Note: Descriptions are shown in the official language in which they were submitted.
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i~377~8
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BACKGROUND OF THE INVENTION
This invention relates to a process for pro-
ducing a non-protein nitrogen (NPN) containing material
useful as a feed supplement for cattle and sheep. More
; 5 particularly, it relates to a process for producing urease-
resistant glucosyl ureides and to the high assay product of
the process.
It is now common to feed cattle and sheep
feed supplements. These supplements provide nitrogen
as a source for meeting the protein needs of the animals.
The most commonly used supplement is urea. When the urea
enters the rumen of the animal, the enzyme urease acts on it
to form ammonia. The microorganisms in the rumen then convert
- the ammonia into a form of protein which can be utilized by
- 15 the animal.
There is a disadvantage in using urea in that
; : ,
it is converted to ammonia at a relatively rapid rate. If
the dosage of urea is too large, the capacity of the micro-
organisms to convert the produced ammonia to protein is exceeded
and the excess ammonia is either converted to urea and ex-
creted by the animal or, if its concentration is high enough,
,, I_ , ~ .
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Recent work has been directed toward develop-
ing "slow release" NPN compounds. The objective is to
:.-
provide compounds which produce ammonia in the rumen at a
reduced rate with which the microorganisms can cope.
One family of compounds, glucosyl ureides,
is known to have the property of slow release of ammonia in
the rumen of cattle or sheep. These compounds have the added
advantage of providing the ammonia in association with an
energy source (glucose), which promotes greater growth of the
microflora in the rumen. A United States patent of interest
in this connection issued to McNeff under No. 3677767. This
patent teaches reacting molasses (which contains glucose
and non-reducing sugars, such as fructose) with urea under
~ 15 acidic conditions to produce a liquid product containing
; glucosyl ureide. The product has been shown to be useful
as a feed supplement for ruminant animals.
Other pertinent prior art is exemplified by
U.S. patents: 2612497, issued to Meijer; 3023205, issued to
Meijer; and 3020273, issued to Steadman.
, ~
SUMMARY OF THE INVENTION
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The present invention is eoncerned with a
- ! known reaction whereby glucose is eombined with urea under
aeidie eonditions to produce glucosyl ureides. However,
in aeeordance with the invention the process is carried out
in a manner sueh that a relatively high degree of eonversion
j of the glucose is aehieved, and the end product contains
¦ a relatively high concentration of ureides.
More particularly, glucose is reacted with
urea under aeidie and drying eonditions to produee a produet,
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preferably solid, containing a relatively hi~h proportion
o~ urease - resistant glucosyl ureides.
- When first considering drying the glucose-urea
Mixture during reaction to drive off water and shift the
equilibrium of the reaction toward producing more ureides, we
anticipated that the concentration of acid would increase,
leading to dehydration and undesired carmelizing or degradation.
Surprisingly, the pH of the acidic mixture actually increases
during the drying process. Carmelizing is not a serious problem.
In retrospect, it appears that ammonia, released from the urea,
or protein, which is present if one uses hydrolyzed grain as
the starting material, buffers the reaction to permit it to
proceed to completion.
Since drying is an integral part of the process,
we prefer to use a glucose-containing starting material which -
is relatively free of other hexoses, which make drying difficult
- and which can result in charring of the final product. We
have found that grain starch, such as that occurring in barley,
wheat or corn, may be hydrolyzed to provide a preferred
starting material containing glucose. If, for example, molasses
is used in the process, it will not readily dry to a useful
product, due to the presence of a mixture of sugars and the
tendency of contained fructose to char. However, we have found
; that some hygroscopic mixtures of sugars can be tolerated in
the starting mixture, provided that inert materials, such as
grain husks, are also present.
~- The product of the process comprises a solid, con-
densation reaction product of (1) glucose-containing hydrolyzate
of grain, selected from the group consisting of barley, wheat
and corn, and (2) urea, in the presence of acid under drying
condition at elevated temperature, said product containing the
major propcrtion of said glucose in the form of glucosyl
ureides.
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The process for producing the desired mixture of
glucosyl ureides comprises the steps of: providing an aqueous ~ ;
` mixture of a starting material containing one or more reducing
carbohydrates as the major dry matter constituent, the major
constituent of said reducing carbohydrates being glucose, and
urea; and reacting the mixture under acidic and drying con~
ditions at an elevated temperature sufficient to convert the
major portion of the glucose and urea into glucosyl ureides ~
and to produce a solid product~ ;
DESCRIPTION OF`THE DRAWINGS
` In the drawing:
' Figure 1 shows a preferred form of the process
outlined in terms of chemical equations for the hydrolysis
and condensation reactions; and
Figure 2 is a plot showing the effect of tempera-
' ture of the reactlon mixture on the hydrolysis reaction.
DESCRIPTION OF T~E PREFERRED E~BODIMENT
The invention will now be described in terms
of providing glucose by hydrolyzing starch present in barley.
' 20 While a number of grains, such as wheat, corn and the like
can be used, barley is preferred as it is commonly used in
Western Canada as cattle feed. '
- - ,
Equipm_ t' _ d`analy's'is pro`c'edur`es
The hydrolysis reaction was carried out in a ' '
:' 25 50 ml reactor provided with stirrer, inlets for introduction
' of reagents and sampling, a mantle, and a temperature controller ~'~
capable of keeping the temperature within'+ .5~C. ~ ''
The glucose analyses were performed by the
; orthotoluidine method and by the use of a Beckman* ~lucose
autoanalyzer, Model ERA-2001*. The first method involves the
reaction of the glucose present with o-toluidine in acetic acid.
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The color is measured spectrophotometrically using 5 determin-
ations for each sample, one for the blank, and one for the
standard. The error of the method was found to be 2%. Time ;
employed for each deter~in~tion: ~15 minutes.
The second method involves the oxidation of
the glucose to gluconic acid by a glucose oxidase. The
oxygen used, proportional to the glucose present, is measured
and expressed in percent glucose. Error similar to the
o-toluidine method; time for analysis: 3 minutes per sample
Imaximum)-
Due to a combination of errors (error of the
analysis of glucose plus error involved in the hydrolysis
reaction) two results that differed from each other to an
extent of 5% were considered identical.
Only in cases where the results were signifi-
cantly different was it concluded that the parameters studied
had an influence on the yield of glucose. r
Urea was analyzed by hydrolyzing it to ammonia
with the enzyme urease, and determining the ammonia either
potentiometrically by the use of an ion specific electrode,
or by colorimetry by reaction of the ammonia with sodium
nitroprusside plus sodium salicylate in the presence of
sodium dichloroisocyanurate.
Using this last method, the ammonia plus free
urea present are analyzed together, and the final result
expressed as percent of free urea present.
Ureides were determined by paper chromatography
and by the use of a Waters liquid chromatograph, Model M6000*
using a system of columns packed with Bondapak X Corasil*, and
as solvent, a mixture made up of four parts of a 2-to-1
mixture of isopropanol ethylacetate and one part distilled water.
Starch determination was done by treatment of
*trade mark
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a known amount of barley suspended in water with 85% phos-
phoric acid (pH 1,5 ~ 2) at 80C overnight. A control
experiment was done at the same time with pure potato starch.
This first treatment liquefied all the starch present. After
cooling and adjusting the pH to 4.5 sodium hydroxide, both
unknown and control were treated with the same amount of
glucoamylase at 62C for 24 hours. The glucose present was
analyzed, and the result obtained from barley corrected
according to the glucose obtained from the control experiment.
The content of starch was found to be 55% on a dry basis.
The Hydrolysis Reaction
There are several known procedures available
for hydrolyzing starch to produce glucose useful for this
invention. For Qxample, one may liquify starch by mixing a
1 15 30% aqueous suspension of starch with 0.07% of ~-amylase at
a pH of 6.9. The temperature is held at 70-75-80-85C for
15 minutes at each temperature. The mixture is then boiled
; for an hour, cooled to 85C, and 0.04% of ~-amylase is added
- and the mixture kept at 82C for 30 minutes. Agitation is
maintained throughout these steps. The reaction mixture is
then cooled to 60C, the pH is adjusted to 4.0 - 4.5 with
; HCl, 0.1% of the enzyme amyloglucosidase (commonly termed
glucoamylase) is added and the product incubated at 60C
for 24 hours to complete conversion of the starch to glucose.
However, we prefer to use a novel process
wherein only one enzyme, glucoamylase, is used under particular
conditions to convert at least 90% of the starch to glucose.
More particularly, an aqueous mash of ground barley is mixed
with an appropriate amount of glucoamylase under acidic condition
, 30 at temperatures within the range 65 - 67C and reacted for
; sufficient time to achieve the desired-degree of conversion.
i In a preferred embodiment, ground barley is slurried with
water ln a proportion of between 140 - 240 parts per 100 parts
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1()3~76~3
of grain. Acid~ usually 85% phosphoric acid, is added to
bring the pH to about 3.0 ~ 4,5, Glucoamylase, preferably
in the form of an aqueous solution containing 100 units per
milliliter, is added and the temperature is then raised
to about 66C. Eighty milliliters of enzyme solution will
hydrolyze 100 pounds of liquified starch in 72 - ~6 hours
at 62C and pH4. The enzyme addition may be from 30 to 60
units per 100 grams of barley. rrhe reaction is carried on
for about 10 to 15 hours. A runny slurry or soup is obtained
containing about 15% glucose. The process converts about
90~ of the starch to glucose.
A number of variables that could influence
the course of the novel hydrolysis reaction have been inves-
tigated. Each one was examined under a set of standard
conditions, so only one variable was changed at a time. The
~- standard conditions were:
- 100 g. of whole barley, crushed to 4/64
inches
- 240 ml. water
- pH 4 - 4.5 by use of 85% phosphoric acid
- 0.6 ml. of glucoamylase added in two batched;
0.3 ml. at zero time and 0.3 ml. four hourslQtQr
- reaction time - 24 hours
It was found that the ratio of 240 ml. of
water/100 g. of barley gave a handleable porridge at the
beginning of the reaction, and a final runny soup that was
also easily handled. A pH close to 4 is the optimum acidity
for the enzyme glucoamylase.
The following variables were found to have
little effect on the reaction: particle size (ranges tried:
2/64 - 4/64 inches); type of water (tap water vs. distilled
¦ water); stirring; whole or dehusked ~arley; type of barley;
! moisture content of the barley; and temperature gradient within
the reactor.
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~ ~03776~
One variable, temperature of the reaction
; mixture, was found to have an effect on glucose yield.
As illustrated in Figure 2, glucose yields were poor outside
the reaction temperature range of 65 - 67C. Yields were
maximized at about 90% within this range.
The process has advantages over the prior
art in that (1) it is a single enzyme treatment as compared
to the double enzyme treatments previously usedi (2) lower
temperatures are used; and (3) the reaction does not require
vigorous agitation. These differences result in a simpler
process.
- The Condensation Reaction
Once the glucose - containing hydrolyzate is
available, it is reacted with urea under acidic drying
conditions to produce gluccsyl ureides. The reaction is
preferably carried out to maximize the yield of ureides, ;
particularly of monoglucosyl ureide, while minimizing the
production of caramel and other products of the decomposition
of glucose. Therefore it is normally conducted under mild
temperature conditions to produce a solid product. At this
time, the process has been developed to the point where yields
1,
in the order of 90% may be achieved.
The hydrolyzate is reacted with an amount
of urea which is preferably slightly in excess of that amount
required stoichiometrically to combine with the glucose to
produce monoglucosyl ureide. We normally use about a 10%
excess of urea. One can, if desired, react the glucose with
less than the stoichiometric amount - this approach has the
advantage of providing free glucose in the supplement,
thereby improving its palatability; however, normally the
objective of maximizing conversion to ureides is overiding.
Alternatively, one can use a larger excess than 10%. However,
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we have not noted any significant increase in yield when
excesses of 200% and 300% were used.
The pH of the starting reaction mixture is
; established at about 2 - 4.
The drying operation is influenced by
~ temperature, retention time and depth of bed. In general,I we seek to maximize conversion by drying to a solid state
';' '
without sianificant charring. One should keep in mind that,
if the temperature is high, charring can occur if the retention
time is too long. Most of our drying investigations have
been carried out on a laboratory batch basis using a thin
layer (about 5/16") of reaction mixture on trays in a hot
air oven. Under these conditions, drying is carried out at
a temperature of about 75-90C for a prolonged periodj
~ 15 usually in the order of 10 - 24 hours. However, we have
.. ' I
also successfully carried out trials in a rotary hot air
drier having an inlet air temperature of 600F and outlet
temperature of 300F. The retention time used was only
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I seconds to produce a solid product.
In the case of barley, the preferred end
product is a solid brown material having the following typical
composition:
monoglucosyl ureide - 21.0%
diglucosyl ureide - 8.0%
Water insoluble matter
from barley - 25.0%
Water soluble matter
from barley - 16.0%
Free glucose - 3.5%
Free urea - 4.4%
Phosphate - 4.5%
Water - 3.0%
Unidentified compounds - 14.6%
(Mostly nitrogenous compounds
derived from glucose and urea)
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The invention will be further understood
; with reference to the following examples:
Example 1
This example provides a detailed description
of a preferred method for carrying out the hydrolysis and
condensation reactions.
.. .. .
The example involved hydrolysis of whole
barley, followed by the reaction of said hydrolyzed barley
; with urea plus phosphoric acid. lOOg of crushed whole barley
with a moisture content of 11%, 240 ml of water, and 0.6g
I of 85% phosphoric acid were mixed together at room temperature.
Thirty units of Diazyme L-1003 were added to the mixture and
heat was applied with stirring so that the reaction mixture
was brought to 65C in approximately 45 minutes. Thre~ hours
after having reached this temperature, 30 more units of
Diazyme L-100 were added and the mixture kept for approximately
10 hours at 65C under stirring.
At this stage, the glucose content of the
barley hydrolyzate was found to be 16.3~ of 90~ of the
theoretical amount.
To lOOg of barley hydrolyzate containing 14g
I of glucose were added 4.68g of urea (no excess) and lg of
85% phosphoric acid. The mixture was spread out on a
plastic tray, the thickness of said mixture being 6 to 7 mm,
and held at 75C for 14 hours. At this time analysis of
glucose by the glucose oxidase method showed 5.5%, which
represents a conversion of 89%. Analysis of the product
showed urea plus ammonia, expressed as urea, 3.36%; ureides
expressed as monoglucosyl urea, 37.1~4; combined nitrogen,
expressed as monoglucosyl urea, 37~; total nitrogen, 7.76~.
3Registered trade mark for glucoamylase manufactured by
Miles Laboratories, Elkhart, Indiana, U.S.A. Eighty units
of said enzyme will hydrolyze 100 pounds of liquefied starch
in 72 to 96 hours at 62C and pH4.
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4A~alyzed by a potentiometric method.
Example II
This example shows the results obtained when
a 10% excess of urea is used.
To lOOg of barley hydrolyzate prepared
according to Example I, containing 15.6g of glucose, were
added 5.7g of urea (10% excess) plus l.lg of 85% phosphoric
acid. The mixture was placed on a plastic tray, the thickness
of said mixture being close to 7 mm, and kept at 75C for
14 hours. At this stage, the solid product showed the following
analysis: Glucose, 1% (conversion of glucose 98%); ammonia,
expressed as urea, 0.46%; ammonia plus free urea, expressed
as urea, 3.74%; ureides, expressed as monoglucosyl urea,
40.3%4; combined nitrogen, expressed as monoglucosyl urea,
43%; total nitrogen, 8.6%.
;' Example III
This example shows the effect of increasing
the temperature during the drying-operation.
' To lOOg of barley hydrolyzate, prepared
" 20 according to Example I and containing 12.6g of glucose, were
added 4.6g of urea (10% excess) plus lg of 85% phosphoric
' acid. The mixture was placed on a plastic tray, the thickness
of said'mixture being 10 mm, and kept at 95C for 5 hours.
At this time the solid material was broken into small pieces
and held at 95C for 2 more hours. The solid material gave
~' the following analysis: glucose, 2% -(conversion of glucose
94~); ammonia, expressed as urea, 0.26%; ammonia plus free
urea, expressed as urea, 2.9%; ureides, expressed as monoglucosyl
urea, 34.4~4; combined nitrogen, expressed as monoglucosyl urea,
' 30 40%; total nitrogen, 8.0%.
Ex-ample''IV
I This example is similar to Example II, however
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a longer retentid~ tlme is used,
To lOOg of barley hydrolyzate, prepared
according to Example I and containing 13g of glucose, were
added 4.76g of urea (10~ excess~ plus 0.95g of 85% phosphoric
acid. lOOg of this mixture were poured into a plastic tray,
the thickness of the said mixture being 7 mm, and kept at
75C for 24 hours. At this stage, 30.6g of solid material
were obtained that gave the following analysis: glucose,
- 3.2% (conversion of glucose 92%); ammonia plus urea, 2.81%
monoglucosyl urea, 18.9%; combined nitrogen, expressed as
monoglucosyl urea, 46~; total nitrogen, 8.67~.
- Example V
This example shows the use of only 85% of the
stoichiometric amount of urea required to react with the
glucose to produce monoglucosyl ureide.
To lOOg of barley hydrolyzate, prepared
according to Example I and containing 13g of glucose, were
added 3.68g of urea (85% of stoichiometric amount) plus 0.73g
; of 85% phosphoric acid. lOOg of this mixture were poured
into a plastic tray, the thickness of the said mixture being
7mm, and kept at 75C for 24 hours. At this time, 30g of
solid material were obtained that gave the following analysis:
glucose, 8.1% (conversion of glucose 80%); ammonia plus urea,
expressed as urea, 2.06%; monoglucosyl urea, 16.7%; combined
nitrogen, expressed as monoglucosyl urea, 36%; total nitrogen,
7.0%.
Example VI
This example shows the effect of using a 100%
' excess of urea.
To lOOg of barley hydrolyzate, prepared according
to Example I and containing 13g of glucose, were added 8.66g
of urea (100% excess) plus 1.73g of 85% phosphoric acid. lOOg
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~ lV377ti~
of this mixture were poured into a plastic tray, the thickness
of the said mixture being 7 mm, and kept at 75C for 24 hours.
At this stage, 32.9g of solid material were obtained that
~ gave the following analysis: glucose, 0.6~ (conversion of
- 5 glucose 98.3~); ammonia plus urea,~expressed as urea, 10.67%
,
: monoglucosyl urea, 27.~%; combined nitrogen, expressed as
- monoglucosyl urea, 47%; total nitrogen, 12.27%.
Example VII
- This example shows the use of corn as the source of starch in the hydrolysis reaction.
lOOg of corn (whole grain, 18.9% moisture)
were mixed with 240 mls of tap water and ground exhaustively.
The pH was adjusted to 4.5 with 85~ phosphoric acid and 30
units of Diazyme L-100 added. Stirring was commenced and
the temperature was raised to 67C. At this time, 30 units
of Diazyme L-100 were added and the reaction was carried on
for 24 hours. A yellow, runny soup was obtained - it contained
14.6% glucose.
Example VIII
This example shows the use of wheat as the
source of starch in the hydrolysis reaction.
lOOg of whole ground wheat (12.4% moisture)
were mixed with 240 mls of tap water. The pH was adjusted
to 4.7 by the addition of 0.6g of 85~ phosphoric acid. 30
units of Diazyme L-100 were added and stirring was commenced.
The temperature was raised to 67C over a period of 1 hour.
The reaction was continued at this temperature and, after
i 3 hours, an additional 30 units of Diazyme L-100 were added.
The reaction was then continued for a total reaction time of
16 hours. 340 g of hydrolyzate containing 14% glucose were
- obtained.
To lOOg of the wheat hydrolyzate were added
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5.2g of urea (10% excess) plus 1~ of 85% phosphoric acid.
The mixture was placed in a plastic tray to a thickness of
7 mm, and kept at 75C for 14 hours. At this stage, the
solid material gave the following analysis: glucose, 4.8%
(conversion 90%); ammonia plus free urea, expressed as urea,
2.39~; ureides, 4 expressed as monoglucosyl urea, 41.63%;
total nitrogen, 8%.
Example IX
This example shows the results of drum-drying.
Barley hydrolyzate containing 13.8~ glucose was rapidly
evaporated using a rotary drum under the following conditions:
Thickness of product: 0.025 inches
Steam pressure: 48 psi
:
Production: 1.45 pounds of dry material per
foot 2 per hour
` Slurry feed rate: 4.55 pounds/hour
Surface area: 0.962 foot
rying time: 37 seconds --
In this way, a rapid evaporation of water was
' 20 achieved, however less glucose was converted to ureides
- ! compared to the previous examples. A solid product was
- obtained that gave the following analysis: glucose, 15%
(conversion 70%); urea plus ammonia, expressed as urea,
5.41%; combined nitrogen, expressed as monoglucosyl urea,
35.0%; monoglucosyl urea, 12.7%; total nitrogen, 8.4%.
! Example X
:., I
This example shows the results of a centrifuge
and evaporation trial.
200 g of barley hydrolyzate (13% glucose)
plus 10~ excess urea were centrifuged at 200 rpm for 20 hours.
105g of liquid and 105g of cake were obtained. The liquid
and the cake were evaporated to yieId 45.15 g of syrup,
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and 55.?g of cake, respectively; the~ were mixed together to
give a mixture with a water content of 35%. This mixture
was dried at 75C for 20 hours. 73g of a solid were obtained
- that gave the following analysis: glucose, 3% (conversion 90%);
ammonia plus urea, expressed as urea, 3.4%; combined nitrogen,
expressed as monoglucosyl urea, 37%, total nitrogen, 8.51%.
Example XI
This example shows the results obtained when
the hydrolyzate was cooked instead of dried in accordance with
the invention
To lOOOg of barley hydrolyzate containing 138g
of glucose, were added 50g of urea (10% excess) plus lOg of
85% phosphoric acid. lOOOg of this mixture were evaporated to
; 395g of solid. This solid was placed in a closed container
and kept at 75C for 14 hours. At this stage no loss of water
was observed. The solid pastry product showed the following
analysis: glucose, 8% (conversion of glucose 77%); urea plus
ammonia, expressed as urea, 3.98%; combined nitrogen, expressed
as monoglucosyl urea, 25%; total nitrogen, 6.76%.
A principal advantage of our product is that
it can be produced with a high total equivalent crude protein
` content, with up to 90% of this crude protein in a slow
... .
ammonia release, urease-resistant form. The following examples
are from animal feeding trials using various forms of our
product and demonstrating the effectiveness of the product.
Example XII
For the purpose of this trial, steers of Here-
ford breeding were divided into two groups; one group fed a
ration which contained no supplemental protein, the other
group fed a ration which contained the solid NPN product
prepared as in Example IV. These mixtures constltuted 33%
of the final ration and were formulated to contain similar
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levels of all nutrients except crude protein. See Table 1.
TABLE 1
- Formulat1on of Diets
Ingredients Control Solid non-protein
ration nitrogen ration
(g~) (%)
~ ,: ~ ' . .. .
Straw (oats) 66.7 66.7
Concentrate 33.3 33.3
Composition of Concentrate
Barley 93.7 61.5
- Ground limestone 1.6 2.4
Trace mineralized salt 1.0 1.0
Vitamin premix 11 2.0 2.2
Barley-based non-protein nitrogen -- 32.8
To supply 2,300, 380 and 2.3 IU of Vitamins A, D3 and E,
respectively, per pound of total ration.
Compo_ltion of Total Ration
Moisture ................. (%) 15.8 15.5
Crude protein............. (%) 5.9 8.9
- Calcium................... (%) 0.3 0.34
Phosphorus................ (~) 0.21 0.26
Digestible energy......... Mcal/pound 1.08 1.05
From Table 2, we see that the steers fed with
the solid non-protein nitrogen supplement gained 20% faster
than did the animals that received the control ration; they
also ate more and needed a lower amount of intake per pound
gained.
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T~BLE 2
Effects of Crude Protein Supplementation
On the Perfoxmance of Growing Steers
- Control Non-protein Nitrogen
Number of steers 16 16
Feeding period days 154 154
Av. initial weight pounds 527 530
Av. final weight pounds 612 632
Av. daily gain pounds 0.55 0.66
Av. daily feed pounds 12.5 13.3
Feed/gain 22.47 21.2
. _ _
12Feed intake is given on an as-fed basis (approximately 84.5%
dry matter).
Example XIII
In this example, three groups of steers were
fed rations supplemented with one of the following: (l)our NPN
product, prepared as in example IV; (2)soymeal (which is a
premium plant source of protein); and (3)urea. A fourth control~
group was not fed any supplement.
The growing ration used was based on corn silage
with a "barley-protein supplemented" concentrate to provide an
overall ration with an energy equivalent of approximately
1.17 Mcal per pound of dry matter. The equivalent crude
protein content of the unsupplemented control was 0.94 pounds
per pound of dry matter, while that of the supplemented rations
was 0.11 pounds per pound of dry matter.
The results of the trial, the compositions of
the concentrates, and the nutrient content of the feed are
given in the following tables:
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TABLE 3
301 302 303 304
(Control)(Soymeal)(Test NPN)(Urea)
(No supplement)
Number of animals 144 143144 144
Days on test 118 105103 119
Pounds Silage consumed
per head/day 47.89 50.27 49.30 48.97
Concentrate consumed
per head/day 2.99 3.13 3.07 3.06
Pounds total dry matter
consumed per head/day14.66 15.39 15.09 14.99
Average total gain
(pounds per head)184.7 185.7184.5195.4
Average daily gain1.56 1.77 1.79 1.64
- Conversion ratio
(dry matter consumed9.37 8.70 8.43 9.13
per pound gain)
~. . _ . __ _
TABLE IV
Concentrate Composition
: .
- 301 304 302 303
Low Soybean Test
Portein Urea Meal NPN
:........................................ _
~ Ingredient:
;: 25 Ground Barley 95.35 92.1271.17 74.01
Urea (45% N) -- 3.23 -- --
Soybean Meal -- --24.58 --
Test NPN -- -- -- 21.56
Dicalcium phosphate -- 0.06 -- --
-30 Ground Limestone 2.00 1.941.84 1.95
Plain Salt 1.32 1.321.35 1.86
Beef Mineral/Vitamin Pre. 0.332 0.332 0.332 0.332
Sodium tripolyphosphate 0.710 0.710 0.430 --
Elemental Sulfur0.286 0.286 0.286 0.286
- Total 100.0 100.0100.0 100.0
Nutrient:
Dry Matter (%) 88.56 88.9588.78 88.96
' Crude Protein (%)11.08 19.7719.75 19.78
i Digestible Protein (%) 6.88 -- 15.47 __
,-40 M.E. Rum. (Mcal/lb)1.144 1.106 1.115 1.101
i TDN (%) 69.56 67.2069.87 66.90
Calcium (%) 0.810 0.797 0.813 0.807
Phosphorus (%) 0.515 0.515 0.519 0.515
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Sodium (%) 0.749 0.749 0.752 0.761
Magnesium (%) 0.092 0.089 0.135 0.096
Potassium (%~ 0.361 0.349 0.740 0.375
Sulfur (%) 0.481 0.477 0.554 0.481
Iron (mg/lb) 46.9 46.1 56.0 54.4
Copper (mg/lb) 14.5 14.4 15.5 30.5
Manganese (mg/lb)22.4 22.2 24.0 22.7
Zinc (mg/lb) 78.2 77.6 74.3 85.2
Crude Fat (%) 1.8 1.8 1.7 1.4
Crude Fiber (%) 4.8 4.6 4.9 3.7
TABLE V
Nutrient Content of a 1:4- Blend of Concentrate
and Corn Silage C.M. (% of DM)
301 304 302 303
Low Soybean Test
Protein Urea Meal NPN
----___ _ _ __
Dry Matter (%) 100.0 100.0 100.0 100.0
Crude Protei~ (%)9.43 11.20 11.20 11.20
M.E. (Rum) (Mcal/lb)* 1.176 1.167 1.177 1.166
;
TDN (%)* 71.55 71.01 71.60 70.95
Calcium (%) 0.379 0.376 0.379 0.377
'-. Phosphorus (%) 0.335 0.334 0.335 0.334
Sodium (%)- 0.211 0.210 0.211 0.213
Magnesium (%) 0.207 0.206 0.175 0.187
Potassium (%) 0.934 0.934 1.01 0.936
Sulfur (%) 0.123 0.122 0.138 0.123
Iron (mg/lb) 115.6 115.40 117.5 117.0
Copper (mg/lb) 5.9 5.9 6.1 9.2
Manganese (mg/lb)20.6 20.5 20.9 20.6
Zinc (mg/lb) 30.5 30.4 29.7 31.9
Fiber (Mg/lb) 22.7 22.7 22.7 22.5
Vitamin A (I.U./lb)3700.03700.03700.0 3700.0
.
; ME - Metabolitable energy
TDN - Total digestible nutrients
From these data, it will be seen that:
(1) in terms of feed conversion ratio, the
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the NPN supplement out-performed all
other groups, followed by the soymeal,
the urea, ancl the control group. The :: ;
observed difference betw~en the soymeal
and the NPN group - and other groups -
was statistically significant at the 95%
; confidence level, while the difference
between the soymeal and the NPN group
was not significant at this level.
(2) the soymeal and the NPN groups gained
~: .
approximately at the same rate, and at
a significantly higher rate than the
control or the urea group. The urea -
; group gained at a significantly faster rate
` 15 than the control group.
(3) in terms of feed intake, the soymeal
group consumed significantly more, while
the control group consumed significantly
~` less feed than the other groups.
While certain examples~ structures~ composi-
tions and process steps have been described for purposes of
- illustration the invention is not limited to these. Varia~
tions and modifications within the scope of the disclosure
and the claims can readily be effected by those skilled in the
art.
SUPPLEM~NTA~Y PISCLOSURE
This supplementary disclosure relates to the
use of starch-containing materials other than grains selected
from the group consisting of barley, wheat and corn in the
process of the Prîncipal Disclosure for producing a non-protein
nitrogen-containing material useful as a feed supplement
for cattle and sheep. The starch-containing materials may
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i include different grains, such as rice and oats, as well as
sources other than grains, such as cassava roots and potatoes.
; The characteristics of various suitable starch sources are
given in Table 7.
TAsLE 7
Starch ~oisture Starch
Source (%) - (%, d.m.b.)
barley 10.8 59.9
sorghum 8.5 57.9
10 tapioca 10.0 96.6
rice 11.9 95.6
oats 10.5 53.3
: corn 9.8 82.1
cassava root 7.6 93.7
15 potatoes 78.4 69.5
The invention is not restricted to materials
which hydrolyze to produce glucose as the major hydrolysis
product. Glycoses, HOCH2 ~CHO~)nCHO, other than glucose can be ~ -
reacted with urea to produce glycosyl ureides. For example,
the spent sulphite liquours of the paper industry containxylose in amounts greater than glucose and can be used in the
process of the present invention.
Broadly stated, the invention is a process for
producing a non-protein nitrogen feed supplement for ruminants
which comprises: providing an aqueous mixture of a starting
- material containing one or more reducing carbohydrates as the
major dry matter constituent, and urea; and reacting the
mixture under acidic and drying conditions at an elevated
temperature sufficient to convert the major portion of the reducing
carbohydrates and urea into ureides and to produce a solid product.
~ .
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1(137 ~
ANALYS I S` PROCEDURES
,
Analysis procedures similar to those described
in the Principal Disclosure were used unless otherwise specified.
The concentration of total ureides was deter-
mined by acid hydrolysis of the product to give glucose and
urea, followed by enzymatic hydrolysis of the urea and by the
~- potentiometric measurement of the resulting ammonia. The -
`, measurement gave the total ammonia concentration; this value
was corrected for the amount of free ammonia and free urea to
~,. 1
~X` 10 obtain the total ureide concentration.
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The moisture content of a starch source was
determined by heating a sample in an oven at 75C to constant
weight.
Starch determination was done by treatment of
a known amount of the starcll source suspended in water with
; 85% phosphoric acid (pH 1.5 - 2.0) at 80C overnight. A
control experlment was simultaneously conducted using pure
potato staxch. After cooling and adjusting the p~I to 4.5 with
sodium hydroxide, the unknown and the control samples were
treated with the same amount of glucoamylase at 62C for 24
hours. The glucose thus produced was analyzed, and the result
obtained was corrected according to the glucose obtained from
the control experlment.
.. .... .. .. ..
THE HYDROLYSIS REACTION
The hydrolysis reaction was carried out in
equipment similar to that described in the Principal Disclosure.
The standard reaction conditions were~
100 g substrate, crushed to 2/64"
24Q ml distilled water
pH 4.0 to 4.5 (by use of 85% phosphoric acid)
1.0 ml Diazyme L-100* added in two batches;
0.5 ml at zero time and 0.5 ml three hours
after reaction temperature reached 65C
reaction time: 24 hours
The relatively low conversions to glucose in
the case of sorghum and corn prompted the pre-treatment of the
substrate at high temperature and acidic conditions overnight,
prior to hydrolysis. In all cases, this procedure substantially
increased the conversion of starch to glucose.
It was also found that the mixing of the sub-
strate with water in a ~aring* Blender prior to the hydrolysis
reaction also increased thé conversion to glucose. This
was tried in the case of sorghum, corn, and potatoes.
.. .
~ *trademark - 22 -
~S~3~'~6~
- Due to the high moisture content, potatoes were
the only starch source which was not crushed prior to reaction.
Instead, the potatoes were cut into small pieces with a knife
and mixed with water in a Waring* slender. The soup obtained
after hydrolysis was extremely runny, indicating that far
less than the standard 240 mls of water could be used. A
reaction using only 120 mls of water was tried; this also pro-
duced a very runny soup.
- The standard reaction time of 24 hours was cut
to 6 hours when it was discovered that ~he heater malfunctioned
overnight. During the 6-hour reaction it was observed that the
controller maintained the temperature constant to within
- 1.0C. The average increase in glucose content between 6 hours
of reaction and 24 hours of reaction was 2.2~ based on
analysis of the first 13 hydrolyses in which the heater was
thought to have Punctioned properly.
THE CONDENSATION REACTION
The condensation reaction was carried out by
mixing the hydrolyzate with a lQ~ stoichiometric excess of
urea and with phosphoric acid ta obtain a pH of 2.5 - 3Ø The
mixture was spread on a plastic tray and heated at a given
temperature to dryness. The product was analyzed for glucose
and monoglucosyl ureides.
The following examples illustrate the use of
various starch-containing materials in the process of the
present lnvention.
Example 14
This example shows the use of sorghum as the
source of starch in the hydrolysis reaction.
: 30 10-Q g of crushed sorghum were mixed with 240 mls
of distilled water. The pH was adjusted to 2.0 with 85~ -
~ .
* trademark - 23 -
~ ()37~
phosphoric acid and the mixture was left at 80C overnight.
After cooling and adjusting the pH to 4.5 with sodium hydroxide,
:.
50 units of Diazyme L-100* were added. Stirring was commenced
and the temperature was raised to 65C. Three hours a~ter
having reached this temperature, an additional 50 units of
Diazyme L-100* were added. The reaction was then continued
for a total reaction time of 24 hours, at which stage the glucose
content of the hydrolyzate was found to be 15%.
5.5 g of urea (a 10% excess) and 2 g of 85%
; 10 phosphoric acid were added to 100 g of the sorghum hydrolyzate.
The mixture was spread in a plastic tray to a thickness of 7 mm, ~ ~ -
and kept at 75C for 2a hours. At this stage~ the solid -
material gave the following analysis: glucose, 3% (93% con~
version); ammonia, expressed as urea, 0.44%; ammonia plus free -
urea, expressed as urea, 5.6%; ureides, expressed as mono-
glucosyl urea, 34.8% com~ined nitrogen, expressed as mono-
glucosyl urea, 42~1%; total nitrogen 8.51%.
Example 15 -
; This example shows the use of oats as the source
of starch in the hydrolysis reaction.
100 g o~ crushed oats were mixed with 24Q mls ofdistilled water. The pH was adjusted to 4.5 with 85%
phosphoric acid and 50 units of Diazyme L-100* were added.
Stirring was commenced and the temperature was raised to
65C. Three hours after having reached this temperature, an
additional 5Q units of Diazyme L-100* were added. The reaction
was then continued for a total reaction time of 24 hours, at
which stage the glucose content of the h~drol~zate was found
to be 15.g%.
5.84 g of urea (a 10% excess) and 2 g of 85%
phosphoric ac~d were added to 100 g of the oat hydrolyzate.
' ~
~ ~ * trademark - 24 -
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7~ 3
The mixture was spread in a plastic tray to a thickness of 7mm,
and kept at 75C for 70 hours. At this stage, the solid
material gave the following analysis: glucose, 1~ (98% con-
version); ammonia, expressed as urea, 0.58%; ammonia plus free
urea, expressed as urea, 5.0%; ureides, expressed as mono-
glucosyl urea, 38.9%; combined nitrogen, expressed as mono-
glucosyl urea, 45.0%; total nitrogen, 9.52%.
Example 16
This example shows the use of rice as the source
of starch in the hydrolysis reaction.
lQ0 g of crushed brown rice were mixed with 240
mls of distilled water. The pH was adjusted to 4~5 with 85%
phosphoric acid and 50 units of Diazyme L-100* were added.
Stirring was commenced and the temperature was raised to 65C.
Three hours after having reached this temperature, an additional
50 units of Diazyme L-100* were added. The reaction was then
continued for a total reaction time of 24 hours, at which
stage the glucose content of the hydrolyzate was found to be
21 4%
- 20 7~84 g o~ urea (lQ% excess) and 3 g of 85% -
phosphoric acid were added to 100 g of the rice hydrolyzate.
The mixture was spread in a plastic tray to a thickness of 7 mm,
and kept at 75~C for 50 hours. At thîs stage, the solid
material gave the following analysis: glucose, 1% (98% con-
version~; ammonia, expressed as urea, 0.78%; ammonia plus free
urea, expressed as urea, 5.5%; ureides, expressed as mono-
; glucosyl urea, 37.0%; combined nitrogen, expressed as mono-
;~ glucosyl urea, 61.5%; total nitrogen, 11.3%.
Example 17
. '.
` 30 This example shows the use of corn as the
source of starch in the hydrolysis reaction.
.' ''~ ` .
, * trademark - 25 -
` '' ,~
3~U~7~
.
lQQ g of crushed corn were mixed with 240 mls ~ -
of distilled water and ground exhaustively. The pH was ad-
justed to 4.5 with 85% phosphoric acid and 50 units of Diazyme
L-100* were added. Stirring was commenced and the temperature
was raised to 65C. Three hours after having reached this
temperature, an additional 50 units of Diazyme L-100* were
added. The reaction was then continued for a total reaction
time of 6 hours, at which stage the glucose content of the
hydrolyzate was found to be 10.8%.
4.34 ~ of urea (10% excess) and 1 g of 85%
phosphoric acid were added to 100 g of the corn hydrolyzate~
The mixture ~as spread in a plastic tray to a thickness of 7
mm, and kept at 75C for 22 hours. At this stage, the solid
material gave the following analysis: glucose, 2~4% (94%
conversion); ammonia, expressed as urea, 0.15%; ammonia plus -
free urea, expressed as urea, 3.0%; ureides, expressed as
monoglucosyl urea,-25.9%; combined nitrogen, expressed as
monoglucosyl urea, 44.5%; total nitrogen, 8.35%.
The results of the hydrolysis of various grains
and the analyses of the corresponding condensation products
are given in Table 8.
TABLE 8
Glucosyl
Glucose Conver- ureide
in hydro- sion to (GU) in Yield Glucose
lyzate glucose product of GU reacted
Substrate ` (~O) (%) (~O~ (%) (%)
barley 17.8 100 14-3 } 16 hrs.
16.3 at 75C
14.5 93.2 19-6 l 24 hrs. 85.1
18.3 J at 60 32.3 84.9
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~ * trademark - 26 -
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.
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1~)37~7f~3
TABLE 8 (continued)
Glucosvl
` Glucose Conver- ureide
' in hydro- sion to (GU) in Yield Glucose
lyzate glucose product - of GU reacted
Substrate(~ ) (%) - (~-) ` (~)
sorghum 3.0 18.03.3 90 hrs. at 23.1 78.4
65
15.0a 93.216.3l 20 hrs. 28.8 93.5
12.5J at 75 22.4 68.1
b
9.6 71.1 4.9 24 hrs. 12.8 95.2
at 60
3.5 19.2 3.4 50 hrs. 24.0 65.1
at 65
4.7c,d 25.8 6.7 16 hrs. 34.5 79.0
at 75
4.2 16 hrs. 21.5 53.6
at 65
.: ' .
tapioca18~2 65.0 25.0 68 hrs 38.6 95.1
at 60
22.2 17 hrs. 34.1 92.8
; at 60-80
; 25 15.6 138 hrs. 23.9 97.4
at 60
20.8 51 hrs. 31.9 97.5
at 65
;;
rice 11.9 43.1 7.2 138 hrs. 15.0 93.8
- at 60
19.6 27 hrs. 42.4 94.1
- at 65 J
18.6a 72.9 <4 5 days ~6 97.9
at 65
21.4 73.8 19.8 50 hrs. 26.8 98.2 '
at 75 `
-:
22.5 75 hrs. 31.8 97.9
at 65
~ ~',' '.
Example 18 ;~
:: :
This example shows the use of a starch-con-
taining material that is not a grain.
;
,
- 27 -
:
~76
- 100 g of crushed cassava root were mixed with 240mls of distilled water. The pH was adjusted to 4.5 with 85%
phosphoric acid and 50 units of Diazyme L-100* were added.
- Stirring was commenced and the temperature was raised to 65C.
Three hours after having reached this temperature, an additional
50 units of Diazyme L-100* were added. The reaction was then
continued for a total reaction time of 24 hours, at which stage
the glucose content of the hydrolyzate was found to be 21.1%.
7.74 g of urea (a 10% excess) was added to 100 g
of the cassava root hydrolyzate, and the pH was adjusted to 2.5
with 85% phosphoric acid. The mixture was spread in a plastic
tray to a thickness of 7 mm, and kept at 60C for 120 hours.
~ At this stage, the solid material gave the following analysis:
.~ glucose, 1% (98% conversion~; ar,~onia, expressed as urea, 0.33%;
ammonia plus free urea, expressed as urea, 5.4%; ureides, ex-
pressed as monoglucosyl urea, 41.4%; combined nitrogen, expressed
as monoglucosyl urea, 57.~P6; total nitrogen, 9.27%.
Example 19
This example shows the use of starch-containing
- 2~ material that is not a grain.
100 g of diced potatoes were mixed with 240 mls
of distilled water an~ ground exhaustively. The pH was adjusted
to 4.5 with 85% phosphoric acid and 50 units of Diazyme L-100*
~; were added. Stirring was commenced and the temperature was
raised to 65C. Three hours after having reached this
temperature, an additional 50 units of Diazyme L-100* were added.
The reaction was then continued for a total reaction time of 6
hours, at which stage the glucose content of the hydrolyzate was
found to be 3.7%.
1.36 g of urea (a 100% excess) and 1 g of 85%
phosphoric acid were added to 100 g of the potato hydrolyzate.
"' ..
~ * trademark - 28 -
;'
The mixture was spread in a plastic tray to a thickness of 7 mm,
and kept at 75C for 18 hours. At this stage, the solid material
gave the following analysis: glucose, 1% (98% conversion);
ammonia, expressed as urea, 0.29%; ammonia plus free urea,
expressed as urea, 1.7%; ureides, expressed as monoglucosyl
urea, 30.7%; combined nitrogen, expressed as monoglucosyl urea,
52.1%; total nitrogen, 8.26%.
Exà~ple 20
.
This example shows the use of spent sulphite
liquor as a source of reducing sugars in the condensation reaction.
The spent liquor used was typical of that obtained from a hard-
wood pulp, and contained approximately 26% xylose on a dry matter
basis.
25 g of spent sulphite liquor (40% moisture)
having a pH of 2.0 were mixed at room temperature with 1.8 g
urea. The mixture was spread on a plastic tray to a thickness
of 7 - 8 ~m, and kept at 75C for 110 hours. At this stage, the
product gave the followlng analysis: free ammonia plus free
- urea, expressed as urea, 5.2%; ureides, expressed as equivalent
~ 20 monoglucosyl urea, 17.8%; total nitrogen, expressed as urea,
-. 10. 0% .
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