This invention relates to methods of producing tissue culture media derived from plant seed material. More particularly, this invention relates to methods of producing tissue culture media derived from plant seed material and leveraging the media to inoculate and cast an engineered substrate into a complex geometry. Still more particularly, this invention relates to the casting of mycological biomaterials.
As is known from published United States Patent Application 2008/0145577, use can be made of a fungus to form composite materials by mixing an inoculum including a preselected fungus with discrete particles and a nutrient material capable of being digested by the fungus.
It is an object of the invention to provide an economical method of producing a tissue culture media for use in making composite materials.
It is another object of the invention to provide an economical method for casting items from a tissue culture media.
It is another object of the invention to provide an economical method for casting engineered substrates from a tissue culture media.
Briefly the invention provides a method of making a tissue culture media comprising the steps of measuring a selected seed matter at specified ratio (m:v) with water to form a mixture; autoclaving the mixture in an autoclavable vessel; inoculating the autoclaved mixture with a cell or tissue strain; and incubating the inoculated mixture for a desired time to form a culture/gelling liquid.
The methods described within explain the practices and materials that may be used to produce tissue culture media comprised of plant seed material and water, as well as potentially supplemental nutrients (minerals, vitamins, and the like). Certain plant seeds and seed matter, upon mixing with water, gelatinize and the residual solids become suspended in solution. Such materials include chia seeds, basil seeds, psyllium seed husks, and the like. The seeds provide the essential nutritive and thickening properties for the media while minimizing the number of ingredients added to water. Industry has found use in purifying the gel-forming fraction of these substances for dietary supplementation. This tissue culture media may be used for applications that currently employ media requiring multiple costly ingredients and chemical inputs.
Scientific literature has reported the successful use of fatty acids in promoting growth of mycelial tissue liquid cultures. The purported mechanism of action for this achievement may be attributed to the uptake of lipids to the cell membranes that then facilitate nutrient transfer from media into cells. Previously work has proven this effective in liquid cultures of yeast, bacteria, and fungal mycelia.
This is relevant to the use of seeds in tissue culture media because seeds are often a valuable source of oils and fatty acids in addition to nutrients and other beneficial compounds (carbohydrates, minerals, and simple sugars). For example, chia seeds have been reported to contain 25-38% oil by weight. These are mostly polyunsaturated fatty acids (alpha-linoleic acid making up 62% of total fatty acids).
Chia seeds, like many other seeds, possess additional nutritive properties that are quite valuable for cell growth and proliferation. The seeds are 19-23% protein and 22.1% dietary fiber. In vivo digestibility has been rated at 93-100%. Several types of antioxidants are present, such as myricetin, quercetin, kaempferol, caffeie acid, and chlorogenic acid. They also provide B vitamins, calcium, phosphorus, potassium, zinc, magnesium, and copper.
1. Tissue Culture Media
2. Gel Assisted Casting of Mycological Substrate
In this example, liquid addition to substrate serves, two primary functions: introduction of mycelial culture to the substrate and introduction of gelling agent to the substrate.
Psyllium Husks
In this example, a liquid solution of nutritional supplements and gelling agents is added to a substrate with grain inoculum.
In this example, a liquid solution of nutritional supplements and gelling agents is added to live, pre-colonized substrate.
In this example, a liquid solution of nutritional supplements and gelling agents is added to live, pre-colonized substrate.
A mixture formed of a culture/gelling liquid and substrate described above may be used in the fabrication of various members, for example, using various types of molding techniques. For example, one method that could be employed is the use of high pressure to compact the materials together. Much in the same way as structural wood composites, such as Medium Density Fiberboard (MDI′) and Oriented Strand Board (OSB), desired shapes could be created using compression to create cohesion between particles or fibers.
The use of high pressure, heat, gels, and waxes or adhesive additives could prove quite useful in primary shaping of a mycological composite product. This would produce a high density part, and due to its small or nonexistent gaps between particles, additional additives or created voids may be needed to allow for respiration.
Temperature changes could be employed to activate temperature set binding agents, such as waxes, however the nutritional agents in the biological substrate may be enough to hold certain shapes together until the fungi fully colonize the part. Steam could be used to activate binding agents as well as clean machined cavities that would serve as molds.
With a highly cohesive substrate, or one with long fibers, a small amount of pressure may be able to cause structural cross-linking of particles or fibers enough to hold a desired shape. Various additives have already proven to serve as functional binding agents including guar gum, heated starch solutions, and chia, seeds. This is referred to as gel-assisted casting. Hand-packing substrate coated in guar gum and calcium into a soft-walled tool and then ejecting the finished member by hand has shown the ability to create a self-supported 3D casted part that over about 5 days became fully colonized with mycelia.
One method of producing such a self-supporting part would be to start with a two part mold, such as a die used in the practice of die-casting of aluminum, or plastic injection molding. This mold could then be closed, and through an inlet cavity, filled with either a ground pre-colonized substrate or a freshly inoculated substrate by way of gravity, pneumatic or hydraulic filtration and mechanical conveyance, such as a peristaltic or piston pump.
In the case of hydraulic or pneumatic conveyance, the cavities could be perforated, and the die could be enclosed in an air-tight container. By removing fluid from the container and negatively pressurizing the container, fluid would rush out of the cavity, pulling in fluid through the injection port, which would in turn pull substrate into the cavity and against the walls of the cavity of the mold. The walls would filter out the substrate from the fluid, which could be either a gas or liquid, and compact the substrate together under the pressure of the flow.
After the cavity is filled by any of the methods described, additional pressure could be applied to the mixture of substrate within the cavities by way of a piston compressing additional material in through the injection port. After the cavity has been sufficiently filled and compacted, the part could be removed by separating the two part die and ejecting the part onto a sanitary conveyor by pushing pressurized air through the holes in the mold.
Use may also be made of a three part tool (not shown) comprised of three independently actuated featured cavities. Two parts form a cavity which is the negative of the desired geometry and the third part serves as a sleeve to affix the two negative-forming parts together. In this embodiment, sterilized and inoculated substrate is applied to the cavity and mechanically actuated to uniformly fill the void and/or modify the density. The material could be compressed with a piston or vibrated into position. After casting of the substrate into a coherent mass, the sleeve is removed from the tool and the two negative-forming parts are retracted to expose the casted substrate. The casted part is then conveyed into an incubation environment or incubation vessel that can control environmental conditions including but not limited to temperature, CO2, and relative humidity.
In another embodiment, use is made of a two part tool wherein the two parts form a cavity which is the negative of the desired geometry and the tool is conveyed on a work surface, such as a conveyor belt. Such a tool may employ injection molding techniques wherein a slurry of the mixture of culture/gelling liquid and inoculated substrate is pumped with a metering pump (peristaltic, diaphragm) into the cavity of the tool. Also, the casting cavity may have multiple chambers to form an engineered substrate of complex geometry. After casting of the substrate into a coherent mass, the sleeve is removed from the tool and the two negative-forming parts are retracted to expose the casted substrate. The casted part is then conveyed into an incubation environment.
Multiple parts may be cast simultaneously, conveyed forward and then pushed into incubation vessels.
Of note, depending of the constituents, a mixture of culture/gelling liquid and inoculated substrate placed in the cavity of a casting tool and held for a time sufficient for the mixture to form a coherent mass that may be subsequently processed out of the tool. For example, some mixtures need beheld for several seconds whereas other mixtures may take several hours. Once removed from the casting tool, the coherent mass is incubated and then dried.
The invention thus provides an economical method of producing a tissue culture media for use in making composite materials.
The invention provides improved methods for casting mycological biomaterials particularly from tissue culture media derived from plant seed material.
This application claims the benefit of Provisional Patent Application 61/573,888 filed Sep. 14, 2011.
Number | Date | Country | |
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61573888 | Sep 2011 | US |