Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
AIIR-32 O
``` ~078766
B~CKGROU~D OF T~IE INVENTION
This invention relates to an improved method of tempering
animal tissue for heparin production. More particularly, this
invention relates to an improved process or raising the
temperature of frozen heparin-bearing animal tissue under
controlled conditions of temperature and time in preparation
for isolation of heparin. The tissue is first particulated, as
; by grinding, chopping or other means, while in a frozen or
partially frozen state, thawed and warmed in a heat exchanger
and thereafter fermented at 45 -95 F. for 2 to 15 hours but not
substantially beyona the time severe foaming or gassing begins.
The resulting tempered tissue may be defatted and dehydrated
with a solvent which forms an azeotrope with the tissue water
and subjected to a heparin recovery procedure or it may be used
directly in heparin recovery processes.
i 15 Heretofore, frozen heparin-bearing animal tissue was
tempered by allowing solidly frozen blocks of the tissue in
bags or boxes to gradually thaw and warm up over periods of
2 to 8 days at ambient temperature of 60 -120 F. while in these
; containers, during which time it had been thought that optimalconditioning of the tissue for heparin release in subsequent
heparin recovery was occurring. However, the tissue is very
sensitive to enzymatic action and subject to decomposition by
undesirable bacterial growth and rotting of tissue. The outside
of a frozen block of tissue subjected to these older tempering
methods could actually putrefy before the interior had reached
a thawed state. This resulted in generation of obnoxious,
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undesirable odors which, during the 2-8 day period, would sprcad
throughout the community surrounding the tempering plant. This
long period of time required for tempering resulted in poor
utilization of space with consequent high overhead expense,
bloody fluid and sewage disposal problems, and tempered tissue
which was not sufficiently uniform biochemically from one lot to
another due to non-uniform tissue breakdown resulting in heparin
unavailability and consequent need for continual adjustment
during later processing in heparin isolation. In addition,
unwanted bacterial decomposition is known to cause high pyrogen
content and increased effort was required for pyrogen removal.
Moreover, when tissues tempered by the above described
prior art procedures were particulated and subjected to azeotropic
solvent processing to remove fat and water, the fat content was
~ not lowered below about 0.5% by weight even under the most
favorable circumstances and more generally ranged from 1.0 to
2.0%. In addition, the defatted-dehydrated tissue particles were
difficult to wet and floated for long periods of time in con-
ventional solutions used in the initial step of the heparin
recovery process and in the mixtures were difficult to handle
and filter subsequently in the process. Tissue tempered according
to the present invention is capable of conversion to desiccated
and defatted tissue of unusually high quality by azeotropic
processing wherein the fat content is generally reduced to about
0.1 to 0.3 weight ~ and the product is readily wetted in the
above described heparin recovery process and mixtures are more
easily filtered. Other indicatio~ of improved quality of such
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dcfatted tissu~s are lighter color, less odor, good texture
and ho~o~eneityas well as co~sistent low levels of fat and
residual solvent. In addition, more fat is recoverable from
the solvent for a given amount of heparin-bearing tissue which
is an advantage. Apparently the novel combination of steps in
the present process of particulating frozen tissue, rapidly
thawing and warming the tissue and fermenting under controlled
conditions is responsible for the increased wettability.
Further and equally important, due to the control of conditions
during tempering, the heparin yield and content of the defatted
tissue having been first tempered by the process of this invention
can be as much as about 70-12~ higher than for the above described
prior art defatted tissue.
As used herein, the term "tempering" refers to raising
the temperature of frozen heparin-bearing animal tissue and
conditioning it for further heparin recovery processing. The
term "heparin-bearing animal tissue" refers to those animal
tissues rich in heparin and suitable for heparin production such
as lung, brain, liver, intestines or inexpensive fleshy parts of
animals. The term'~articulate" or derivatives thereof pertains
to the divided state of the tissue; i.e , size up to about 1/4
inch mesh, or the act of dividing larger pieces which have been
pre-broken or flaked. The term "fermentation" refers to the
combined action of enzymes present in the tissue and enzymes
generated by growth of bacteria or microorganisms either present
in the animal tissue or added enzymes or bacteria to speed the
liberation of heparin. The term "azeotropic processing" refers
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to the subjection of the tissue to the boiling action of a solvent which
forms an azeotrope with water to substantially remove it and which extracts
tissue fat into the solvent stream and thereafter collecting and washing the
dehydrated tissue on a filter with solvent.
SUMMARY OF THE INVENTION
The present invention therefore resides principally in the discovery
that improved tempering of frozen or partially frozen heparin-bearing animal
tissue can be effected by particulating and pumping to a heat exchanger where,
under controlled conditions, it is thawed and warmed and thereafter fermented
at controlled temperature for a period of time to substantially effect
enzymatic conditioning of the tissue to improve heparin availability.
According to the present invention; there is provided a process for
tempering frozen heparin-bearing animal tissue in preparation for isolation
of heparin which comprises the steps of ~-
1) particulating said tissue,
2) thawing and warming the particulated tissue to a temperature within
the range of 45 to 95F in a heat exchanger, and
3) fermenting the thawed tissue from step 2 at a controlled temperature
within the range of 45 to 95F for a period of time of 2 to 15 hours to
substantially effect enzymatic conditioning of said tissue to improve heparin
availability.
The process provides improved biochemical uniformity from one lot
to another, exceptionally high availability of heparin due to control of time
and temperature throughout the processing of each lot, and conditioned
tissues which are very low in pyrogen content.
The process of the invention may be represented diagrammatically
as follows:
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Flaked or Prebroken
Frozen Tissue
.
Particuiate
Thaw an d Warm .
in ~eat
EXch2 nger ,
Ferment .
45-95 F-
. .
. To Heparin Isolation
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TEMPERI~G PROCESS FOR HEPARD~ TISSUE
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1078766
The preferred process comprises grinding frozen or partially
frozen tissue and rapidly thawing and warming the thawed tissue in a heat
exchanger and thereafter fermenting the tissue at temperatures within the
range of 45 to 95F for a period of time to optimize heparin availability,
length of time required being dependent on temperature. Generally speaking,
the higher the temperature within this range the shorter will be the time
required. For some unexplained reason,severe evolution of gas and foaming
signals the end of the desirable fermentation phase.
The heparin in the tissue tempered as in this invention may be ~-
recovered by a number of techniques including the method of U. S. Patent
No. 2,797,184. The tempered product may be dehydrated and defatted first by
azeotropic processing procedures such as in U. S. Patent No. 2,539,544 to
provide a solid heparin source which is exceptionally low in fat and readily
wetted by solutions used in heparin separation such as are described in
U. S. Patent No. 2,797,184 or 2,954,321 with superior recovery of heparin
obtained as compared with conventionally tempered tissue which has also been
defatted by azeotropic means.
The present invention, generally stated, provides an improved
process for tempering frozen heparin-bearing animal tissue prior to heparin
recovery and isolation procedures, particularly wherein the process eliminates
the unsanitary conditions of rotting and obnoxious odor which are attendant
in prior art methods.
The present invention also provides a process for tempering
frozen heparin-bearing animal tissue under controlled conditions as to pre-
cise time and temperature at all times including a controlled fermentation
step wherein the total microbiological population is evenly distributed in
the substrate and then growth promoted allowing the action of both endogenous
and exogenous enzymes on the substrate, which produces a uniformly condi-
tioned product having a more consistent biochemical content, which is lower
in pyrogen content and high in available heparin content, and which may be
advantageously employed to prepare a dehydrated and defatted heparin tissue
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by azeotropic processing exceptionally low in fat content and easily wetted
and easily suspended in liquid involved in heparin recovery processing.
There follows a more detailed description of the best mode of carrying out
the invention.
DETAILED DESCRIPTION OF THE INVENTION
The preferred process of the present invention for tempering
frozen heparin-bearing tissue in preparation for isolation and recovery of
heparin comprises the steps of
1) particulating the frozen tissue to any size up `~
to about 1/4 inch mesh size, preferably 1/8 to
1/4 inch mesh size, preferably using a grinder,
2) thawing and warming the particulated tissue from
step 1 to a temperature within the range of 45
to 95F, preferably 65 to 85F, using a heat
exchanger, preferably a shell and tube heat
exchanger, and
3) fermenting the warmed tissue from step 2 while
maintaining it at a temperature within the range
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3' A of 45 to 95F, preferably ;4~ to 85F for a
period of 6 to 8 hours time but not substantially
beyond the time foaming starts due to gas liberation.
The heparin-bearing animal tissue for the process of this
invention originates at the meat packing plant where it is cut
froc anical carcasses and handled according to specified
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procedures for preserving and enhancing heparin values, boxed
or bagged and deep frozen. As a consequence, the animal parts
a~rive at the heparin recovery plant as blocks of one kind of
frozenJ agglomerated animal part such as, for example, lung in
the size and shape of the containers. Usually, the size of the
blocks of frozen animal parts is too large for direct grinding
in the size grinder feasible for this art and it is therefore
necessary to reduce the size of the blocks by some means prior
to feeding to a grinder. To accomplish this the deep-frozen
blocks of tissue may be cracked or prebroken mechanically in a
'~ device known as a PrebreakerTM Preferably, however, the blocks
of tissue are partially defrosted for about 8 hours at ambient
temperatures of 80-100 F. and thereby brought from their deep-
frozen state to a softer state by raising the temperature of
the blocks to about 20-~2 F. after which time the softened
tissue may be chipped or flaked in preparation for grinding.
When the flaking or chipping procedures are followed the prefer-
able temperature to which the blocks of tissue are raised is
about 26F. as the tissue is rigid enough to be flakedJ yet in
a somewhat softened condition feasible for the flaking operation.
A suitable flaking machine is the Hydrauflaker produced by
the General Machinery Corp., SheboyganJ Wis. In any caseJ the
tissue should not be so cold that the gound tissue in the next
step refreezes into balls and clumps which prevent pumping. In
general, the particle size of prebroken or flaked frozen tissue
can vary from 1/8 inch to 2 inches in diameter.
In step 1 of the pxocessJ grinders operate to reduce
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the si~e o~ the prebroken or flaked, frozen, or partially
frozen tissue to that ranging from that present in a puree up to
a maximum dimensional mesh size of about 1/4 inch, preferably
up to 1/8 inch mesh size. Grinders which are suitable are
the Comitrol produced by Urschel Laboratories of Valparaiso,
Ind. and the Autio Grinder produced by the Autio Company,
Astoria, Oregon.
In step 2, the ground frozen or partially frozen tissue
is introduced by means of a pump such as a Moyno pump to a
heat exchanger which operates to thaw and warm the tissue in
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~0 minutes or less, preferably within about 5 minutesJ to 45 -
` 95 F. using a heat exchange surface temperature not to exceed
140 F. Above about 3~ minutes too much variation is introduced
` in later processing. Heat exchange surfaces having a higher
temperature than 140 F. cause fouling of the surfacesJ denaturing
of protein and microbiological kill-off. Shell and tube heat
exchangers with tissue passing through the tube are highly
satisfactory and preferred but wiped surface heat exchangers
may be also used. The preferred shell and tube heat exchangers
will range in tube size of about ~/4 inch diameter to about one
inch in diameter and will consequently have surface to volume
ratios of about 50 - 75 ft ? per ft.3. Surfaces of tubes in this
size range remain unfouled.
In step 3, the warm tissue is held in a vessel having an
inert surface such as a stainless steel tank at a temperature
A f 45 to 95 F., preferably ~ to 85 F., for a period of time
sufficient to condition the tissues as a result of a fermentation
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involving enzymes already present and enzymes produced by
growing microorganisms. Above about 95 ~., heparin values are
rapidly 105t and below about 45F., the fermentation step is
ineffective. Two to 15 hour~ fermentation time is required at
45 to 95OF. and for some unknown reason the completion of the
beneficial fermentation is signalled by severe gas liberation
and rising in the holding tanks and further fermentation
decreases the yield of heparin. ~he holding period should be
terminated then or just preceding this indicator according to
previous experience as to time requirement for a particular
temperature. Illustrative of the time temperature relationship
are the following coordinates obtained by trial and error at
~ a ss~
A~ which the frothing or ga~ing phase had begun.
~ime,hr. Temp.~ F.
9-10 75
8 80
6 85
It is not necessary to wait until gassing occurs to obtain the
superior product of this invention. Generally, there is some
variation of microorganisms in lung tissue taken from individual
animals; howevex, grinding and mixing of hundreds of lung lobes
assures that the fermentation will eventually proceed. When it
is desirable to speed the fermentation, the necessary micro-
organisms may be added as, for example, by seeding the tissue
at the beginning of the holding period with tissue which has
already been fermented.
l`he conditioned product of this invention is ideally suited
for use in azeotropic desiccating and defatting processes to
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further enhance heparin availability and separation such as
are disclosed in U. S. Patents 2,619,425 and 2J5~9J544.
PreerablyJ the azeotropic dehydrating-defatting operation is -;
conducted at atmospheric pressure using ethylene dichloride at
a temperature not exceeaing 180Y. Defatted tissue so obtained
is characterized by itæ low fat content of about 0.1 to 0.3
weight % and by its excellent permeability as measured by
wettability and suspendability.
The following are specific examples of the process of
this invention.
` ExamPle 1 (Improved Method)
Partially defrosted frozen beef lung at 25-30 F. in amount
, of 15,190 lbs. was flaked using a Hydrauflaker (Model FS-6)
to a size range of 1/8 inch to 1/4 inch thick and up to 4 inches
long. The flaked frozen lung was then ground with a Comitrol
Grinder (Model 2100) having o.o6 inch size opening. The ground
frozen or partially frozen lung was pumped with a Moyno pump
through 3/4 inch diameter tubes of a shell and tube heat exchanger
to thaw ana warm the lung to 68 -7~ F. residence time in the heat
exchanger being about 4 minutes. The warm ground lung was then
held in a stainless steel tank for 6 hours at 70-75 F. No
external heat was needed to maintain the temperature during the
fermentation and a slight rise in temperature due to heat of
reaction was also noted. There was obtained about 15l000 lbs. of
tempered lung suitable for heparin recovery processing. ~o
undesirable odor was present during the processing.
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Exam~le 2
Partially defrosted frozen beef lung at 26 F. in amount
of 16,200 lbs. was flaked and ground as in Example 1, but
using 0.120 inch openings on the grinder. The ground lung was
thawed and warmed to 830F. in a heat exchanger as in Example 1,
residence time in the heat exchanger being about 4 minutes.
The warm ground lung was then held in a stainless steel tank
A ~ for~ hours at 8~-85 F. No undesirable odor was present during
the processing. There was obtained 15,800 lbs. of tempered
I 10 lung suitable for heparin recovery processing.
¦ Example ~
Frozen bee~ lung (12,000 lbs.j at 25-30 F. was flaked and
ground as in Example 1 and thawed and warmed to 40-50F.,
residence time being about 2 minutes in the heat xchanger. The
ground lung was held at 40 -52 F. for 6 hours. There was
obtained about 11,900 lbs. of tempered beef lung.
; Example 4
Frozen beef lung (12,000 lbs.) at 25 -~0F. was flaked and
; ground as in Example 1 and thawed and warmed to 90-110 F. in
the heat exchanger. The warmJ ground lung was held at 90 -110F.
for 6 hours. There was obtained about 11,900 lbs. of tempered
beef lung.
Example 5
Frozen lung (16~200 lbs.~ at 26 F. was flaked ana ground
as in Example 1 and thawed and warmed to 79 F. in the heat
exchanger. The warmJ gound lung was held at 79-80 F. for
8 hours. There was obtained 15,850 lbs. of tempered bee~ lung.
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~ZETROPIC EXTRACTION OF TEMPERED TISSUE
The tempered lung product prepared in Examples l to 5 were
separately subjected to azeotropic distillation and extraction
with ethylene dichloride at atmospheric pressure, collected on
a filter, washed with ethylene dichloride and dried to remove
residual ethylene dichloride. The dried and defatted lung
products were processed for their heparin contents by an
identical procedure. Comparative data are in Table l.
Table 1
Lunq Fermentation! Extraction and Heparin Isolation
%
yield of %
- Dried Fat in Crude
Fermentation Defatted Dried Heparin Heparin
Ex. Conditions Lung Defatted Yield Potency
No. Temp, F. Time,hr. (a) Lung (b) (c)
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70-75 6 18 . 4 0 . 18 i58 79
2 85 6 17 . 4 o . 30 153 59
3 40-50 6 17 . 1 o . 12 164 48
4 9o-110 6 1 ~ . 9 o . o8 86 74
8 18.0 c0.2 155 96
(a) Wt. % based on starting lung.
(b) Units heparin x 103 ~g. desiccated and defatted lung.
(c) Units per mg. in crude heparin.
COMPARISO~ OF WETTABILITY OF TEMPERED-
DESICCATED AND DEFATTED TISSUE
Comparison of wettability was made of azeotropically defatted
tissue prepared from tissue tempered by the new improved process
of this invention with that of the defatted tissue prepared from
tissue tempered by the old methoa, wherein the lung was tempered
about 4 days at ambient temperature of 80-100 F. For this
comparison, a 20 gram sample of the defatted product was stirred
in 200 ml. water until the particles appeared wet on the outside
1078~7~6
and the stirring stopped. The time required for the bulk of
the particles to sink was then recorded. Data are in Table 2.
Table 2
Wettability comParison
Time ~or Particles
Method o~ to Sink to Bottom,
TemPerinq Seconds
Old >180
New < 10
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