Patent Application
20230392109
The present invention relates to an apparatus and methods for inoculating liquid culture, for example for spawn production.
Cultivation of fungi, and in particular mushrooms, may include a step of culturing mycelium in a liquid nutrient broth or on solid agar. In liquid culture, once the mycelium has sufficiently expanded, the mycelium rich suspension may be inoculated into spawn bags, i.e., sealed bags containing sterilized grains. The grains provide a nutrient surface upon which the mycelium can further colonize before being introduced to a substrate where the mushroom is ultimately grown.
Single use needle syringes are typically used to inject the mycelial liquid culture into spawn bags. When inoculating multiple spawn bags in succession, contamination may occur each time the syringe is refilled. Refilling the syringe also takes time. While larger syringes may be used, these may be awkward to handle and settling of the mycelial suspension can occur within the syringe, resulting in uneven rates of inoculation. Further, maintaining a desired temperature of mycelial liquid culture may be hampered by the constant refilling, the shape, and the limited volume capacity of single use syringes.
Improved apparatus and methods for producing and inoculating liquid culture are desirable.
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
One aspect of the invention provides an inoculation apparatus comprising: a vessel for containing liquid mycelium culture comprising: an outlet port; an injection port; and a breather port; an auto-fill syringe comprising a nozzle and an inlet port; a connection line with a first end connecting to the outlet port and a second end connecting to the inlet port; and an injection means connected to the nozzle.
The first end of the connection line may comprise a female keg fitting and the outlet port may comprise a male keg fitting with a stem extending into the vessel, the male keg fitting connected to the female keg fitting. The injection port may comprise an aperture covered by a self-healing elastomer. The injection means may comprise a needle. The needle may comprise a sealed end and a plurality of radially arranged holes. The injection means may comprise a second nozzle comprising a distal face having plurality of angled holes. The vessel may comprise a draft column and aeration means disposed in the draft column. An air pump may be connected to the vessel and in fluid communication with the aeration means. A temperature regulating means connected between the air pump and the aeration means may be provided. The temperature regulating means may comprise an inline heater. The vessel may comprise a detachable lid, the detachable lid comprising the outlet port, the injection port and the breather port. The vessel may comprise a blender attachment lid interchangeable with the detachable lid, the blender attachment lid comprising a rotatable blade assembly, wherein the blender attachment lid is coupleable to a blender rotary drive for actuating the rotatable blade assembly.
Another aspect of the invention provides a method of inoculating liquid culture for spawn production. The method comprises: a) providing an inoculation apparatus as described herein; b) injecting mycelial liquid culture into the vessel; c) growing the mycelial liquid culture to a desired maturity; d) providing a plurality of spawn containers containing sterilized grains; e) serially inoculating the plurality of spawn containers by injecting the mycelial liquid culture on to the sterilized grains in the spawn container.
The spawn container may comprise a spawn bag and the injection means may comprise a needle, and wherein step e) may comprise inoculating the spawn bag with a dose of the mycelium liquid culture equivalent to multiple volumes of the auto-fill syringe without withdrawing the needle from an injection port of the spawn bag by allowing auto-filling of the syringe with the mycelium culture from the vessel between injections.
The injection means may comprise a second nozzle comprising a plurality of holes and wherein step e) comprises inoculating the sterilized grains with simultaneous multiple streams of mycelium liquid culture.
Step c) may comprise providing temperature regulated aeration to the culture.
After step c) may comprise exchanging the lid of the vessel with a lid with a blender attachment lid and blending the culture into a slurry.
Step e) may comprise inoculating at a rate of 20 to 60 mL of the mycelium liquid culture per pound of the sterilized grains.
The grain may be selected from the group consisting of rye, barley, rice, millet, wheat, and popcorn.
Another aspect of the invention provides a method of inoculating plant roots, the method comprising: a) providing an inoculation apparatus as described herein, wherein the vessel contains rhizobial liquid culture; and b) serially inoculating a plurality of plants by injecting the rhyziobial liquid culture adjacent the plant roots.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.
In drawings which illustrate non-limiting embodiments of the invention:
Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
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Inoculation apparatus 10 allows for serial inoculation of several spawn bags in rapid succession without manual refiling of a syringe after each injection and/or between spawn bags. In particular a user can inoculate a first spawn bag 48 by piercing its injection port with needle 32 of injection assembly 19 of inoculation apparatus 10. Mycelial liquid culture LC is injected into first spawn bag 48. Multiple loads of auto-fill syringe 20 can be effected by the user by manually “pumping” the syringe (here, squeezing the V-shaped handle) multiple times to allow a greater volume of liquid culture LC to be injected into spawn bag 48, as well as to allow subsequent injection of spawn bags 48′, 48″ and 48′″ in rapid succession. In some embodiments, 5 to 100 mL, or 10 to 80 mL, or 15 to 70 mL, or 20 to 60 mL of mycelial liquid culture LC may be injected per pound of sterilized grains 48. In some embodiments grains 48 may be provided in a spawn container other than a spawn bag, such as sterile jars and the like.
Body 55 includes an attachment means 64 for attaching to auto-fill syringe 20. Attachment means 64 may for example be a luer lock fitting. In the case of a spawn bag 48, instead of piercing with a needle, spawn bag 48 may be opened in a sterile environment and nozzle 54 aimed to provide a split stream of liquid culture LC to sterilized grains 48. In the case of jar inoculation, the jar may be opened in a sterile environment and nozzle 54 aimed to provide a split stream of liquid culture LC to sterilized grains in the jar. The double napped cone shape of body 55 and angled holes 60 of nozzle 54 work together to guide liquid culture LC into multiple streams from nozzle 54 that substantially spread from a horizontal to vertical when viewed from the side, and substantially 360 degrees around when viewed end-on.
The multiple streams created by nozzle 54 and needle 70 create multiple inoculation sites in grains 48 for more effective inoculation.
In some embodiments, before and/or during inoculation, vessel 12 of inoculation apparatus 10 may be placed on a magnetic stirrer 54, with sterilized stir bar 46 inside vessel 12 to mix mycelial liquid culture LC to ensure a consistent amount of mycelium is being inoculated per load of auto-fill syringe 20. In some embodiments, where a particular temperature of mycelial liquid culture LC is desirable during inoculation, vessel 12 may be kept warmer by suitable means such as warming plate (not shown) or may be kept cooler by suitable means such as at least partially submersion in a cooler (not shown), to maintain the desired temperature of mycelial liquid culture LC throughout the inoculation process.
Once liquid culture LC is sufficiently mature, draft column 270, tubing 273 and aeration means 272 and lid 213 may be replaced with a sterilized blender attachment lid 274. Blender attachment lid 274 includes a rotatable blade assembly. Blender attachment lid 274 is connected to a blender rotary drive (not shown) and mycelial pellets produced in vessel 212 are blended into a liquid culture LC slurry. Blending the liquid culture LC into a slurry reduces the occurrence of clogging of injector assembly 19 during inoculation. The blender attachment is then replaced with lid 213 and the inlet/outlet port is suitably connected by connection line 26 to an injection assembly 19. The foregoing steps would typically be conducted in a sterile environment, e.g. in a laminar flow cabinet or the like.
In some embodiments, inoculation apparatus 10 allows for serial inoculation of rhizobial liquid culture for plant roots in a similar manner as described herein for mycelial liquid culture for spawn bags.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.
This application claims priority from, and for the purpose of the United States of America the benefit under 35 U.S.C. § 119 in connection with, U.S. application No. 63/348,401 filed 2 Jun. 2022 and entitled APPARATUS AND METHODS FOR LIQUID CULTURE PRODUCTION AND INOCULATION which is hereby incorporated herein by reference for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63348401 | Jun 2022 | US |
