"REPRODUCED WITH THE PUBLISHER'S PERMISSION"

[Reprinted from THE JOURNAL OF THE AMERICAN OIL CHEMISTS' SOCIETY, August, 1956 Issue, Vol. XXXIII, No.8, pp. 360:-363]

Urease Activity in Soybean Meal Products 1

ALLAN K. SMITH, PAUL A BELTER,2 and ROBERT L. ANDERSON/

Northern Utilization Research Branch/ Peoria, Illinois

HE PROCESSING OF SOYBEANS for oil and meal by Neither the investigations of Sizer and Tytell nor
T solvent extraction includes the important opera-
tion of steaming or "toasting" (1, 6) of the meal
those of Hofstee included data on soybean meal.
Therefore our investigations were made on the effect
for development of optimum nutritional value. The of adding oxidizing and reducing agents and on pre-
toasting operation follows removal of the hydrocarbon heat treatment of the meal as well as other factprs
solvent which usuallY involves the use of steam. Be- affecting the assay of urease in soybeans.
cause too much moist heat is detrimental to the nutri-
tive value of the protein, the time and temperature of Method
toasting must be controlled within optimum limits. The assav method used for this determination was
lVIanv test"methods for control of toasting' have been' a "contil1l~ous titration" of ammonia method men-
propos~d (4), but the two which have beeI~ used most tioned previously by Croston et al. (4), Th~s meth?d
are 1) the modified Caskey-Knapp (2,3), an assay maintains the pH of the urease-urea reactIOn at ltS
for urease, and 2) the nitrogen-solubility method, a optimum activity of about 6.8 during th~ .reacti.on
measurement of the water-soluble nitrogen compounds period to prevent a decrease in urease actIvIty WIth
(8). often referred to as the nitrogen-solubility index increasing alkalinity. This was accomplished by add-
or NSI value. The modified Caskey-Knapp (2) is a ing' standard hydrochloric acid from a burette and
method which measures a change in the pH, for a us'ing bromothymol blue or a pH meter to indicate
given reaction time, of a weakly buffered system of the desired end-point. In routine work the time used
sovbean oil meal and urea. Recently Croston, Smith, in titration can be largely eliminated by use of auto-
al~d Cowan (4) proposed a method based on an il:- matic titrating equipment. Unless otherwise indicated,
crease of the electrical conductivity of a soybean 011 all samples ,vere prepared by grinding the dehulled
meal svstem similar in composition to that used in the and defatted flakes in a haml11er mill to pass an 80-
Caskey-Knapp method. The modified Caskey-Knapp mesh screen. Size of the sample was reduced from
and electrical conductivitv methods are useful only 200 mg'. used bv Croston ·for feed meals to 20 mg.
for toasted meals which ar'e produced for use in feeds, This SI~1all samp'le was shown to be representative by
in which an estimated 99.5 to 99.950/0 of the urease a series of check determinations made on different
activity has been destroyed. and in which the changes days. All reactions were carried out in a constant
in pH'or conductivity ~re \'ery smalL tel;lperature bath at 40°C. except as indicated.
The increasing' utilization of sovbean meal in special The reagents used were (1) 0.1 N HCl, (2) 0.1 N
food products a~d for industrial 'purposes has created NaOII, (3) phosphate buffer of pH 6.8 made up of
a need for another analvtical control method. These 0.025 mole of ILHPO" 0.025 mole I\TL,PO~, and 0.8 g.
meals prepared for spe~ialty uses receive much l.ess of glutathione per lifer, and (1) buffered urea SOI~l­
steam treatment than feed meals and have a lllg-h tion made up daily by dissolving 6 g. C.P. urea 111
urease activity. This report deals primarily with the 100 m1. of buffer solution number (3) without the
development of an assay method for urease in these g·lutathione.
soYbean meals of high activity. Included in the in- ~ Proeedure. Assays were carried out in beakers in a
ve'stig'ation are data on the rate of urease inactivation constant temperature bath at 40°C. For each deter-
with ~<;team treatment at atmospheric pressure, a com- mination of highly active meal a 20-mg. sample (unless
parison of urease activity in several varieties of soy- othenvise indicated) v.-as added to 5 m1. of the buffer
beans, the activity of immature and frost-damaged solution (3) contai~ling the glutathione and was al-
beans, and several factors affecting the precision of the lowed to stand for 30 min. in the 40°C. bath; 5 m1. of
assaI' method. the buffered urea solution (4) were then added to start
Il~ a 1941 study of'the activity of a crystalline ure- the reaction. The pH of the reaction was maintained
ase svstem Sizer and Tytell (7) showed that activity is at approximately 6.8 by slowly adding 0.1 N HCl and
modified by the oxidation-reduction potential of a llsing bl'omthymol blue in its green color range to
svstem as ~ontrolled by added ox'idizing or reducing indicate the desired end-point. At the end of 30
compounds and that a certain optimum potential min. the reaction was terminated by rapidly adding
favors a maximum activity value. additional 0.1 N HCl to a total of 10 mI., or more, as
Using an assay method in which the reaction was required for very highly active meals. The system
carried out at 20°C., Ho£stee (5) demonstrated in was then titrated with 0.1 N NaOH to pH 4.7. A pH
1949 that. when crystalline urease or a water extract meter indicated the end-point. A control was run
of the jack bean was stored under refrigeration tem- parallel with each sample. For the control the urease
peratures PI' in the frozen state, the assay results were was inactivated by. addinz the hVClfochloric acid to
verI' low. However if the same urease samples, after the sample before' adding 'buffers' (3) and (4). The
sto;ag'e at reduced temperature, were heated to 60°C. difference between the control and sample in milli-
for 5~ min., the assay results on a given sample were liters of 0.1 jV HCl or its ammonia equivalent was
uniformly high. Assay values for heat-treated samples taken as the urease activity of the meal.
were as much as five times those 'without the treatment. Thl? principal differen~es between this procedure
----;:-p~nted at the meeting of the American Oil Chemists' Society, and that described bv Croston et al. are the soaking of
Honston. Tex.. Apr. 23-25, 1956.
2 Present address: University of Michigan, Ann Arbor.
the sample for 80 min. at 40°C. and the use of a re-
:J In military service.
-lOne of th~e Branches of the Agricultural Research Service, U. S.
ducing agent. The need for thes~ modifications as .,~ell
Department of Agriculture. as other factors affecting the assay of urease actlnty
 in untoasted and lightly toasted meals is described in pH 6.8 and comparing the results with samples having
the following section. no glutathione. All samples were presoaked for 30
min. at 40°0.
Factors Affecting Urease Activity The results with glutathione are not uniform; for
Soaking Time and Effect of Mild Stea.m Treatment. most samples the glutathione increased the assay
The effects of soaking raw meal and lightly steamed values whereas for others the results were unchanged.
meal in a pH 6.8 phosphate buffer (without reducing The effects of cysteine and hydrogen peroxide also
agent) for various periods of time are shown in: Fig- were investigated. The cysteine at double the concen-
ure 1. The solid lines are for raw or unsteamed meal, tration of glutathione gave about the same general
type of results whereas the values obtained "\vith per-
7.00 oxide were low, indicating an inactivating effect. The
effect of peroxide was also lacking in uniformity.
w
a::::; ----
Ao_ _-----.:::HAROSOY 1954
Although the magnitude of the effects of glutathione
and of hydrogen peroxide on activity 'were non-
U1
>- 0- uniform, the general trend was for reducing agents to
t: E
>0 increase the assay values and for oxidizing agents to
;::'" HAWKEYE 1954
decrease them. Since enzyme activity is g'enerally
<.>
<t :0
wOo
(fl
<t:I:'"
wZ
~~~~~---~-------
-- -- -- ..... measured at optimum conclitions, the l;se of~a redu~­
ing agent (glutathione) and the 30-mill. presoaking
0:
::>z
ci
.............. _- ----
FLAMBEAU 1954 period were made a part of the assay procedure. In
the adopted procedure the concentration of gluta-
E thione was 0.08% by weight during the 30-mill. pre-
WITHOUT REDUCING AGENT soaking period and 0.04% during the reaction period.
In subsequent experiments the effect of glutathione
15 30 45 60 75 90 105 120
on the assay of toasted meals was not detectable. How-
ever its use is recommended for all highly active meals
SOAKING TIME, MINUTES
since the amount of toasting required before reducing
FIG. 1. Effect on urease activity of time of waking of raw
and of lightly steamed soybean meals at 40°0. in pH 6.8 agents lose their activating effect "\vas not determined.
phosphate buffer for various periods. Solid lines are for raw Toasting may be expected to increase the number of
meal, and broken lines are for very lightly steamed meal. No exposed sulfhydryl groups of the protein and lessen
reducing agent present in these assays. the need for reducing agents.
Our results, like those obtained by Hofstee and by
Sizer and Tytell on crystalline urease and extracts of
and the broken lines are for the same meal treated in jack bean, show that soaking the soybean meal at an
an autoclave with live steam for 5 min.; the steam
elevated temperature prior to the assay as well as the
treatment started with a cold autoclave. The differ-
use of a reducing agent are important factors in ob-
ences in results for raw and lightly steamed meals at taining optimum and uniform results. To determine
zero soaking time show an effect similar to but less whether the 40 0 0.-30-min. treatment is adequate, a
than those found by Hofstee in his pre-heat treatment comparison was made with the 5- andIO-min. soaking
of crystalline urease and jack bean extracts. These treatments at the 60° temperature used by Hofstee.
results also show that soaking the raw meal for about Table I shows our urease values for three varieties of
30 min. at 40°0. increases the activitv to about the
same value as for the lightly steamed ineal.
Reducing Agents. The effect of glutathione on the TABLE I
assay of laboratory samples of raw meals prepared Effect of Time and Temperature of Presoaking and of Glutathione
on Urease Activity. All Assays at 40°0. for 30 Min.
from several varieties of soybeans is shown in Fig'-
ure 2. These assays were m~de by adding increasil{g Assay yalue of meals
amounts of glutathione to the samples buffered to Sample Added
Variety gln ta thione 1 T_re,a,tm_e_n_t_o_f_m-,-ea_ls _
No.
cone. wt. 60'C. for 160'C. for 1 40 'C. for
5 min. 10 min. 30 min.
0.1 N NIL,/20 mg. of sample
8'00[

7.00._ _• •
,DUNFIELD 1941 >.---------.
,HAWKEYE 1948

ROANOAKE 1954

LINCOLN 1954

HAWKEYE 1952
soybean meal. The comparisons were made with and
FLAMBEAU 1954 without glutathione in the buffer solution used in the
presoaking period. The assays for all samples were
made at 40° for 30 min. The data show that o'luta-
thione increases the assay values for all three ~neals
4.000 6.02 0.04 0.06 O.OB 0.10 0.12 treated at 60°0. but foi, onlv one meal treated at
. GLUTATHIONE, PERCENT WEIGHT OF SOLUTION 40°0. In comparing the thre'e methods of pretreat-
FIG. 2. Effect of various concentrations of glutathione (0 ment, the 40 0 0.-30-min. method gives values equal to
to 0.12% on solution volume) on urease activity of soybean or somewhat higher than the 60°0. treatment. These
meal for several varieties of soybeans. experiments further confirm that a reducing agent

7
and presoaking period are essential for optimum
assay values and that the 40°C.-30-min. treatment is
the inost effective.
Effect of Wa.ring Blenclor. A decrease 'in the 30-min. ,.. e 11502
11501 HAWKEYE
t: A LINCOLN
soaking period would be desirable for a routine assay. >
;:: 0 11503 HAROSOY
<> \
An attempt was made to obtain the same -results by <l \
treatment of the sample in a 'Varing Blendor 5 for 11;2 70 \
...J
<l \
min. The Blendor treatment, made without a reduc- z
c; \
ing agent in the buffer, had an inactivating effect on
the urease and caused a decrease of more than 50% in
cr0
u.
60
\
\
,
\0
the assay values. Use of glutathione in the sample 0
50
\
during treatment in the Blendor reduced the rate of I-
Z
w
e\
inactivation but did not completely inhibit the effect. <>
a:
\
\
w 40
The inactivation of urease by the Blendor appeared a. \ 0
to be an air-oxidizing effect accelerated by the cavitat- ,..'t: '{l
ing action of the Blendor and is further confirmation
~
30
""- .... NITROGEN SOLUBILITY INDEX
of the need to control the oxidation-reduction poten-
tial of the system during the assay.
<l
w 20
. . J--- 0

Effect of A.utoclave Treatment on Disappearance of

(/)
<l
w
a:
-------- l!J

Vrea.se. Table II and Figure 3 show, for several ::>

10

TABLE II
Effect. of Increasing Periods of Heat Treatment in Autoclave on o 5 10 15 25 25 30
Urease Activity of Soybean Meal, Expressed in :Mil1iliters TIME IN AUTOCLAVE, MINUTES
of 0.1 N Ammonia. Note the Increase in Size of Sample
for 15· and 30·Min. Treatments FIG. 3. Effect on urease activity of steam·treating soybean
meals in an autoclave at atmospheric pressure in the range of
Time in a autoclave. min. o to 30 min. and a comparison with the rate of change of
solubility of nitrogenous compounds in water for the same
0 5 10 15 30
Sample No. time. 'l'he ureaS,g values are calculated on a percentage basis,
Sample size, in mg. using the original raw meals as 100%.
20 20 20
I 20 20 I 100 200
method over the other, the variation in urease activity
m!.h mI.h m!.c Im!.b ro1. c m!.h mJ.C
11503·Harosoy occurring in plant operation, which might be ex-
1954....................... 7.4 6.4 5.7 4.7 4.5 1.0 0.11
11504·Clark pected to be less than in a laboratory investigation
1954....................... 5.6 3.8 3'.8 3.2 3.1 0.3 0.03 of this type, should be determined. Because com-
11501·Hawkeye
1954....................... 5.4 4.0 3.6 3.0 2.9 0.15 0.08 mercial beans are composited to some extent, they
11502·Lincoln
1954....................... 5.2 14.1 3.5 3.2 2.8 0.15 0.02 may show less variation in urease activity than the
:l Autoclave at atmospheric pressure. selected samples used in this investigation. Figure 3
b
C
One day after treatment in autoclave.
Seven da:ys after treatment in autoclave.
also shows a comparison of the urease activity method
with the nitrogen solubility method of measuring the
rate of change in the meal with steam treatment.
varieties of soybean meal, the approximate rate at There is a great dissimilarity in the curves represent-
which urease activity decreases when the meal is ing the two methods of measurement.
heated in an autocla;'e at atmospheric pressure. In Figure 4 is a gra.ph of the experimentally deter-
this series of experiments the autoclave was thor-
oughly heated before introducing the meal, therefore
the increase in activity after 5 min., which was noted 8.00 e 11501 HAWKEYE
in Figure 2, is not apparent. The more vigorous steara A 11502 LINCOLN
o 11503 HAROSOY
action in this experiment reduced the urease activity iii 11504 CLARK
below the point where the heat-activating effect can
be observed. The data in Table II are the experi-
mentally determined values calculated to milliliters
of 0.1 N ammonia per 20 mg. of sample whereas the '"0.
urease values in Figure 3 are calculated on a percen- :c
" 5.00
tage basis with the raw meal, of each variety assigned z
a value of 100%. This latter method of calculation z 4.00
d
starts all urease values at the same point on the graph,
namely 100%, but at the end of 5 and 10 minutes of 'E 3.00
autoclaving there isa substantial difference in the ,.."
t:
;::
calculated assay values. However as inactivation in- ~ 2.0.0
creases, the measured values approach each other. It
is suggested that the method of using a percentage ~
~ 1.00
decrease in urease activity of the meal may have an a:
::>
advantage over using the directly determined values.
This method of calculation would require a urease o 5 10 15 20 25 30
measurement prior to desolventizing as the 100% TIME, MINUTES
value. Before deciding on the advantages of one FIG. 4. Urease activity as affected by time of treatment in
autoclave at atmospheric pressure for meals obtained from
'Mention of trade·named products does not imply that they are
endorsed or recommended by the Department of Agriculture over other four varieties of soybeans. Results are expressed as 0.1 N NH.
firms or similar products not mentioned. per 20 mg. of meal.
 6.00 nearly twice as much activity as the cotyledons. The
10-15
5.50
urease activity contributed by the hulls, germ, and
cotyledons to the activity of the whole meal is ap-
~ 5.0.0 proximately 0.2~c, 3.8%, and 96%, respectively.
:::;: These data indicate that removal of hulls, as is done
>-- Co
!:: E
4.50 in some commercial processing, would favor an in-
>0 creased urease value for the combined cotyledon-germ
t; N 4.00
<t ~ fraction. However since in commercial operation the
w ~ hulls are incompletely removed and part of the germ
~ £ 3.50
wz
co usually separates with the hulls, the urease activity of
::;) z 3.00 the dehulled meal may not be significantly different
from that of the whole meal. In laboratory operation
E 2.50 it was noted that, when grinding whole meal in a
hammer mill, the hulls resisted grinding and consti-
tuted a major portion of the last fraction remaining
in or leaving the mill. It was found that this fraction
~ ro w ~ ~ ~ M m 0 gave 30 to 40% lower urease values than the" through
FROST DAMAGE, ESTIMATED PERCENT fraction" and that it was essential to ha.ve all of the
FIG. 5. The urease activity of immature soybeans damaged sample pass through the mill and screen in order to
(0-100%) by freezing. Month and day of harvest of each obtain uniform assays.
sample is shown on each graph. Frost Damage am'd Jiaturity. Preliminary experi-
ments were made on the urease activity of immature
mined urease values in terms of 0.1 N ammonia for and frost-damaged beans. The frost damage was
the three meals shown in Figure 3 and the additional affected by freezing the immature bean pods in dry
meal No. 11504. "VIlereas the values for three of the ice at the time of harvest. Two series of beans were
four meals are reasonably close to each other, the investigated. The first was an Adams variety which
high results of the fourth meal indicate that the was harvested in 19:51 and had been used for oil-color
unusually high activity of some meals must be con- studies at our laboratorv: thev had been stored at
sidered in using urease activity as a processing ooe. until August, 1955. The se~ond series was Hawk-
control test. eye beans harvested at various periods in September,
YariaNon w#h Ya.riety and Location. In this limited 1955. One portion of the 1955 beans was frozen in
investigation the urease activity of raw meals derived dry ice at the time of harvest and the other portion
from different varieties of soybeans ranged between was assayed without freezing. All samples were air-
4.5 and 7.6 ml. of 0.1 N ammonia per 20 mg. of meal. dried, defatted, and ground for the assay.
Stated in other units, 1 mg. of meal liberates from 0.38 Figure 5 shows the urease -activity plotted against
to 0.65 mg. of ammonia from urea. The maximum the percentage of frost damage, which is approxi-
varietal variation of the samples tested was about mately equal to immaturity. These results show that
70%. How'ever most values were in a narrower range urease content of soybeans decreases rapidly with
of 5.5 to 6.5 mI., or a variation of about 2010; this is increasing frost damage or immaturity. The urease
a variation which decreases with increasing heat values for the Hawkeye beans assayed without freez-
treatment. Because of the limited number of assavs ing are not shown separately on the graph since they
performed and absence of agronomic data there "is were the same as for the frozen samples thus indi-
insufficient information to account for the urease vari- cating there is no difference in urease activity between
ation. Location appeared to be as much of a factor as frost-damaged and unfrozen soybeans of'the same
variety; for example, Earosoy samples collected at maturity.
several locations gave activity values of 7.6. 6.7. 5.5. Summary
and 4.9. This is about the same range foune1 fo~ tll(~ A. method which uses direct titration of ammonia as
different varieties. A more extensive studv of varietv a measure of urease activity was nwdified for use in
and location might be expected to show ~hether soil the assay of raw and of slightly denatured soybean
fertility or climatic conditions are important influ- meal. The modifications which were adopted were
ences on urease content of the sovbean. the use of glutathione, a presoaking of the sample for
Distribution of Urease in Seed Parts. One hundred 30 min. at 40 0 e., and making of the assay at 40 0 e.
grams each of Lincoln (11502) and Harosoy beans Data on the urease activitv of several varieties of
(11503) were cracked between steel rolls and sepa- soybeans, and for immatur~ and frost-damaged soy-
rated by hand into cotyledons, hulls, and germ. beans were determined.
Before removal of the oil the approximate division
of the beans in~o their three parts was 90%, 8%, REFERENCES
and 2%, respectIvely. The cotyledon and germ frac- 1. Belter, P. A.• and Smith, A. K., J. Am. Oil Chemists' Soc., 29,
170-174 (1952).
tions were defatted with hexane. and all three frac- 2. Bird. H. R., Boucher, R. 0., Caskey. C. D. Jr .. Hayward, J. W.,
tions were ground for urease .determination. The and Hunter, J. E., J. Asso. Official Agr. Chern., 30, 354-64 (1947).
3. Caskey, C. D. ,Jr., and Knapp, Frances C., Ind. En". Cliem.,
urease values in ml. of 0.1 N ammonia per 20-mg. Anal. Ed., 16, 640-641 (1944). ~
4. Croston, C. B., Smith, A. K., and Cowan, J. C., J. Am. Oil Chern.
samples were as follows: ists' Soc., 32, 279-282 (1955).
Defatted 5. Ffofstee, B. H. J., J. Gen. Physiol., 32, 339-349 (1949).
Hulls Cotyledon Germ W1lOle Meal 6. I\.lose. A. A., Hill, Barbara, and Fevold H. L. Food Technol. 2
Lincoln 0.1 1 (1948). '" ,
6.1 11.2 6.0
Harosoy 0.2 7. Sizer, 1. W.. and T)-tell. A. A., J. BioI. Chern., 138, 631-642
7.6 13.3 7.4 (1941).
8. Smith. A. Ie, Belter. P. A.. and Johnsen, V. L., J. Am. Oil
These data sho\y that the hulls are very low in Chemists' Soc., 29, 309-312 (1952).
urease activity and the germ, on a weight basis, has [Received :March 19, 1956]

GPO 818049'