rice
were ground to .02 inch in a barley mill. The ground rice
was doughed into 4 liters of pH 6 extraction buffer, and the
suspension was stirred for 3 hours at 50C.
The pH of the extract was adjusted to 5.0, and the
resulting supernatant was clarified by centrifugation. The
pH of the supernatant was readjusted to 6Ø
For long-term storage, it was desirable to obtain
the preparation as a salt-free powder. This was accomplished
_g_
2~
by diafiltering the supernatant from the pH adjustment step
vs. 0.1 M NH4HCO3. This salt was chosen since: (1) the
preparation requires salt to remain in solution; and (2)
NH4HCO3 sublimes and is removed by subsequent freeze drying.
After diafiltration vs. 4 volumes of 0.1 M NH4HCO3,
the retentate was freeze dried. ~he results of this extrac-
tion are summarized in Table 3.
Example 4. ~H optimum of rice pullulanase. The
p~ optimum range was determined on rice pullulanase isolated
by the procedure outlined in Example 3. The following
buffer systems were used: (1) pH 4.0-5.5--0.1 M acetic acid
ad~usted to the appropriate pH with NaOH; (2) pH 6-7 0.1 M
KH2PO4 adjusted to the appropriate pH with NaOH. Stock
pullulan (10% w/v in H2O) was diluted to 1% w/v in the
appropriate buffer. Rice pullulanase was then assayed over
the pH range 4-7 under standard conditions. The results
indicated that optimal activity is obtained in the pH range
of 5-6.5.
Example 5. Temperature optimum of rice
pullulanase.
(A) In the absence of substrate. Rice pullulan-
ase prepared as described in Example 3 was made to a final
concentration of 2 mg/ml in 0.1 M acetate buffer, pH 5Ø
Aliquots of this mixture were incubated in the temperature
range 40-70C for times ranging from 10-60 min. Aliquots of
the incubates were withdrawn, cooled, and subjected to the
standard pullulanase assay. The results illustrated in Fig.
3 show that the enzyme was rapidly inactivated at tempera-
tures in excess of 40C. Complete inactivation occurred at
60C after 10 min.
(B) Presence of substrate. Rice pullulanase
prepared as described in Example 3 was made to a concen-
-10-
11~2104
tration of 1 mg/ml in 0.1 M acetate pH 5Ø Ali~uots of
this preparation (sufficient to yield a final concentration
of 0.2 mg/ml incubate) were delivered into tubes containing
pullulan and 0.1 M acetate buffer, pH 5.0 which had been
equilibrated to the desired temperature. At each tempera-
ture (ranging from 40C-70C), l-ml aliquots were withdrawn
after 10, 20, and 30 minute incubations and inactivated by
delivering them into the dinitrosalicylic acid solution used
for color development. The reducing sugars were determined
in the standard manner.
From the results of these experiments is is ap-
parent that the enzyme is stable in the presence of sub-
strate up to 60C for 30 min. This is in marked contrast to
the temperature stability of the enzyme alone as described
in Example 5-A.
Examples 6-15 will illustrate the application of
rice debranching enzyme (pullulanase) to the brewing process.
Examples 7-12 will illustrate the use of the enzyme in com-
bination with various alpha 1,4 carbohydrases with ferment-
ing beer, while Examples 13 and 14 will illustrate its use
prior to fermentation. In all cases/ the wort used was
mashed as an all-malt wort and was adjusted to about 12 to
about 15P with a commercial converted corn-derived syrup,
prior to fermentation. In the examples which follow the
original gravity was constant. The worts were pitched with
a stock brewing culture of S. uvarum to a final concentra-
tion of 1 x 107 cells/ml and fermented at 15C.
Example 6. Preparation of grain diastases for
use with rice pullulanase.
(A) Malt diastase. High-gib distiller's malt was
ground in a standard barley mill. The powder (150 g) was
doughed into 1.5 liters 0.1 M acetate buffer, pH 5Ø The
slurry was stirred for 2 hr. at 50C and the supernatant
recovered as described for rice crude extract (Example 1).
The enzyme was further purified by adding
(NH~)2SO4 to a final concentration of 40 g/100 ml. The
precipitate was harvested by centrifugation and resuspended
in 0.1 M acetate buffer pH 5Ø The suspension was clari-
fied by diafiltration vs. the same buffer, concentrated and
stored at 4C.
(B)_ Preparation of malt beta-amylase. Malt
diastase prepared as in Example 6-A contains both alpha- and
beta-amylase, with alpha~am~lase in greatest concentration.
Malt alpha-amylase can be seiectively inactivated at acid pH
(9). The pH of a portion of the malt diastase, prepared as
described in Example 6-A, was adjusted to 3.6 and incubated
at 35C for 2 hr. The solution was clarified by centrifuga-
tion, and the pH of the supernatant was readjusted to 5Ø
(C) Preparation of soybean diastase. Whole
soybeans were ground in a Wiley mill using a 20 mesh screen.
Ten gm of powder were stirred in 100 ml .01 M acetate buffer,
pH 5.2 at 55C for 1 hr. The solution was clarified by
centrifugation followed by filtration using a filter aid.
The supernatant was diluted 4-fold, diafiltered vs. H2O, and
concentrated to the original volume. The concentrate was
stored at 4C.
(D) Isolation of wheat diastase. Wheat diastase
was isolated from pearled hard winter wheat ground as de-
scribed in Example 6-B for soybean diastase. The powder (50
g) was doughed into 100 ml 0.1 M phosphate buffer--0.1 M
NaCl, pH 6.0, and the suspension was stirred for 3 hr at
50C. The suspension was clarified as described in Example
1 for the rice enzyme.
The supernatant was dialyzed vs. .02 M phosphate
buffer--0.2 M NaC1 and then water.
-12-
2~
Table 4 summarizes the amylase activity of the
grain amylases described in Example 6.
The glucoamylase used in the experiments described
below was Novo 150 obtained ~rom Novo Industries, Wi.lton,
Connecticut.
Example 7. Superattenuation of fermenting beer
using rice pullulanase-malt diastase. The wort formulated
as described above was fermented: (l) with no enzyme addi-
tion (Beer #l) to establish the atten~ation limit; ~2) with
the addition of glucoamylase (Beer #2) to establish the
superattenuation limit; and (3) with the addition of rice
pullulanase and malt diastase (Beer #3). In all cases, the
worts were pitched and aerated as described above after
which the appropriate enzymes were added. The beers were
fermented at 15C. Table S summarizes the enzymes used as
well as their concentration in the fermenting beer in U/1.
Table 6 lists the properties of the end-fermented
beers described above. The enzyme-free control contained
0.5-0.6 g/100 less alcohol than did either Beer #2 or #3
which were superattenuated with glucoamylase and rice pull-
ulanase/malt diastase, respectively. When packaged at 3.3%
ethanol the real extract in Beer #3 was reduced by about 1.0
g/100 over that in Beer #1 and was nearly identical to that
obtained when glucoamylase was used (Beer #2). At this
alcohol concentration Beers #2 and #3 would contain 92-93
cal/12 oz as opposed to 108 cal/12 oz for Beer #l.
The carbohydrate profiles summarized in Table 7
show that Beers #2 and #3 have nearly identical carbohydrate
compositions at end-fermentation and that in both Beers #2
and #3 the nonfermentable sugars ~greater than DP-3) are
substantially reduced over that obtained in Beer #1.
Example 8. Superattenuation of fermenting beer
using rice pullulanase-soybean diastase. The wort was
aerated and pitched as in Example 7. Rice pullulanase and
soybean diastase were added according to the schedule listed
in Table 5. The beer was fermented as described in Example
7.
The end-fermented beer (Beer #4, Table 6) super-
attenuated to about the same degree as the glucoamylase
control (Beer #2), yielding a beer of 93.4 cal/12 oz when
packaged at 3.3 g/100 ethanol. The carbohydrate profile is
given in Table 7 (after 12 days of fermentation) and shows
that the nonfermentable fraction was nearly identical to
that of the glucoamylase control (Beer #2).
Example 9. Superattenuation of fermenting beer
using rice pullulanase-wheat diastase. The wort was aerated
and pitched as described in Example 7. Rice pullulanase and
wheat diastase were added as shown in Table 5. The beer was
fermented at 15C as described in Example 7.
Reference to Table 6 shows that this beer (Beer
#5) superattenuated to the same level as the glucoamylase
control (Beer #2). When packaged at an alcohol concentra-
tion of 3.3 g/100 ethanol, the beer would contain 92.5
cal/12 oz. The carbohydrate composition shown in Table 7
shows that the nonfermentable fraction was nearly eq~al to
that of Beer #2.
Example 10. Superattenuation of fermenting beer
with rice pullulanase-malt beta-amylase.
The wort was aerated and pitched as described in
Example 7. Rice pullulanase and malt beta-amylase (Example
6-B) were added as described in Table 5 and the beer fer-
mented at 15C in the normal manner.
The results in Table 6 show that this beer (#6)
end-fermented in 8 days, which was faster than the gluco-
amylase control ~Beer #2) or any of the beers formulated
-14-
with rice pullulanase in conjunction with the other grain
diastases. Again, the carbohydrate composition was similar
to that of the glucoamylase control as shown in Table 7. A
beta-amylase appears to be superior to any of the diastases
(containing both alpha- and beta-amylase) used in the previ-
ous examples.
Example 11. Superattenuation of fermenting beer
with rice and malt flours. Polished #4 brewer's rice and
high gib distiller's malt were ground to 20 mesh in a Wiley
mill. They were added to the wort aerated, and pitched as
described above. One wort (Beer #7, Table 6) contained 5.2
g rice flour and 0.12 g malt flour/liter, while the other
(Beer #8) contained twice as much of each flour. The worts
were fermented as described above. The grain additions to
Beer #7 were calculated to yield 15.4 units pullulanase per
liter and 140 units of malt diastase per liter based on
extraction as illustrated in Examples 3 and 6-A, respec-
tively.
The data presented for Beers #7 ~nd #8 in Table 6 shows
that both beers attenuated to the same level as the gluco-
amylase control (Beer #~) and the beers illustrated in
Examples 7-11 in which enzyme extracts were employed.
Example 12. Rice pullulanase used with glucoamylase
to shorten fermentation time. The wort was used to set up 5
separate fermentations. All the worts were aerated and
pitched after which rice pullulanase and glucoamylase were
added as described in Table 8. From the results in Table 9,
it can readily be seen that rice pullulanase significantly
shortened the fermentation time over th~ glucoamylase control
even at reduced glucoamylase (Beers #11 and 12) or pullulan-
ase concentration (Beer #13).
Example 13. Conversion of all-malt wort prlor
to fermentation with rice pullulanase-malt beta-amylase. An
1~21(~4
all-malt wort was obtained following kettle-boil. Three
wort samples were converted with malt beta-amylase in con-
junction with decreasing concentrations of rice pullulanase.
Another sample of the wort was converted using rice pullul-
anase in conjunction with glucoamylase.
In all cases the procedure was the same. The
worts were equilibrated at 60C with stirring in a water
bath. The enzyme concentrations were adjusted to the levels
shown in Table lOA. Incubation was allowed to continue for
30 minutes after which they were delivered into a flask
contained in a vigorously boiling water bath. They were
al'owed to remain there for 2 hr to inactivate the enzymes.
The worts were then cooled, and the resulting trub was
removed by centrifugation.
The malt to syrup adjunct ratio was adjusted to
the same level as the wort described in Examples 7-12. The
worts were then pitched, aerated, and fermented as described
in Example 7.
The alcohol and real extract are summarized in
Table 11. The beers were superattenuated relative to the
no-enzyme control (Beer #1, Table 6~. When packaged at 3.3
g/100 ethanol, the caloric content of these beers would be
about 98 calories, some 10 calories less than Beer #1 for-
mulated with no enzyme addition.
; Example 14. Addition of grain amylases to pre-
converted beers Since the beers cited in Example 13 did
not superattenuate to the same level as the beers cited in
Examples 7-12, various enzymes were added to see if the
attenuation limit could be decreased. The yeast was removed
from Beer #14 by centrifugation, and the clarified beer was
split into two equal portions designated 14A and 14B. Both
beers were repitched and received the enzymes listed in
Table lOB. Fermentation was then continued at 15~C.
-16-
~21~
Beers #15A and #16A were not repitched. Instead,the enzymes were injected directly into the fermenting beers
as described in Table lOB.
The results of the secondary fermentation are
listed in Table ll. Addition of the alpha 1,4 carbohydrase,
malt beta-amylase, did not significantly reduce the specific
gravity (Beer #14A), suggesting that most of the nonferment-
able sugars contained alpha 1,6 linkages. In contrast, malt
beta-amylase in conjunction with rice pullulanase ~Beers
#14B and #16A), or rice pullulanase alone (Beer #15A),
superattenuated the beers to the same level as the gluco-
amylase control (Beer #2) or the beers described in Examples
7-12.
Example 15. Addition of rice pullulanase to
w~rt ~t ~a~h in. Rice pullulanase, prepared as described in
Example 3, was added to 400 ml foundation water to a final
concentration of 520 U/1 at 46C. Then, 129.6 g pale malt
were doughed in, and the mash was subjected to the following
mash cycle^ (1~ 46C for 30 min; and (2) 60C for 120 min.
The brew was mashed-off at 77C after which the spent grain
was removed by filtration through cheesecloth. The first
wort was clarified by centrifugation, diluted, and placed in
a boiling water bath for 2 hr. All subsequent steps were as
described in E~ample 11.
This beer end-fermented to a final specific
gravity of 1.0019 ~0.49P) as opposed to 1.0029 (0.75P) for
Beer #1, the no enzyme control cited in Example 7. Thus,
incorporation of rice pullulanase in the mash reduced the
attenuation limit by 0.26P
104
REFERENCES
l. Miller, G . Anal. Chem. 31, 964, 1959.
2. Bernfield, P. Advances in Enzymology XII (Nord,
F., ed.) 379, Intersciences Publishers, New York,
1951.
3. Pazur, J. Methods in Enzymology XXVIII, Ginsberg,
V. (ed.~ 931, Academic Press, 1975.
4. Kneen, E. (ed.). "Alcohol Determined Refracto-
metrically" in Methods of Analysis of the
American Society of Brewing Chemists, 7th Revised
edition, published by the Society, 1976.
5. Olshausen, J. Brewers Digest 27, 45, 1952.
6. Olshausen, J. Brewers Digest 27, 53, 1952.
7. Helbert, J. R. J. Amer. Soc. Brew. Chem. 36,
66, 1978.
8. Martinelli, L. (Chairman) ASBC Journal 35,
p.104, 1978.
9. Scobell, H., Brobst, K. and Steele, F. Cereal
Chem. 54, p.905, 1975.
10. Greenwood, C. and A. ~acGregor. J. Inst. Brew.
71, 408, 1965.
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Amylase Specific Activity U/mg Protein
. .
Malt diastase 81.1
Malt beta-amylase13.9
Soybean diastase 9.0
Wheat diastase 31.5
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The rice which may be used as the source of the
enzyme of the present invention is food-grade rice which has
been treated at conditions mild enough to preserve the
enzymatic activity. Either seed rice or polished dry milled
rice may be used. The enzyme may be extracted from a wide
variety of rice, including LaBelle, LeBonnet, Nato, Star-
bonnet, or Brazos. However, commercially polished dry
milled brewer's rice is preferred.
If the enzyme is extracted from rice prior to use in
this process, the spent rice from which the enzyme has been
extracted can be utilized as a starch source in mashing or
for adjunct syrup formulation to make the use of the rice
enzyme more economical.
Although the use of the rice debranching enzyme
which has been described is its use in preparing a low
calorie or superattenuated beer, it might possibly have
other applications. For example, a mixture of the rice
debranching enzyme and a grain diastase may be advantag-
eously used to prepare a starch conversion product having a
high maltose content. Because of its natural origin, the
debranching enzyme from rice would no doubt be approved for
use in food products without too much difficulty.
It will be apparent to those skilled in the art
that a number of modifications and changes can be made
without the departing from the spirit and scope of the
present invention. Therefore, it is intended that the scope
of the invention be limited only by the claims which follow.
