CA1078766A - Process for tempering tissue for heparin production - Google Patents
Process for tempering tissue for heparin productionInfo
- Publication number
- CA1078766A CA1078766A CA264,455A CA264455A CA1078766A CA 1078766 A CA1078766 A CA 1078766A CA 264455 A CA264455 A CA 264455A CA 1078766 A CA1078766 A CA 1078766A
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- tissue
- heparin
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- heat exchanger
- temperature
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
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- Molecular Biology (AREA)
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- Biochemistry (AREA)
- Materials Engineering (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Materials For Medical Uses (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
IMPROVED PROCESS FOR TEMPERING TISSUE
FOR HEPARIN PRODUCTION
ABSTRACT OF THE DISCLOSURE
An improved method of tempering frozen heparin-bearing animal tissue is disclosed wherein the frozen tissue is particulated, thawed and warmed in a heat exchanger and fermented at temperatures ranging from 45°-95°F. for 2 to 15 hours. Putrefaction and odor are avoided, the tissue has improved uniformity in biochemical content and is capable of high heparin yields when processed according to known pro-cedures. When the tempered tissue is defatted with an azeotropic solvent, more of the fat is removed and the defatted tissue is more permeable to heparin recovery solutions and heparin isolation is facilitated.
FOR HEPARIN PRODUCTION
ABSTRACT OF THE DISCLOSURE
An improved method of tempering frozen heparin-bearing animal tissue is disclosed wherein the frozen tissue is particulated, thawed and warmed in a heat exchanger and fermented at temperatures ranging from 45°-95°F. for 2 to 15 hours. Putrefaction and odor are avoided, the tissue has improved uniformity in biochemical content and is capable of high heparin yields when processed according to known pro-cedures. When the tempered tissue is defatted with an azeotropic solvent, more of the fat is removed and the defatted tissue is more permeable to heparin recovery solutions and heparin isolation is facilitated.
Description
``` ~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
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, -~
-` 1078766 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 :.. : .. . ...
..
" 1078766 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 .:: . : . :, . . .
:`` 1078766 -:
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
-` 1078766 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 :.. : .. . ...
..
" 1078766 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 .:: . : . :, . . .
:`` 1078766 -:
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:
1~
~ 5 -. ~
: ., ,. ~ :
.
iO~78766 Flaked or Prebroken Frozen Tissue .
Particuiate Thaw an d Warm .
in ~eat EXch2 nger , Ferment .
. .
. To Heparin Isolation \ /
TEMPERI~G PROCESS FOR HEPARD~ TISSUE
- 5a -.. . ..
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 '~
1(378~66 .
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 ,s'S- '' 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 , , ~ :. ',. . ' :
1(:97876~
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 :: .: . : ,, . :
. .
' 1078766 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 . . .
~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 .' ' ., ' ''' .
10787~;6 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.
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 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.
~ . ; :
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.
~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) . _ .. . . _ 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
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:
1~
~ 5 -. ~
: ., ,. ~ :
.
iO~78766 Flaked or Prebroken Frozen Tissue .
Particuiate Thaw an d Warm .
in ~eat EXch2 nger , Ferment .
. .
. To Heparin Isolation \ /
TEMPERI~G PROCESS FOR HEPARD~ TISSUE
- 5a -.. . ..
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 '~
1(378~66 .
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 ,s'S- '' 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 , , ~ :. ',. . ' :
1(:97876~
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 :: .: . : ,, . :
. .
' 1078766 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 . . .
~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 .' ' ., ' ''' .
10787~;6 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.
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 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.
~ . ; :
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.
~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) . _ .. . . _ 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 _ .- - ,. : ~ . : :
(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 _ .- - ,. : ~ . : :
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. 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 95°F in a heat exchanger, and 3) fermenting the thawed tissue from step 2 at a controlled temperature within the range of 45° to 95°F for a period of time of 2 to 15 hours to substantially effect enzymatic conditioning of said tissue to improve heparin availability.
2. A process according to claim 1 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 65° to 85°F in a heat exchanger, and 3) fermenting the warmed tissue from step 2 within a temperature range of 65° to 85°F for a period of 6 to 8 hours.
3. A process of claim 2 wherein the heparin-bearing animal tissue is beef lung.
4. A process of claim 1 wherein the heat exchanger is a shell and tube heat exchanger, said tissue being moved through the tube.
5. A process of claim 1 wherein in step 3 the fermentation is aided by seeding the warmed tissue with bacteria to speed up the fermentation.
6. A process of claim 1 wherein in step 1 the starting tissue is in a flaked condition.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US62780775A | 1975-10-31 | 1975-10-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1078766A true CA1078766A (en) | 1980-06-03 |
Family
ID=24516213
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA264,455A Expired CA1078766A (en) | 1975-10-31 | 1976-10-29 | Process for tempering tissue for heparin production |
Country Status (8)
| Country | Link |
|---|---|
| AU (1) | AU512447B2 (en) |
| CA (1) | CA1078766A (en) |
| DE (1) | DE2646677C2 (en) |
| DK (1) | DK490476A (en) |
| FR (1) | FR2329677A1 (en) |
| GB (1) | GB1505238A (en) |
| IE (1) | IE44542B1 (en) |
| IT (1) | IT1075975B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886397A (en) * | 2016-06-30 | 2016-08-24 | 江苏东元畜产品有限公司 | Enzyme membrane coupled enzymolysis device for efficiently extracting heparin sodium |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HU177887B (en) * | 1979-03-21 | 1982-01-28 | Richter Gedeon Vegyeszet | Process for preparing a raw material containing heparin |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2410084A (en) * | 1943-12-09 | 1946-10-29 | Upjohn Co | Recovery of heparin |
| US2623001A (en) * | 1949-04-07 | 1952-12-23 | Bengt E G V Sylven | Preparing heparin |
| US2539544A (en) * | 1950-01-03 | 1951-01-30 | Levin | Simultaneous defatting and dehydrating of fatty substances |
| US2884358A (en) * | 1957-04-22 | 1959-04-28 | Southern California Gland Co | Process for preparing crude heparin |
| FR1334096A (en) * | 1962-09-14 | 1963-08-02 | Forest Laboratories | Process for preparing heparinoids |
-
1976
- 1976-10-15 DE DE19762646677 patent/DE2646677C2/en not_active Expired
- 1976-10-22 IE IE233576A patent/IE44542B1/en unknown
- 1976-10-25 GB GB4425276A patent/GB1505238A/en not_active Expired
- 1976-10-27 FR FR7632418A patent/FR2329677A1/en active Granted
- 1976-10-29 DK DK490476A patent/DK490476A/en not_active Application Discontinuation
- 1976-10-29 IT IT6961076A patent/IT1075975B/en active
- 1976-10-29 CA CA264,455A patent/CA1078766A/en not_active Expired
- 1976-11-01 AU AU19190/76A patent/AU512447B2/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886397A (en) * | 2016-06-30 | 2016-08-24 | 江苏东元畜产品有限公司 | Enzyme membrane coupled enzymolysis device for efficiently extracting heparin sodium |
Also Published As
| Publication number | Publication date |
|---|---|
| IE44542B1 (en) | 1981-12-30 |
| GB1505238A (en) | 1978-03-30 |
| IE44542L (en) | 1977-04-30 |
| IT1075975B (en) | 1985-04-22 |
| AU512447B2 (en) | 1980-10-09 |
| FR2329677B1 (en) | 1980-06-06 |
| DK490476A (en) | 1977-05-01 |
| DE2646677C2 (en) | 1985-11-07 |
| DE2646677A1 (en) | 1977-05-05 |
| FR2329677A1 (en) | 1977-05-27 |
| AU1919076A (en) | 1978-05-11 |
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