New insights on the urease activity range for soybean meal:

a worldwide opportunity for the poultry industry

Nelson Ruiz, PhD
Nelson Ruiz Nutrition, LLC
Suwanee, GA 30024
nelsonruiz313@gmail.com

Introduction
Since the discovery that the heat treatment of soybeans (Osborne and Mendel, 1917) improved its
nutritional value the identification, quantification and reduction of antinutritional factors have played a
major role in the definition of quality in processed soy products in general, and soybean meal (SBM) in
particular. By 1944 Casky and Knapp devised a method to detect inadequately heated SBM using the
activity of the naturally occurring enzyme, urease, present in soybeans. As a consequence of this initial
report a fairly simple, straightforward and consistent method was developed, standardized, and published
by the major professional chemists associations (AOCS, 2011a; AACC, 1995). Basically the industry
adopted the delta pH or pH rise range between 0.05-0.20 pH units as the range of adequacy of heat
treatment of SBM for monogastric animals. For decades, the range of change in pH units from 0.05-0.20
has served as an indicator of adequate SBM processing. When the change in pH was below 0.05 units
there were concerns that the SBM was overheated and above 0.20 pH units the SBM was considered
underheated and plenty of antinutritional factors were still active.
Historically, the first challenge to the range came from the work by Dale and co-workers in 1987 when
they demonstrated that the lower limit of the range was for practical purposes baseless and that the urease
activity (UA) test was not the correct method to test for overheated SBM. They documented an in vitro/in
vivo correlation between the protein solubility of SBM in KOH and broiler chick performance for
adequately and overheated SBM. Therefore, for practical purposes a value of 0.000 pH units doesn't
imply overheated SBM, just zero UA.
Although the upper limit of the range has remained accepted over the years, Hayward in 1975 proposed
an upper level of 0.30 pH units while Waldroup et al. (1985) suggested that an upper level of 0.50 pH
units was still adequate. In Brazil animal feed manufacturers use 0.30 pH units as the upper limit to
indicate adequacy of SBM processing according to Penz and Brugalli (2000).
The purpose of this presentation today at the Arkansas Nutrition Conference is to present and discuss the
evidence to propose to the oilseed and the poultry industries a new range for the UA test: 0.000-0.050 pH
units.
Correlation between trypsin inhibitors and urease activity
Before tackling the challenge to the upper limit of the UA test it is important to recall that the UA test
exists because it is a fairly quick method to measure residual antinutritional factors after the processing of
SBM and full-fat soybeans. When the relationship between urease and trypsin inhibitors in SBM is
critically examined, a biological relationship between the two cannot be found. Even though it is well
established that the anti-nutritional factors found in soybeans have detrimental effects on poultry
performance, urease is not a toxic protein to poultry and is not of much concern to monogastric animal
nutritionists. There is only a casual relationship because as heat is applied in the presence of adequate
moisture trypsin inhibitors and urease are deactivated. Urease, unlike trypsin inhibitors, is easy to
measure and, because of it, it is used as a "marker" of trypsin inhibitor activity. While the UA test is
available since the 1940s, an analytical method to determine trypsin inhibitors in soy was not published
until 1969 (Kakade et al, 1969) modified later by Hamerstrand et al. (1981), and standardized by the
International Organization for Standardization in 2001.
Two independent data sets are presented to illustrate the correlation between trypsin inhibitors and urease
activity. In Table 1 the data set published by Mustakas et al. (1981) in a paper on critical processing

factors in SBM and consisted of 24 samples of a single soybean cultivar subjected to different processing
conditions in a pilot plant. The second set is made of 104 commercial SBM samples analyzed by
Nutrianlisis Ltda. (Belalczar and Otlora, 2012) between 2008 and 2012 that were of different origins
(U.S., Argentina, Brazil, and Bolivia). It is evident that the correlation (R-square) between trypsin
inhibitors and urease activity within each set is high. These data confirm that by measuring UA in SBM
an acceptable estimation of the trypsin inhibitor content in soy processed products is possible.
Table 1. Correlation between trypsin inhibitors and urease activity in 24 samples of soybean meal
(Mustakas et al., 1981)
Trypsin
Inhibitors,
mg/g
3.3
2.6
3.7
4.3
5.2
8.5
4.1
6.1
2.8
6.1
5.7
2.7

Urease
Activity,
pH units
0.03
0.27
0.35
0.20
1.61
1.92
0.36
1.04
0.02
1.36
0.33
0.02

Trypsin
Inhibitors,
mg/g
4.8
2.7
8.2
4.6
2.9
3.4
3.7
7.7
1.6
8.3
7.5
3.6

Urease
Activity,
pH units
1.24
0.17
1.83
1.05
0.15
0.11
0.45
1.18
0.05
1.97
1.69
0.21

R-square = 0.8053

Why the upper limit of the range needs to be revised and lowered to 0.050 pH units?
Since 2005 it has been reported that high trypsin inhibitors in SBM are correlated with rapid feed passage
outbreaks in broilers (Ruiz and Belalczar, 2005). Rapid feed passage is defined here as the condition in
which broiler droppings lose their normal shape and consistency, do not display the characteristic white
uric acid cover, contain undigested feed that is visible to the naked eye, usually have a yellowish-orange
color, are frequently watery and contain sloughed intestinal tissue (see photos below). Broilers in a flock
experiencing a rapid feed passage outbreak have extremely dirty feathers, lack body weight uniformity
and display poor pigmentation.
As a consequence, the litter becomes wet and slippery, feed conversion is negatively affected, body
weights are lower than the desired standard and considerable economic losses may be realized.
Ruiz and Belalczar concluded in 2005 on the basis of six rapid feed passage events in the field that
trypsin inhibitors above 3.5 mg per gram of SBM were associated with rapid feed passage at inclusion
levels of SBM of about 20% in broiler feeds. A detailed description of the data presented in 2005 was
published in Feedstuffs in January of 2012 (Ruiz, 2012). However, data accumulated over the years
(2005-2012) from a large number of rapid feed passage events in the poultry industry in the north of
South America support the concept that the optimum residual concentration of trypsin inhibitors in
commercial SBM is below 2.0 mg/g, provided the solubility of the protein in KOH is at least 78%.
What is then the relationship between the upper limit of the UA range and rapid feed passage?

It is very simple in retrospect. Table 3 summarizes the UA and trypsin inhibitors analyses of commercial
SBM lots involved in six outbreaks of rapid feed passage in broiler flocks as well as the same analyses of
SBM lots that performed adequately around the same times and geographies.

These outbreaks occurred in 4 countries between 1998 and 2004. The dotted line in Table 3 shows that in
this data set urease activity values above 0.06 pH units rapid feed passage was observed in the field. The
current upper limit of the UA test is 0.20 pH units, meaning that up to that value the quality of SBM is
adequate for broiler feeding. Since the final amount of trypsin inhibitors present in the feed is dependent
on the inclusion level of SBM in the formula (and full-fat soybeans if available in the market) and their
trypsin inhibitor content, a reasonable approach is to formulate for a given maximum concentration of
trypsin inhibitors in the feed. For a SBM lot containing 2 mg of trypsin inhibitors per gram and
formulated for a 25% inclusion level, and assuming that there is no other trypsin inhibitors source in the
feed, the trypsin inhibitors content per gram of feed is 0.50 mg. Over the years I have learned that 0.540.58 mg/g is the tolerable amount by 40-42 day-old broiler chickens. Therefore, in terms of UA it means
that SBM should not be above 0.050 pH units.
A possible explanation of why a UA range that worked for decades now it doesn't
For nearly 70 years the UA test has served the animal feed industry and has basically gone unchallenged
in the quality evaluation of billions of tons of SBM. It has been a very useful test to the industry.
However, nearly everything has changed in the last 70 years, and broiler chickens are not the exception.
Havenstein et al. (2003) conducted a comparison of broilers that were available to the poultry industry in
1957 with broilers being used in 2001. They fed 1957 broilers diets that, according to historical records,
were typical of 1957 and also fed them typical broiler diets available in 2001. A similar approach was
conducted with a typical breed of broiler chickens available to the poultry industry in 2001. What is of
interest here is what would had been the intake of trypsin inhibitors in 1957 birds fed the 1957 diets
versus 2001 birds fed the 2001 diets. Of course, the authors could not predict at the time that the UA
range was going to be questioned, and they did not determine UA and trypsin inhibitors in the soybean
meal used in their simulations. But their data on feed intake and diet composition are sufficient to
simulate what trypsin inhibitor intakes were occurring in 1957 birds versus those for 2001 birds.

Table 2. Correlation between trypsin inhibitors and urease activity in 104 samples of soybean meal
(Belalczar and Otlora, 2012)
Trypsin
Inhibitors,
mg/g
1.75
1.81
2.02
1.81
2.00
1.70
1.92
3.56
3.60
3.71
1.90
2.10
3.56
3.40
1.82
1.88
2.15
2.25
2.30
1.95
1.95
2.10
2.82
2.18
2.12
2.02
2.09
1.80
2.09
1.80
1.78
1.75
1.65
4.50
3.05
2.85
1.70
2.10
3.80
1.80
1.76
2.15
1.55
1.50
1.62
1.72
1.85
1.88
3.50
2.10
3.80
3.96
1.75
1.70
2.14
3.20

Trypsin
Inhibitors,
mg/g
5.50
7.00
7.50
6.00
6.00
9.00
4.00
6.20
5.50
5.20
7.50
8.00
9.50
9.00
7.90
8.20
7.00
6.50
7.25
6.90
6.50
6.00
5.50
7.90
5.82
5.75
5.50
6.80
5.95
7.00
6.85
6.92
7.12
5.92
5.40
5.00
6.52
6.20
6.90
4.20
4.07
6.40
4.00
9.50
8.24
7.20
6.95
9.20

Urease
Activity,
pH units
0.015
0.024
0.040
0.011
0.023
0.005
0.032
0.120
0.120
0.129
0.021
0.032
0.108
0.127
0.020
0.014
0.025
0.020
0.024
0.020
0.050
0.049
0.050
0.060
0.047
0.020
0.031
0.020
0.031
0.020
0.007
0.010
0.000
0.198
0.080
0.030
0.000
0.060
0.090
0.020
0.020
0.050
0.000
0.000
0.000
0.060
0.016
0.032
0.107
0.009
0.139
0.142
0.006
0.001
0.024
0.072
R-square = 0.9748

Urease
Activity,
pH units
0.292
0.382
0.450
0.290
0.276
0.530
0.152
0.321
0.285
0.257
0.430
0.502
0.550
0.510
0.452
0.445
0.412
0.320
0.355
0.402
0.482
0.320
0.300
0.450
0.320
0.290
0.310
0.405
0.300
0.480
0.350
0.420
0.440
0.325
0.285
0.312
0.425
0.355
0.415
0.250
0.157
0.335
0.146
0.600
0.480
0.420
0.380
0.520

Trypsin inhibitors vs. Urease activity

(Belalczar and Otlora, 2012)

0.700

Urease activity (pH units)

0.600

y = 0.0738x - 0.1224
R = 0.9748

0.500

n = 104

0.400
0.300
0.200
0.100
0.000
0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Trypsin inhibitors (mg/g)

Table 3. Urease activity and trypsin inhibitors in SBM from six rapid feed passage outbreaks.
Country
ECUADOR
COLOMBIA
ECUADOR
COLOMBIA
COLOMBIA
VENEZUELA
PERU
COLOMBIA
PERU
COLOMBIA
ECUADOR
VENEZUELA
COLOMBIA
COLOMBIA
COLOMBIA
COLOMBIA

Urease Activity
pH units
.01
.02
.03
.03
.05
_ _ _ _ _ _ .06 _ _ _ _ _
.08
.08
.09
.14
.17
.19
.25
.28
.29
.33

Trypsin Inhibitors
mg/g
1.90
2.20
2.40
2.40
2.60
_ _ _ _ _ 2.80_ _ _ _ _
4.00
4.04
4.20
4.82
5.50
5.60
6.00
6.80
5.90
7.29

Rapid Feed
Passage?
NO
NO
NO
NO
NO
_ _ _ _ _ _NO_ _ _ _ _
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
(Adapted from Ruiz, 2012)

Table 4. Calculated trypsin inhibitor (TI) intake by "1957" broiler chickens fed 1957 diets versus "2001"
broiler chickens fed 2001 diets1
Broiler
chicken
genetics

Average
body wt.
&
g

Average
FCR
&

1957

539

2.34

Average
feed
intake
&
G
1261

2001

2672

1.63

43553

TI
content in
SBM 48
mg/g

Expected
UA in
SBM 48
pH units2

2
3
4
5
6
2
3
4
5
6

0.000-0.055
0.062 -0.136
0.136-0.210
0.210-0.284
0.284-0.357
0.000-0.055
0.062-0.136
0.136-0.210
0.210-0.284
0.284-0.357

Average
Average
diet TI cumulative
content
TI intake
mg/g4,5
mg/bird
0.476
0.714
0.952
1.190
1.428

600
900
1200
1500
1800
20075
3011
4014
5018
6021

Performance at 42 d and diet composition data taken from Havenstein et al., 2003
Extrapolated using data from Table 2
3
See note 5 for feed intake distribution from starter, grower 1 and grower 2
4
For the 1957 diet the SBM inclusion was 23.8% in the starter diet and assumed as the only diet fed to
42 d.
5
For the 2001 diet the calculations of TI intake at the 2 mg/g concentration in SBM are as follows:
Starter, containing 30% SBM, 1000 g x 0.600 mg/g = 600 mg
Grower 1, containing 23.05% SBM, 2000 g x 0.461 mg/g = 922 mg
Grower 2, containing 17.9 % SBM, 1355 g x 0.358 mg/g = 485 mg
TOTAL cumulative intake per bird= 2007 mg
2

Trypsin inhibitor intake calculations were made for the following trypsin inhibitor concentrations in
commercial SBM: 2, 3, 4, 5, and 6 mg/g. Using the data presented in Table 2, which includes a wide
range of trypsin inhibitors and their corresponding urease activity values, the expected corresponding
intervals of UA values associated with those trypsin inhibitor concentrations were established.
The estimates in Table 4 indicate that a 1957 broiler chicken consuming a feed containing SBM that
could have been analyzed and found to have a UA range of 0.150-0.200 pH units would not have even
consumed the amount of trypsin inhibitors that a 2001 broiler chicken would have consumed at the lower
concentration of 2 mg/g of SBM.
In other words, a 1957 chicken fed a diet containing SBM in the upper limit of the current UA range, that
is about 0.20 pH units, would have consumed about 1200 mg of trypsin inhibitors during its entire cycle
of 42 days. A 2001 chicken fed a diet containing SBM in the lower limit of the UA range, that is about
0.05 pH units, has already not only consumed 1200 mg of trypsin inhibitors, but also an additional 807
mg for a total of 2007 mg during the entire cycle of 42 days.
A major component responsible for the advancement and success of the poultry industry in existence
today is, without doubt, genetics. The broiler that is used today by the world-wide industry is considered
to be an eating machine. In order for these modern broilers to express their full genetic potential for
maximizing tissue growth they must each and every day consume enormous quantities of feed so as to
meet their energy and nutrient requirements. Thus, feed intake of these broilers far exceeds that of broilers
available decades ago. Therefore, it can be concluded that the upper limit of the UA range was valid when
it was established in the 1940s and continued to be valid for a good number of years. However, it seems
that somewhere prior to the 1990s this upper limit became invalid, rapid feed passage outbreaks started to

occur in different geographies (Kouwenhoven et al., 1992; Lpez et al., 1998; Butcher et al., 1999; Miles
and Butcher, 2002) without an immediate obvious explanation. In Latin America the syndrome is known
as "trnsito rpido", in Europe and other countries may be called "disbacteriosis". The data presented
since 2005 (Ruiz and Belalczar, 2005; Ruiz et al., 2008; Ruiz, 2012) are carefully analyzed field
observations concluding that excess intake of trypsin inhibitors delivered to the feed through SBM high in
trypsin inhibitors is at least one of the explanations for the occurrence of rapid feed passage in
commercial broiler operations. The analytical tool to estimate trypsin inhibitors in a reasonable time, the
urease activity test, continues to be an excellent one, but the interpretation of the range of adequacy has to
be changed to 0.000-0.050 pH units, provided the solubility of the protein in KOH is at least 78%. The
usage of three figures after the point is to improve the correlation with the trypsin inhibitor analysis.
Implications to the poultry industry
A new urease activity range has several implications. First of all, it is time for the poultry industry to
adjust quality standards to today's reality. Broiler producers are losing money every time they have to deal
with a rapid feed passage outbreak in the field. Poultry companies know how much it costs them and the
industry each year if a rapid feed passage outbreak results in a loss of just one point of feed conversion.
The losses don't stop with poor feed conversion because the more trypsin inhibitors present in the feed,
the wetter the litter is going to be. Poor quality litter relates to poorer air quality due to ammonia, poorer
flock uniformity, downgraded carcasses due to blistered breasts and loss of the paws to their specific
markets.
Second, this is an opportunity for both the oilseed and poultry industries to come together and discuss the
reasons why such a change is needed with respect to the adequacy of the urease activity range which is
presently being used. Trading on the basis of quality is a win-win situation for all concerned. Of course,
a win-win attitude on both sides is the best approach when entering into discussions on topics as in this
case of SBM quality. Such a proposed change in the urease activity range does not necessarily mean that
it has to be set in concrete. Both sides may have to give and take. Lets just assume that the trading range
is 0.000-0.050 pH units as proposed above. Then lets propose a margin of variation of up to 0.070 pH
units, but after 0.075 pH units a discount table is established or negotiated in a manner similar to those
used in discounts for crude protein and moisture. This just makes good sense and it is time that it be
discussed and implemented. Alternatively, a premium may be negotiated for the new range (again,
provided solubility of the protein in KOH is at least 78%) with the premium being discounted beyond
0.075 pH units, for instance.
Third, a considerable amount of work has being published in reference to the toxicity of trypsin inhibitors
(and other antinutritional factors in raw soybeans) which clearly document that birds (broilers, layers,
breeders) do not adapt to high trypsin inhibitors in the diet (Nesheim and Garlich, 1966; Ruiz et al.,
2008). Therefore, it would be advantageous to continue to develop commercial soybeans that have low
concentrations of or no trypsin inhibitors and market them because of their added value.
A final note in reference to the units used to express trypsin inhibitors
The predominant method in the US industry is the AOCS method (2011b) which expresses trypsin
inhibitors as TIU (trypsin inhibitor units) per gram. The predominant methods used out of the United
States (Hamerstrand et al. 1981; ISO, 2001) express trypsin inhibitors as mg of trypsin inhibitors per
gram. Given the strong correlation between trypsin inhibitors and urease activity and considering that
both units of expression come from the same basic method developed by Kakade et al (1969) it should not
be difficult to develop a table such as Table 2 in this paper to relate trypsin inhibitors expressed as TIU/g
to urease activity. Unfortunately, no serious effort has been made to establish a statistically valid
conversion between both units of expression.

Acknowledgements
Many thanks are expressed to Fabiola de Belalczar and her staff at Nutrianlisis (Bogot, Colombia) for
all the excellent analytical work over the years. Special thanks are also extended to Lloyd Sauer, Director
of ContiTec for his encouraging support. I also would like to express my appreciation to Dr. Richard
Miles, Emeritus Professor at the University of Florida for reviewing the manuscript, and many thanks to
the Continental Grain Company for covering a large part of the cost of this project.
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