CA1125202A - Method and apparatus for sterile cultivation of cells on solid substrates - Google Patents
Method and apparatus for sterile cultivation of cells on solid substratesInfo
- Publication number
- CA1125202A CA1125202A CA340,713A CA340713A CA1125202A CA 1125202 A CA1125202 A CA 1125202A CA 340713 A CA340713 A CA 340713A CA 1125202 A CA1125202 A CA 1125202A
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- Prior art keywords
- blender
- set forth
- air
- connector means
- sterile
- Prior art date
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Abstract
ABSTRACT OF THE INVENTION
The invention disclosed comprises a process and apparatus for the cultivation of cells, and having particular application to the production of mushroom spawn, in which a particulate substrate, water and, in some cases, chalk are introduced into a rotary blender and thoroughly sized and sterilized at a temperature of about 250°F. The mixture is cooled in the blender and a cell line inoculum is sterilely introduced into the mixture and thoroughly blended with the substrate, The blended inoculum and substrate are transferred in a sterile air flow from the blender to sterile containers which include a breathing strip in which the cells lines are incubated. The apparatus includes the combination of blender, transfer means and a clean room for filling the sterile containers.
The invention disclosed comprises a process and apparatus for the cultivation of cells, and having particular application to the production of mushroom spawn, in which a particulate substrate, water and, in some cases, chalk are introduced into a rotary blender and thoroughly sized and sterilized at a temperature of about 250°F. The mixture is cooled in the blender and a cell line inoculum is sterilely introduced into the mixture and thoroughly blended with the substrate, The blended inoculum and substrate are transferred in a sterile air flow from the blender to sterile containers which include a breathing strip in which the cells lines are incubated. The apparatus includes the combination of blender, transfer means and a clean room for filling the sterile containers.
Description
~ ~1~52~2 FIELD OF THE INVENTION
The present invention relates to a method and apparatus for the production of cell lines in pure culture on a solid substrate and, in particular, to the sterile cultivation of mushroom spawn. The invention is also contemplated to be useful in the production of metabolites ànd antibiotics from other cell lines as well as in the cultivation of tempeh and miszo.
BACKGROUND OF Tl!E INVENTION
The production of mushroom spawn is old and well known. For example, dry grain is placed in a bottle with water and calcium carbonate and closed with a cotton plug and sterilized. After sterilization, the bottle is cooled and the grain is inoculated with mycelium, reclosed and permitted to stand for the period of mycelium incubation. U.S.
Pat. No. 1,869,517. ln the cultivation of mushroom spawn, sterilization of the nutrients and growing containers is very necessary in order to avoid the growth of other bacteria or molds which would inhibit the mushroom spawn.
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~5 The production of tempeh also has been known, particularly in the oriental culture, for centuries. Tempeh is fermented from soybeans inoculated with several species of Rhy~opus. Recent techniques permit tempeh to be rapidly fermented in plastic bags in which the mold forms 8 mycelial mass binding the soybeans into a cake-like product.
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!0 Improvements in~ growing mushroom spawn include pre-cooking and swelling the grains to prevent their caking d~ring sterilization, U.S. Pat. No. 2,530,318; using wood charcoal ~ranular bases having a cereal substrate coating for growing the spawn, U.S. Pat.
No. 2,677,917; and using sterilizable plastic bags for~ incubation of myceli~m and shippin thereof, U. S . P~t . No . 2, 851, 821. : ~ ~ ;
!5 With respect to the method described in U.S. Pat. No. 2,851,821, elongated plastic bsgs are filled with the desired substrate and closed~usmg~ ~ breathable filtering : ~ :
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The present invention relates to a method and apparatus for the production of cell lines in pure culture on a solid substrate and, in particular, to the sterile cultivation of mushroom spawn. The invention is also contemplated to be useful in the production of metabolites ànd antibiotics from other cell lines as well as in the cultivation of tempeh and miszo.
BACKGROUND OF Tl!E INVENTION
The production of mushroom spawn is old and well known. For example, dry grain is placed in a bottle with water and calcium carbonate and closed with a cotton plug and sterilized. After sterilization, the bottle is cooled and the grain is inoculated with mycelium, reclosed and permitted to stand for the period of mycelium incubation. U.S.
Pat. No. 1,869,517. ln the cultivation of mushroom spawn, sterilization of the nutrients and growing containers is very necessary in order to avoid the growth of other bacteria or molds which would inhibit the mushroom spawn.
. .
~5 The production of tempeh also has been known, particularly in the oriental culture, for centuries. Tempeh is fermented from soybeans inoculated with several species of Rhy~opus. Recent techniques permit tempeh to be rapidly fermented in plastic bags in which the mold forms 8 mycelial mass binding the soybeans into a cake-like product.
. ~ :
!0 Improvements in~ growing mushroom spawn include pre-cooking and swelling the grains to prevent their caking d~ring sterilization, U.S. Pat. No. 2,530,318; using wood charcoal ~ranular bases having a cereal substrate coating for growing the spawn, U.S. Pat.
No. 2,677,917; and using sterilizable plastic bags for~ incubation of myceli~m and shippin thereof, U. S . P~t . No . 2, 851, 821. : ~ ~ ;
!5 With respect to the method described in U.S. Pat. No. 2,851,821, elongated plastic bsgs are filled with the desired substrate and closed~usmg~ ~ breathable filtering : ~ :
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material in the mouth of the bag. The bag and contents are therenfter sterilized in much the same manner as the predecessor glass bottles, or by irradiation ~nd inoculated with the desired inoculum and incubated. The advantages of this process include the elimi-nation of glass bottles which were costly, fragile and cumbersome and permitted all process steps to be carried out in the bag and a~ter incubation sllowed the spawn to be transported to the growing site withou~ removing the culture or spawn to an intermediate container. Inasmuch as plants for cultivation are relatively expensive to build and operate, they are few and far between which necessitates transporting spawn considerable distances The advent of the pl~stic incubation bag represented a substantial improvement in the cult;vation and transportation of mushroom spawn. Further improvements to that method were also suggested for trRnsporting spawn. For example, U.S. Pat. No. 3,335,521 taught that the champignon spawn could be shipped in an -air-tight container with ice.
Even today the preferred method of inoculating and incubating mycelium takes place in flexible plastic containers. Accordingly many advancements in the design and construction of containers have been suggested including British Patent 1,176,188 in which the filtering element is incorporated into the wall of the container. See also U.S.
Pat. No. 3,938,658 which teaches the use of a porous filter media over slits formed in the walls of a plastic bag. And, most recently, U.S. Pat. No. 4,063,383 which teaches growing ~O mushroom spawn in a plastic bag having a microporous breathing panel that acts as a lining for perforations in the bag.
` In conventional methods OI cultivating the mushroom spawn, whether using glass bottles or plastic bags, the nutrient or substrate is normal]y sterilized in the container to prevent the growth of unwanted bacteria or other undesired microorganisms~
~5 These methods, however, are labor intensive. The sterilization of nutrient filled bottles or bags, the inoculation of the sterilized nutrient containers and the agitation of the individual nutrient containers to mix the inoculum during incubation requires numerous manual steps. Also, it had been heard that attempts many years ago by Somycel Company in France to sterilize and inocillate the substrate in large batches prior to filling the bottles were unsuccessful and abandoned because of contamination problems.
The present invention overcomes the problems inherent in prior flrt methods nnd reduces the labor involved in the sterili~ation and inoculation processes as well as minimizes the possiblity of contamination during inocu~ation and subsequent bag filling operations. It is also an object of the present invention to eliminate the mixing steps previously required after inoculation of the nutrient containers. A further object of the invention is to provide a method for producing metabolites on ~ solid media rather than in a liquid fermentation process. The invention is also useful in the preparation of cultures for fermentation of food products such as tempeh. In addition to the method of the present invention, it is also an object to provide apparatus for c~rrying out such processes.
Moreover, the invention provides apparatus wherein the sterilized nutrient is bulk inoculated, blended and sterileIy discharged into sterile containers for incubation. The apparatus and method of the present invention overcome many of the obstacles en-countered in cultivating cell lines on solid substrate under sterile conditions.
SVMMAR'L' OF THE IN~!ENTION
Generally, the method of the present invention comprises introducing a mixture of nutrients into a blender, preferably a rotary blender, which is capable of achieving and maintaining temperatures and pressures required for sterilization of the mixture. In the case of mushroom spawn, a typical mixture consists of 50 to 60% by weight grain or cereal, 40 to 50% water and 1% chalk. The mixture is blended and steri-lized by heating the blender to R temperature of about 252F or more. Preferably, steam at 15 psig is introduced into the blender during sterilization and blending. Thetemperature and pressure are maintained for between 35 and 120 minutes, preferably 45 minutes, to achie~le adequate sterilization.
The Mender and mixture are thereafter permitted to cool under R positive pressure of sterile air to a temperature of about 80 F and the inoculum is steril~y transferred into the blender and thoroughly mixed with the nutrient mAterial. After mixing is complete, the blender is connected to a bag filling station by means of a sterile connector which includes measuring means for allocating the desired amount of inoculated nutrient into bags. The bag filling station is positioned in laminar flow of sterile air to .
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prevent contaminants from enterin~ the bag or connector means. Sterilized bags, each of which includes a breathing strip, are posit;oned at the discharge end of the connector means and filled. The filled bags are immediately sealed for incubation and delivery.
The preferred method of the present invention eliminates the difficult step S of inoculating individual bags or bottles as had been done heretofore in the cultivation of mushroom spawn. Further, the method in combination with the preferred apparatus provides a sterile en~ironment for the blending of the nutrient and inoculum. This is important in the preparation of mushroom spawn, cultures for fermentation products such as tempeh, and in growing various other cell lines. .
Other advantages OI the present invention will become apparent from a perusal of the following detailed description of the best mode of cultivating mushroom spawn which is to be taken in connection with the accompanying drawings.
DESCRIPTJON OF THE DRAWINGS
. _ _ Figure 1 is a diagrammatic elevation of the appQratus of the present inven-tion and Figure 2 is a schem atio- diagram of the Air/Steam delivery system.
DESCRIPTION OF THE INVENTION
; . -With reference to Figure 1, a rotary blender 10 is shown. Blender 10 is preferably of the Sype manufactured by Patterson-Kelly having a V-configura~ion and including conventional fluid jackets ~not shown). Other types of commercially available blenders or mixers are suitable for use such as the double cone-type blenders; however, ribbon blenders ~nd the like are not preferred because of the difficulty in cleaning and sterilizing them. Blender 10 rothtes on axis ll and includes ~n inletloutlet port 12 through which the nutrient is introduced apd discharged.
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As shown in Figure 1, blender 10 is provided with two openings, inlet/outlet port 12 and steam/air line 33, through which inoculum can be introduced. If the inoculum is solid or particulate in form, it is sterilely transferred through port 12 into blender 10 by gravity. Preferably port 12 is positioned at ~n angle above the horizontal plane through the axis of Mender 10. Alternatiyely~R liquid inoculum is sterilely trsnsferred through line 33 into blender 10. In both methods of inoculation, all of the connecting pipes and valves must be sterile prior to the inoculation of ehe nutrient to prevent contamination of the batch.
Port 12 is adapted to communicate and connect with connector means 13 n through an airtight rreans or seal such as a Kamlok quick coupling. Any such airtight means or seal must be capable of being sterilized and of sealing pressures up to about 15 psig. Connector means 13 both measures and transports inoculated substrate to. incubation bags. Connector means 13 is preferab~ made from stainless steel tubing and includes first and second valves 14 and lB, preferably butterfly valves. The space between valves 14 and 16 defines measuring chamber 17 having a volume which is substantially the same as the volume of the bag to be filled for incubation.
Connector means 13 is shown extending through floor 18 above ~illing station Z0. ~illing`stat`ion 20 is located in`a làminar ~low of steril-izeq air, preferably in an enclosed commercially available class 100 clean ~0 room. Filling station 20 includes a removable cap 21 adapted to fit over the end 19 of connector means 13 to contain steam injected into means `13 for sterilization. After connecting means 13 is sterilized, cap 21 is removed and sterilized ba~s 22 (sllown in dashed line~ are placed over dischar~e end 19 for filling.
Positioned adjacent to the discharge end of connector means 13 is a bag sealer 23 for heat ~5 sèaling the bags closed immediately after filling.
Since the method of the present invention does not require that bags 22 undergo autoclaving, it is preferable that they be made from a polyethylene film which can be gas, electron beam or radiation sterilized. Such materisl is substantially less expensive than the polypropylene or nylon bags of the prior art. Each bag includes A strip :~
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made from a synthetic fiber material such as Tyvek to permit the cells to breathe. A
Tyvek strip is positioned on each bag in a manner well known to the art.
Referring to Figure 2, a sterile air and steam system 26 is shown having a source of air 27 under pressure of about 15 psig and a source of steam 28. Steam source 28 S is connected through steam trap 29 to blender 10 via line 31, valve 32 and steam inlet 33 of blender 10. Inlet 33 includes a rotary connector means 34 positioned on the axis of blender 10. Air supply source 27 is connected to line 36 through valve 37~ Positioned in line 36 is filter 38 designed to sterilize the air from source 27. Preferably filter 38 is a sterilizable filter such as a Domnick Hunter Bio-x Filter, a Psll Ultipor Filter or the like. The discharge end of filter 38 is connected to line 39 which is connected to blender inlet line 33 through valve 41 and to measuring chamber 17 through valves 42 and 43. Air/Steam system 26 also includes lines 44 and 46 and associated ~alves 47 and 48 for supply sterilizing steam to connector means 13 and air filter 38, respectively. As part of system 26, cap 21 of filling stations 20 includes a drain 51 and steam trap 52.
In the preferred method oî cultivating mushroom spnwn, blender 10 is charged through port 12 with a solid substrate such as rye grain, ch~lk and water in conventional portions. The blender may be cold or preheated at the time of charge;
however, ~s blending begins the jQcket is he~ted to bring the mixture to a temperature of at least 252 F. At the same time valve 32 is opened to introduce steam at a pressure of ~O about 15 psig into blender 10. Blending is continued at 252F for between 35 and 125 minutes and preferably 45 minutes. As is known in the artJ the minimum time necessary to achieve sterilization of the nutrient mixture is desirable because any extra steriliz~tion slows spawn growth.
' ~ ~ .' At the completion of steriliz~tion, the introduction of steam into the ,5 blender is discontinued and the jacket of the blender is cooled to bring the rnixture to between 50 and 90 F. Preferably, the steam is released to f~cilit~ cooling the blender.
When the steam is released sterile air is introduced into the blender through irtlet 33 from source 27 and filter 38 to maintain the positive pressure within the Mender. After cooling, inoculum is introduced into the blender. Preferably, inoculum is steril~ly trans-. ' ; : ~ ~ :
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ferred to the blender by gravity feed through port 12 if the inoculum is solid or through line 33 if it is a liquid. In sterilely transferrilig through port 12, the opening must first be sterilized and a stream of sterilized air flowing out of port 12 is necessary~ It is also possible to transfer sterilized solid inoculum by means of a sterilized bomb which would attach to port 12, in which case a flow of sterilized air would not be required. Olher suitable culture transfer methods may also be used.
The inoculant is thoroughly mixed with the sterilized nutrient. After mix;ng has been completed, connector means 13 is connected to port 12 and sterilized. Steri-liæation is achieved by closing discharge end 19 with cap 21 and introducing steam into connector means 13 through valves 43 and 47 and line 44. After sterilization has been completed the cap is removed and bag placed over discharge end 19. Port 12 is thereafter opened together with first vRlve 14 while second valve 16 is closed. The inoculated sub-strate'is allowed to fill the volume of chamber 17 and first valve 14 closed. Valve 16 is then opened to discharge the contents of chamber 17 into bag 22 positioned over discharge end 19. Simultaneously with opening of first valve 16, sterile air is introduced into chamber 17 to fill the space of the exiting substrate. It is a]so desiraMe to introduce sterile air into blender 10 to maintain a slight positive pressure to fili the void remaining after discharge of the mixture from the blender.
Filling of bags 22 preferably takes place in a clean room as described above, however it is possible to position filling station 20 in a laminar flow of sterilized nir to prevent entrainment of any contaminants into the bag. After each bag has been filled it is sealed so that only air passing through the breathing strip enters during subsequent incubation. ' ~.
While presently preferred embodiments of the best mode of the invention have been shown and described, it may be otherwise embodied within the scope of the appended claims.
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material in the mouth of the bag. The bag and contents are therenfter sterilized in much the same manner as the predecessor glass bottles, or by irradiation ~nd inoculated with the desired inoculum and incubated. The advantages of this process include the elimi-nation of glass bottles which were costly, fragile and cumbersome and permitted all process steps to be carried out in the bag and a~ter incubation sllowed the spawn to be transported to the growing site withou~ removing the culture or spawn to an intermediate container. Inasmuch as plants for cultivation are relatively expensive to build and operate, they are few and far between which necessitates transporting spawn considerable distances The advent of the pl~stic incubation bag represented a substantial improvement in the cult;vation and transportation of mushroom spawn. Further improvements to that method were also suggested for trRnsporting spawn. For example, U.S. Pat. No. 3,335,521 taught that the champignon spawn could be shipped in an -air-tight container with ice.
Even today the preferred method of inoculating and incubating mycelium takes place in flexible plastic containers. Accordingly many advancements in the design and construction of containers have been suggested including British Patent 1,176,188 in which the filtering element is incorporated into the wall of the container. See also U.S.
Pat. No. 3,938,658 which teaches the use of a porous filter media over slits formed in the walls of a plastic bag. And, most recently, U.S. Pat. No. 4,063,383 which teaches growing ~O mushroom spawn in a plastic bag having a microporous breathing panel that acts as a lining for perforations in the bag.
` In conventional methods OI cultivating the mushroom spawn, whether using glass bottles or plastic bags, the nutrient or substrate is normal]y sterilized in the container to prevent the growth of unwanted bacteria or other undesired microorganisms~
~5 These methods, however, are labor intensive. The sterilization of nutrient filled bottles or bags, the inoculation of the sterilized nutrient containers and the agitation of the individual nutrient containers to mix the inoculum during incubation requires numerous manual steps. Also, it had been heard that attempts many years ago by Somycel Company in France to sterilize and inocillate the substrate in large batches prior to filling the bottles were unsuccessful and abandoned because of contamination problems.
The present invention overcomes the problems inherent in prior flrt methods nnd reduces the labor involved in the sterili~ation and inoculation processes as well as minimizes the possiblity of contamination during inocu~ation and subsequent bag filling operations. It is also an object of the present invention to eliminate the mixing steps previously required after inoculation of the nutrient containers. A further object of the invention is to provide a method for producing metabolites on ~ solid media rather than in a liquid fermentation process. The invention is also useful in the preparation of cultures for fermentation of food products such as tempeh. In addition to the method of the present invention, it is also an object to provide apparatus for c~rrying out such processes.
Moreover, the invention provides apparatus wherein the sterilized nutrient is bulk inoculated, blended and sterileIy discharged into sterile containers for incubation. The apparatus and method of the present invention overcome many of the obstacles en-countered in cultivating cell lines on solid substrate under sterile conditions.
SVMMAR'L' OF THE IN~!ENTION
Generally, the method of the present invention comprises introducing a mixture of nutrients into a blender, preferably a rotary blender, which is capable of achieving and maintaining temperatures and pressures required for sterilization of the mixture. In the case of mushroom spawn, a typical mixture consists of 50 to 60% by weight grain or cereal, 40 to 50% water and 1% chalk. The mixture is blended and steri-lized by heating the blender to R temperature of about 252F or more. Preferably, steam at 15 psig is introduced into the blender during sterilization and blending. Thetemperature and pressure are maintained for between 35 and 120 minutes, preferably 45 minutes, to achie~le adequate sterilization.
The Mender and mixture are thereafter permitted to cool under R positive pressure of sterile air to a temperature of about 80 F and the inoculum is steril~y transferred into the blender and thoroughly mixed with the nutrient mAterial. After mixing is complete, the blender is connected to a bag filling station by means of a sterile connector which includes measuring means for allocating the desired amount of inoculated nutrient into bags. The bag filling station is positioned in laminar flow of sterile air to .
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prevent contaminants from enterin~ the bag or connector means. Sterilized bags, each of which includes a breathing strip, are posit;oned at the discharge end of the connector means and filled. The filled bags are immediately sealed for incubation and delivery.
The preferred method of the present invention eliminates the difficult step S of inoculating individual bags or bottles as had been done heretofore in the cultivation of mushroom spawn. Further, the method in combination with the preferred apparatus provides a sterile en~ironment for the blending of the nutrient and inoculum. This is important in the preparation of mushroom spawn, cultures for fermentation products such as tempeh, and in growing various other cell lines. .
Other advantages OI the present invention will become apparent from a perusal of the following detailed description of the best mode of cultivating mushroom spawn which is to be taken in connection with the accompanying drawings.
DESCRIPTJON OF THE DRAWINGS
. _ _ Figure 1 is a diagrammatic elevation of the appQratus of the present inven-tion and Figure 2 is a schem atio- diagram of the Air/Steam delivery system.
DESCRIPTION OF THE INVENTION
; . -With reference to Figure 1, a rotary blender 10 is shown. Blender 10 is preferably of the Sype manufactured by Patterson-Kelly having a V-configura~ion and including conventional fluid jackets ~not shown). Other types of commercially available blenders or mixers are suitable for use such as the double cone-type blenders; however, ribbon blenders ~nd the like are not preferred because of the difficulty in cleaning and sterilizing them. Blender 10 rothtes on axis ll and includes ~n inletloutlet port 12 through which the nutrient is introduced apd discharged.
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As shown in Figure 1, blender 10 is provided with two openings, inlet/outlet port 12 and steam/air line 33, through which inoculum can be introduced. If the inoculum is solid or particulate in form, it is sterilely transferred through port 12 into blender 10 by gravity. Preferably port 12 is positioned at ~n angle above the horizontal plane through the axis of Mender 10. Alternatiyely~R liquid inoculum is sterilely trsnsferred through line 33 into blender 10. In both methods of inoculation, all of the connecting pipes and valves must be sterile prior to the inoculation of ehe nutrient to prevent contamination of the batch.
Port 12 is adapted to communicate and connect with connector means 13 n through an airtight rreans or seal such as a Kamlok quick coupling. Any such airtight means or seal must be capable of being sterilized and of sealing pressures up to about 15 psig. Connector means 13 both measures and transports inoculated substrate to. incubation bags. Connector means 13 is preferab~ made from stainless steel tubing and includes first and second valves 14 and lB, preferably butterfly valves. The space between valves 14 and 16 defines measuring chamber 17 having a volume which is substantially the same as the volume of the bag to be filled for incubation.
Connector means 13 is shown extending through floor 18 above ~illing station Z0. ~illing`stat`ion 20 is located in`a làminar ~low of steril-izeq air, preferably in an enclosed commercially available class 100 clean ~0 room. Filling station 20 includes a removable cap 21 adapted to fit over the end 19 of connector means 13 to contain steam injected into means `13 for sterilization. After connecting means 13 is sterilized, cap 21 is removed and sterilized ba~s 22 (sllown in dashed line~ are placed over dischar~e end 19 for filling.
Positioned adjacent to the discharge end of connector means 13 is a bag sealer 23 for heat ~5 sèaling the bags closed immediately after filling.
Since the method of the present invention does not require that bags 22 undergo autoclaving, it is preferable that they be made from a polyethylene film which can be gas, electron beam or radiation sterilized. Such materisl is substantially less expensive than the polypropylene or nylon bags of the prior art. Each bag includes A strip :~
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.
~~ .
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made from a synthetic fiber material such as Tyvek to permit the cells to breathe. A
Tyvek strip is positioned on each bag in a manner well known to the art.
Referring to Figure 2, a sterile air and steam system 26 is shown having a source of air 27 under pressure of about 15 psig and a source of steam 28. Steam source 28 S is connected through steam trap 29 to blender 10 via line 31, valve 32 and steam inlet 33 of blender 10. Inlet 33 includes a rotary connector means 34 positioned on the axis of blender 10. Air supply source 27 is connected to line 36 through valve 37~ Positioned in line 36 is filter 38 designed to sterilize the air from source 27. Preferably filter 38 is a sterilizable filter such as a Domnick Hunter Bio-x Filter, a Psll Ultipor Filter or the like. The discharge end of filter 38 is connected to line 39 which is connected to blender inlet line 33 through valve 41 and to measuring chamber 17 through valves 42 and 43. Air/Steam system 26 also includes lines 44 and 46 and associated ~alves 47 and 48 for supply sterilizing steam to connector means 13 and air filter 38, respectively. As part of system 26, cap 21 of filling stations 20 includes a drain 51 and steam trap 52.
In the preferred method oî cultivating mushroom spnwn, blender 10 is charged through port 12 with a solid substrate such as rye grain, ch~lk and water in conventional portions. The blender may be cold or preheated at the time of charge;
however, ~s blending begins the jQcket is he~ted to bring the mixture to a temperature of at least 252 F. At the same time valve 32 is opened to introduce steam at a pressure of ~O about 15 psig into blender 10. Blending is continued at 252F for between 35 and 125 minutes and preferably 45 minutes. As is known in the artJ the minimum time necessary to achieve sterilization of the nutrient mixture is desirable because any extra steriliz~tion slows spawn growth.
' ~ ~ .' At the completion of steriliz~tion, the introduction of steam into the ,5 blender is discontinued and the jacket of the blender is cooled to bring the rnixture to between 50 and 90 F. Preferably, the steam is released to f~cilit~ cooling the blender.
When the steam is released sterile air is introduced into the blender through irtlet 33 from source 27 and filter 38 to maintain the positive pressure within the Mender. After cooling, inoculum is introduced into the blender. Preferably, inoculum is steril~ly trans-. ' ; : ~ ~ :
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ferred to the blender by gravity feed through port 12 if the inoculum is solid or through line 33 if it is a liquid. In sterilely transferrilig through port 12, the opening must first be sterilized and a stream of sterilized air flowing out of port 12 is necessary~ It is also possible to transfer sterilized solid inoculum by means of a sterilized bomb which would attach to port 12, in which case a flow of sterilized air would not be required. Olher suitable culture transfer methods may also be used.
The inoculant is thoroughly mixed with the sterilized nutrient. After mix;ng has been completed, connector means 13 is connected to port 12 and sterilized. Steri-liæation is achieved by closing discharge end 19 with cap 21 and introducing steam into connector means 13 through valves 43 and 47 and line 44. After sterilization has been completed the cap is removed and bag placed over discharge end 19. Port 12 is thereafter opened together with first vRlve 14 while second valve 16 is closed. The inoculated sub-strate'is allowed to fill the volume of chamber 17 and first valve 14 closed. Valve 16 is then opened to discharge the contents of chamber 17 into bag 22 positioned over discharge end 19. Simultaneously with opening of first valve 16, sterile air is introduced into chamber 17 to fill the space of the exiting substrate. It is a]so desiraMe to introduce sterile air into blender 10 to maintain a slight positive pressure to fili the void remaining after discharge of the mixture from the blender.
Filling of bags 22 preferably takes place in a clean room as described above, however it is possible to position filling station 20 in a laminar flow of sterilized nir to prevent entrainment of any contaminants into the bag. After each bag has been filled it is sealed so that only air passing through the breathing strip enters during subsequent incubation. ' ~.
While presently preferred embodiments of the best mode of the invention have been shown and described, it may be otherwise embodied within the scope of the appended claims.
Claims (14)
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for cultivation of cells on a solid substrate in a blender capable of being heated and pressurized, said method comprising:
(a) bulk blending and sterilizing in said blender a mixture of moist nutrient particles for a time and at a temperature sufficient to sterilize all of said mixture to form a uniform sterile solid substrate;
(b) cooling the sterilized substrate;
(c) while in said blender inoculating the bulk sterilized substrate with inoculum;
(d) bulk blending the inoculum and sterilized substrate in said blender to prepare an inoculated mixture; and (e) discharging said inoculated mixture from said blender into at least one sterile container for incubation, steps (c), (d) and (e) being carried out under sterile conditions.
(a) bulk blending and sterilizing in said blender a mixture of moist nutrient particles for a time and at a temperature sufficient to sterilize all of said mixture to form a uniform sterile solid substrate;
(b) cooling the sterilized substrate;
(c) while in said blender inoculating the bulk sterilized substrate with inoculum;
(d) bulk blending the inoculum and sterilized substrate in said blender to prepare an inoculated mixture; and (e) discharging said inoculated mixture from said blender into at least one sterile container for incubation, steps (c), (d) and (e) being carried out under sterile conditions.
2, The method set forth in claim 1, including the step of dividing the inoculated mixture into separate portions and placing each portion in a sterilized container for incubation.
3. The method as set forth in claim 1, wherein said blending and sterilization is from between 35-and 120 minutes at a temperature of about 252°F.
4. The method as set forth in claim 2, wherein said inoculated mixture is placed in said container for incubation while causing a laminar flow of sterile air to flow around each container during placement.
5. The method as set forth in claim 2, wherein said nutrient particles consist of grain or cereal and chalk.
6. The method as set forth in claim 2, wherein said incubation containers comprise flexible plastic bags having a breathing strip thereon.
7. The method as set forth in claim 5, wherein said inoculum is mushroom spawn.
8. Apparatus for the preparation and cultivation of cells on a solid substrate, comprising in combination:
(a) a rotary blender having means to heat and maintain any contents thereof to a temperature of at least 252°F, said blender including an inlet/
outlet port and at least one fluid inlet;
(b) connector means adapted to sealingly connect with the port of the blender and including first and second valve means wherein the space between said valves defines a measuring chamber adapted to proportion contents discharged from said blender, said connector means including a fluid inlet;
(c) cap means adapted to fit over the discharge end of the connector means for containing a fluid within said means between said first valve and cap means;
(d) means for sterilizing air;
(e) an air source adapted for communication to said fluid inlets of said blender and connector means through said air filter; and (f) a steam source adapted for communication to said fluid inlets of the blender and connector means.
(a) a rotary blender having means to heat and maintain any contents thereof to a temperature of at least 252°F, said blender including an inlet/
outlet port and at least one fluid inlet;
(b) connector means adapted to sealingly connect with the port of the blender and including first and second valve means wherein the space between said valves defines a measuring chamber adapted to proportion contents discharged from said blender, said connector means including a fluid inlet;
(c) cap means adapted to fit over the discharge end of the connector means for containing a fluid within said means between said first valve and cap means;
(d) means for sterilizing air;
(e) an air source adapted for communication to said fluid inlets of said blender and connector means through said air filter; and (f) a steam source adapted for communication to said fluid inlets of the blender and connector means.
9. Apparatus as set forth in claim 8, including means for creating a laminar flow of sterile air adjacent to and around the discharge end of the connector means.
10. Apparatus as set forth in claim 8, wherein said blender com-prises a V-configuration rotary blender having a heating jacket.
11. Apparatus for the preparation and cultivation of cells on a solid substrate, comprising in combination:
(a) blender means being adapted to heat and maintain any contents therein at a temperature and for such a time as to effect sterilization, said blender including at least one port and at least one fluid inlet;
(b) connector means adapted to sealingly connect said port and having a fluid inlet and a closeable discharge end;
(c) means for filter sterilizing air;
(d) means for connecting said means for filter sterilizing air to said fluid inlets; and (e) a steam source connected to said fluid inlets.
(a) blender means being adapted to heat and maintain any contents therein at a temperature and for such a time as to effect sterilization, said blender including at least one port and at least one fluid inlet;
(b) connector means adapted to sealingly connect said port and having a fluid inlet and a closeable discharge end;
(c) means for filter sterilizing air;
(d) means for connecting said means for filter sterilizing air to said fluid inlets; and (e) a steam source connected to said fluid inlets.
12. Apparatus as set forth in claim 11, wherein said discharge end of said connector means includes (f) a bag filling station.
13. Apparatus as set forth in claim 12, wherein said bag filling station includes means for positioning bags to the discharge end of said connector means.
14. Apparatus as set forth in claim 12, wherein said bag filling station is in communication with said means for filter sterilizing air.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA340,713A CA1125202A (en) | 1979-11-27 | 1979-11-27 | Method and apparatus for sterile cultivation of cells on solid substrates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA340,713A CA1125202A (en) | 1979-11-27 | 1979-11-27 | Method and apparatus for sterile cultivation of cells on solid substrates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1125202A true CA1125202A (en) | 1982-06-08 |
Family
ID=4115704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA340,713A Expired CA1125202A (en) | 1979-11-27 | 1979-11-27 | Method and apparatus for sterile cultivation of cells on solid substrates |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1125202A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115024161A (en) * | 2022-07-27 | 2022-09-09 | 大连卓兴科技发展有限公司 | Production and use method of granular strain with standard specification |
-
1979
- 1979-11-27 CA CA340,713A patent/CA1125202A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115024161A (en) * | 2022-07-27 | 2022-09-09 | 大连卓兴科技发展有限公司 | Production and use method of granular strain with standard specification |
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