Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
8~7
This invention relates to a new and improved method
in the production and use of a fun~al enzyme system described
in ~anadian Patent No. 822,079 issued to the present applicant
on 2-September 1969, and naming J. M. Van Laren and M. B. Smith
as inventors; (and in U. S. Patent 3,418,211 issued 24 December,
1968). More particularly, the invention is concerned with the
control of the pH of a cereal ~rain medium during the enzyme
cultivation in order to retain the alpha amylase (enzyme
No. 3.2.1.1) which is naturally produced together with
glucamylase (enzyme No. 3.2.1.3). This alpha amylase would
otherwise be destroyed by the reduction in pH occurring during
the cultivation.
Prior art (U. S. Patents Nos. 2,~51,567; 2,881,115;
2,893,921; and 3,012,944) reveals that a number of species and
strains of the Aspergillus niger group are capable or producing
starch-hydrolyzing enzymes under submerged, aerobic growth
conditions. For the most part, the enzyme of interest in this art
is that which converts starch, dextrins, and maltose substrates
to dextrose and which has been variously nam~ glucamylase, amylo-
glucosidase, glucoamylase, gl~cogenic activity, and maltase.The activity of this enzyme may be measured by the rate of con-
version of one of the indicated substrates to dextrose. Enzyme
activity may also be measured by the rate of yeast fermentation
and the consequent yield of alcohol from these substrates. The
term "glucamylase" will be used hereinafter to describe the starch-
hydrolyzing enzyme produced in the process and "glucamylase
ferment" to describe the whole final ferment containing enzymes
which attack starch and proteins.
¢;i 7~
In the prior art, a selected or derived culture of
Aspergillus has been combined with particular substrate and
growth conditions ~o yield ferments of high glucamylase enzyme
activity. In aforementioned Canadian Patent No. 822,079, two par-
ticular very active Aspergillus cultures are combined with sub-
strates which are preferably of the nature hereinafter described
for the production of glucamylase and other enzymes essential to
near-maximum rates of yeast fermentation and to near-complete
conversion of starch to fermentable sugars. One o~ the other
types of enzyme is produced in substantial amounts in the process
and which is especially important in grain yeast fermentation is
proteinase. This enzyme makes available yeast nutrients from
grains which promote yeast growth and fermentative activity. As
a result, the glucamylase ferment that results from the practice
of the process of Canadian Patent No. 822,079 possesses exceptional
activity as a fermentation supplement even in instances where
conventional malt fermentation is conducted.
In general, an enzyme system for saccharifying and fer-
menting grain mashes can be produced in accordance with Canadian
Patent No. 822,079 in the following manner: In the preferred
approach, a mediul~ is prepared from ground cereal grain, other
beneficial nutrient supplements and water, and the pH of the medium
is adjusted to 7. The medium is then sterilized and cooled to
between ~7 and 35 C. Finally, the sterilized medium is inocu
lated with a growing culture o one of the highly active strains
under submerged, aerobic conditions (i.e., preferably vigorously
aerating and agitating the medium for a period of 3-7 days at
27-35 C.) To obtain high enzyme potencies the grain concen-
tration should be relatively high as compared to prior art prac-
tices and should pre~erably range bet~Jeen 12 and 20 g. grain per
7?7
100 ml of medium. The nutrient supplements that have been dis-
covered to greatly increase the rate o~ glucamylase production
comprise grain stillage (the dealcoholi~ed, liquid-grain residue
from a previous grain alcohol fermentation) and distillers dried
solubles (the product obtained by removing alcohol and grain
screenin~s from grain stillage to produce thin grain stillage and
then evaporating and drying the fraction). These supplements may
be used on a dry substance basis at the rate of from 1 to 10 per-
cent based on the total weight of the final medium, with the pre-
ferred level being 3 to 6 percent of the total weight of the finalmeidum.
To achieve high yields of glucamylase, aeration and agi-
tation must be of such an intensity to provide an excess (i.e.,
3 ppm or more) of dissolved oxygen in the ferment throughout the
fermentation cycle. This is accomplished by an aeration rate
between 0.25 and 1 volume of air pex volume of medium per minute
and by agitation corresponding to that provided by a power input
of from 0.5 to 2.5 horse power per 100 gallons of medium. Pure
culture conditions should be employed at all stages of inoculum
development and fermentation, and the air used should be steri-
li~ed by a suitable means.
-- 3 --
7~
To determine activity in the saccharification and fer-
mentation of grain mashes the following procedure may be used.
Grain meal with about 0.5% by weight of barley ~alt (premalt) is
slurried in water and cooked at 95-120 C. to gelatinize the
starch. The mash is cooled to about 65 F. and saccharified with
glucamylase ferment at the rate of about 500 ml per bushel of grain
or with the same amount of glucamylase ferment and malt at the rate
of 0.5 to 2~ by weight of grain. Other microbial amylases may be
used in place of malt to liquefy the starch. The mash is then
cooled to 27-32 C~, inoculated with an active culture of yeast,
and allowed to ferment. The activity of different glucamylase fer-
ments may then be determined by weighing the flasks at intervals
(weight losses due to carbon dioxide evolution being measures of
fermentation rates) and by determining alcohol at intervals of
incubation and after fermentation is essentially completed.
Two strains of Aspergillus niger have been found to be
.
most suitable for the indicated saccharification and fermentation,
namely, Aspergillus niger NRRL 3112 and Aspergillus nlger NRRL 3122.
~hese two cultures are mutants that were obtained by irradiation.
When grown in the media and under the conditions described herein,
ferments of these cultures are more active and efficacious than any
previously utilized in the production of grain alcohol, as is more
fully indicated by Examples 1-6 of Canadian Patent No. 822,079, to
which readers are referred.
77
The present inventors have found that it is possible to
retain alpha amylase together with glucamylase for subsequent use
in the liquefaction, saccharification and ~ermentation of cereal
grains, starch and starch products to grain spirits, by carefully
controlling the pH of the cultivation medium during the aerobic
cultivation of such strains. Such an enzyme system as obtained by
the controlled pH cultiviation eliminates the requirements of sup-
plemental starch liquefying enzymes such as those found in barley
malt, bacterial amylases, etc. The inventors have also found that
such controlled pH cultivation results in higher concentration Of
glucamylase.
In one aspect of this invention there is provided a
process of producing an enzyme system characterized by high gluc-
amylase and alpha amylase potency comprising the cultivation under
submerged aerobic conditions of a strain selected from the group
consisting of Aspergillus niger strains NRR~ 3112 and 3122 in a
cereal grain medium of high concentration, the pH of said medium
being maintained within a range between 4.4 and 6.The pH level of
the medium is preferably between 4.5 and 5.5.
2~ In another aspect of this invention, there is provided a
process of producing an enzyme system characterized by high gluc-
amylase and alpha amylase potency comprising the cultivation under
submerged aerobic conditions of the enzyme system produced by the
process mentioned above in a cereal grain medium of high concen-
tration, the pH of said medium being maintained within a range
between 4.4 and 5.5.
In a further aspect of this invention, there is provided
a process of conducting liquefaction, saccharification and fermen-
tation of a starch product to an alcohol product comprising the
addition to the starch product of an enzyme system characterized
by high glucamylase and alpha amylase potency produced by the cul~
tivation under submerged aerobic conditions of a strain selected
from the group consisting of Aspergillus niger strains NRRL 3112
and 3122 in a cereal grain medium of high concentration, the pH of
said medium bein~ maintained within a range between 4.4 and 6 during
the cultivation.
In still a further aspect there is provided a process
of conducting liquefaction and saccharification of a starch product
to yeast mash comprising the addition to the starch product of an
enzyme system characterized by high glucamylase and alpha amylase
potency which is prepared by the cultivation under submerged aerobic
conditions, the enzyme system produced in accordance with the process
as defined above, in a cereal grain medium of high concentration,
the pH of said medium being maintained within a range between 4.4
and 5.5.
In order to maintain the desired level of acidity of the
cultivation medium during the cultivation period, a culturally
acceptable basic agent is added to the medium as needed. The agent
may be any basic agent so long as it is culturally acceptable and
it is suited in view of end use of the cultivation producks. Gaseous
ammonia is preferred as it also serves as a nitrogen source. Gaseous
ammonia is preferably introduced i~to the medium by adding it into
the air stream to be fed into the fermentor. A solution of sodium
or ammonia hydroxide or the like may be used in place of the ammonia
The pH of the medium may go up to about 5.7 to 6 during
subsequent use of the enzyme system. However excessive pH rise
should be avoided as much as possible, or should be kept to as short
a time as possible.
Examples 1 and 2 ln Canadian Patent 822,079 show gluc-
amylase yields, respectively without and with grain stillage as a
nutrient. Neither Example 1 nor Example 2 involved a controlled
pH level of the cultivation medium. The enzyme systems obtained in
accordance with these examples are said to be superior to commer-
cially used fungal amylase ferments as the greatly increased enzyme
potency of the enzyme systems results in reduction in the amount of
erments to be required and high yield of alcohol.
The following examples of the present invention are set
forth to show superiority of the present invention ovex the prior
art including the above-mentioned Canadian Patent No. 822,079.
Example 1 involves pH level controlling during the cultivation and
a substantial amount of alpha amylase is retained. Example 2 sho~s
that the enzyme system obtained in Example 1, which is character~-
ized by a high concentration of glucamylase and alpha amylase, can
be used to produce a similar enzyme system without malt.
Example 3 demonstrates that an enzyme system of either
Example 1 or Example 2 can be used in place of barley malt in the
production of yeast. Example 4 further shows the use of an enzyme
system according to the present invention in the fermentation of
a grain mash to alcohol compared with regular glucamylase plus
malt and compared with malt converted grain mesh.
Glucamylase activity in ferments may be determined by
the method outlined in U. S. Patent 3, 418, 211, Process of
Producing Glucamylase and an Alcohol Product.
Alpha amylase activity in ferments may be determined
by the methods outlined in the 12th edition of the AOAC, page 184
(1975)-
7~
Example 1
This experiment demonstrates the effect of maintainingthe glucamylase fermentation pH between 4.7 and 5.15 during the
entire fermentation cycle. A glucamylase medium consisting of
115 lbs. corn, 5 lbs. dried grains, 32 gal. tap water, 0.5 lb.
premalt, and 1 lb. conversion malt was prepared for fermentation
as outlined in Example 1 of aforementioned Canadian Patent No.
822,079. In this example, anhydrous ammonia was introduced into
the air stream into the fermentor about 18~1/2 hours after set at
a rate sufficient to maintain the pH between 4.7 and 5~15. Other
basic agents can be used, but the simplicity of the addition of
ammonia, along with any potential advantages of its nitrogen source,
favors its use. The results of this glucamylase fermenation are
shown in Table I.
Table I
Hours of GlucamylaseAlpha Amylase
Fermentation pH units/ml units/ml
_ _ ,
Set 5.15 - -
18 5.15
24 4.8 - -
44 4.7
4.85 5.6 ~7.0
20 89 4.95 8.8 81.7
113 5.15 12.7 114.7
137 5.05 15.6 142.2
161 5.15 20.0 160.0
Note: Set Solids - 18.2
7~
A comparison of this table to Tables I and II of
Canadian Patent No. 822,079 markedly demonstrates advantages to
pH control. The alpha amylase naturally produced during the fer-
mentation is retained for use in the liquefaction of mash in sub-
sequent alcoholic fexmentations. Therefore, the maintenance of
a higher pH in the glucamylase fermentation eliminates the require-
ments for any malt in grain alcohol fermentations (or glucamylase
~ermentations), as will be demonstrated in subsequent examples.
Another advantage of the presently disclosed pH control
during the glucamylase fermentation is higher yields of glucamylase.
Example 2
~his example demonstrates tha~ high yields o~ glucamylase
and alpha amylase can be made with the use of previously made en-
zyme system of high glucamylase and alpha amylase and no barely
malt for liquefaction is needed. A glucamylase medium, consisting
of 100 lbs. corn meal and 32 gallons tap water and 1 pint of an
en7.yme system of Example 1 (in place of 0.5 lbs. premalt) and 3
pints of the enzyme system (in place of 1 lb. conversion malt),
was prepared and set according to procedures outlined in Example
20 1 of aformentioned Canadian Patent No. 822,079. The pH was con-
trolled with gas~ous ammonia and the results are shown in Table II.
Table II
Hours of GlucamylaseAlpha Amylase
Fermentation pH units/ml units/ml
Set 4.65
17-3/4 5.6
41-3/4 4.75 0.65
66-3/4 4.85 - 25.g
113-3/4 4O00 10.3 58.2
137-3/4 5.25 12.7 82.6
161-3/4 5.25 16.0 106.7
Note: Set Solids = 19.7%
7~9
A satisfactory high glucamylase and alpha amylase was
produced without the use of barley malt. Canadian Patent No.
82~,079 describes the beneficial effects on the use of stillage
nutrients on glucamylase yields in Example 2 where ammonia was not
used. In the present example good glucamylase yields were ob-
tained without the use of stillage when ammonia was used, indica-
ting beneficial effects of the ammonia.
Example 3
This example demonstrates that an enzyme system of high
glucamylase and alpha amylase produced as described above can be
used in place of about 1~-1.5% barley malt plus glucamylase at the
rate of about 1 pt.-l qt. per bushel in a corn yeast mash and
still achieve the yeast growth and viability described in U.S.
Patent 3,868,307, issued February 25, 1975 to the present applicant
and naming James M. Van Lanen, Merritt B. 5mith and Weldon F.
Maisch as inventors.
One corn mash consisting of 98.5~ corn, 0.5% premalt,
1% malt plus glucamylase at 1 qt. per bushel was prepared along
with a corn mash consisting of corn plus 1/2 qut./bu. of an enzyme
system of hi~h glucamylase and alpha amylase (in place of premalt)
and 1-1/2 pints/bu. of the enzyme system of high glucamylase and
alpha amylase (in place of conversion malt plus 1 qt.~bu. regular
glucamylase). This conver~ion takes place at the natural pH o
the corn mash, which is normally between 5.5 and 5O7. Both mashes
were soured with the same lactic acid culture and were then
sterilized and cooled. A yeast culture was added and the mashes
were incubated at a same temperature until the Balling was reduced
4. The mashes were then cooled to 16 C. and held, and viable
yeast numbers were measured. The counts during the next 110 hours
were comparable as set forth in the table. Both are considered
normal for distillery mashes.
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Table III
Enæyme System of High
Hours after 4 Glucamylase and Alpha
Balling Drop Amylase Malt-Glucamylase
0 110 110
14-1/2 240 170
38 310 250
62 280 320
86 342 392
110 350 37
Example 4
This example demonstrates the usefulness of an enzyme
system of high glucamylase and alpha amylase as dascribed above
as a means of eliminating the requirement of saccarifying malt in
grain alcohol fermentations of corn. Three corn mashes were pre-
pared for fermentation using standard procedures with the only
differences being in the saccarifying agents. The first mash con-
tained 0.5~ pre-malt and 2.5~ conversion malt. The second mash
contained 0.5~ premalt, 1.0% conversion malt plus 1 pt./bu. glucamylase
with no alpha amylase in it. Tha third mash con~ained 0.5% of an
enzyme system of high glucamylase and alpha amylase in place of
premalt and was converted with 1 pt./bu. of the same enzyme system
of high glucamylase and alpha amylase (106.7 alpha units/ml). This
conversion takes place at the natura~ pH of the corn mash, which
is normally between 5.5 and 5.7.
Table IV
Alcohol ~ by Weight
Saccharifying Agent _24 hr. 48 hr. 72 hr.
Malt, 2.5% by wt. of Grain 3.2 7O37 8.27
Malt, 1% by wt. of Grain and
1 pt./bu. Glycamylase 3.49 8.23 8.59
1 pt.~bu. Enzyme System of
High Glucamylase and
Alpha Amylase 3.12 8.31 8.55
,. ,~
77
It is apparent from these results that enzyme systems of
high glucamylase and alpha amylase perform in mash in the absence
of saccharifying mal-t just as well as regular glucamylase plus
saccharifying malt, and both perform better than malt alone.
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