Patent Application
20130309755
This invention relates to a fungal leachate and to a method of making a fungal leachate.
It is common practice to culture fungi in liquid culture media. Typically, such media are comprised of water, a water-insoluble material, a sugar such as sucrose, and other nutrients. This is well documented in such patents as U.S. Pat. No. 2,693,665 and U.S. Patent Application Pub. No.: US 2003/0070351. These processes typically utilize complex combinations of specifically produced nutrient components, which requires extensive processing and mixing and can be prohibitively costly at scale.
The practice of utilizing waste streams from various substrates and manufacturing processes to produce liquid media for cultivating fungal biomass has been well explored. One characteristic common amongst waste fluid and manufacturing by-product derived fungal growing media is their dependence on economically important crops and processes. U.S. Patent Application Pub. No.: US 2008/0153149 provides a good example describing the use of thin stillage from dry-grind corn milling within ethanol production processes as a growing medium for fungi.
Other liquids and liquid by-products, such as the wash water from rice and wheat production, as well as coconut water have been shown to be viable liquid media for growing fungi as well. None of these examples utilize a fungus as the primary agent for processing lignocellulose into water soluble components that can be used to produce liquid culture media.
A process is also known to produce a spent mushroom bed extract that can be used to cultivate fungi. This process focuses on extraction of enzymes and other bioactive compounds utilizing a complex extraction process dependent on homogenization, centrifugation, and filtration.
Accordingly, it is an object of the invention to employ leachate as a liquid culture medium for cultivating fungi.
It is another object of the invention to provide a fungal leachate that does not require additional nutrient components.
It is another object of the invention to provide a process for producing leachate in a less energy intensive manner than prior art processes.
Briefly, the invention provides a fungal leachate solution containing a water soluble fraction of fungally colonized and decomposed lignocellulosic substrate.
In addition, the invention provides a method of producing a fungal leachate solution comprising the steps of obtaining a feedstock of lignocellulosic substrate; colonizing the substrate with a selected fungus; and adding water to the colonized substrate to form a liquid medium containing at least one of sugar alcohol, a phenolic compound and a fatty acid.
In accordance with the method, a lignocellulose substrate—which has been colonized by a species within Basidiomycetes, Ascomycetes, or Zygomycetes—is leached in water to obtain a combination of water soluble products from the lignocellulose substrate, fungal biomass, and products specifically resulting from the fungal decomposition of the substrate, resulting in a leachate that contains all the nutrition necessary to be utilized as a culture medium for fungi.
This leachate (hereinafter referred to as “leachate” or “fungal leachate”) can be used for the same applications and processes that traditional fungal culture media (such as malt extract agar) are used. This functionality of leachate is specifically dependent on the by-products of fungal decomposition of lignocellulose. Application examples would include cultivation of sheets of mycelium, gelatinized leachate to produce a tissue suspension medium for obtaining 3D colonization through the volume of the fluid, or used as an additive to solid substrate.
In accordance with the invention, the method may be varied to include 1) a process whereby the pre-colonized lignocellulose feedstock both produces the leachate medium and the inoculum (via mycelial fragments and/or asexual spores) in a single process, and 2) leachate derived from rye straw (colonized and decomposed as described above) which demonstrates a specific effectiveness at stimulating germination of asexual spores, namely chlamydospores.
These and other objects and advantages of the invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings wherein:
Referring to
In step 2, the colonized feedstock is added to water (or water is added to the feedstock) within a container to form a liquid medium.
The substrate is soaked in the water at room temperature for a period sufficient to leach water soluble compounds from the feedstock and form a liquid medium, i.e. a feedstock-water slurry. The water soluble compounds have been found to include (1) sugar alcohol, such as glycerin, (2) a phenolic compound, such as 2,6 Dimethoxy phenol and (3) a fatty acid, such as pentadecanoic acid. The compounds may also include at least one of a carbonyl and a phenyl wherein the carbonyl is one of heptanal and pentanal and the phenyl is benzoic acid.
Thereafter, in step 3, the feedstock-water slurry is poured through a sieve/filter to separate the solids from the liquid leachate.
In step 4, the liquid leachate is removed and in step 5 is subjected to heat for pasteurization or sterilization particularly where a specific application of the leachate requires such.
Optionally, the feedstock substrate may be soaked in water heated to a temperature of at least 180° F. In this case, the obtained leachate need not be subjected to heat for pasteurization or sterilization.
Also, as an option, the solid substrate need not be removed from the liquid leachate.
Referring to
As above, in step 2, water is added to the colonized feedstock in a container and the feedstock soaked at room temperature for a period sufficient to leach water soluble compounds from the feedstock and to form a feedstock-water slurry.
In step 3, the slurry is then agitated vigorously to release and distribute fragments of mycelium and/or asexual spores from the feedstock into the water/leachate, for example, in experimental trials, a flask containing the slurry was vigorously shaken for this purpose.
In step 4, the slurry is poured through a sieve/filter to separate the leachate from the solids.
Next, in step 5a, the solid feedstock with living fungus is removed from the liquid leachate and, in step 6a, the feedstock is then incubated (i.e. post-leaching) to achieve a desired colonization. In this regard, the fungus is allowed to continue growing in the feedstock in order to bind loose particles of substrate into a solid mass.
In step 5b, the leachate is removed and, in step 6b, is then incubated to allow the fungus to continue growing. The fragments of mycelium/asexual spores present in the leachate (provided by the fungus in the feedstock via agitation) will initiate growth, eventually forming a solid sheet of mycelium on the surface of the leachate and/or growing through the volume of the leachate, if gelatinized, to obtain 3D colonization.
In another embodiment, a leachate may be specifically derived from rye straw colonized and decomposed by a primary saprophyte. This leachate medium shares the same general functionality of other leachate media described here, but has a unique stimulatory effect on the germination of chlamydospores (an asexual spore produced in the mycelium of many fungi).
Chlamydospores in the presence of this specific leachate medium germinate at percentages of over 90% by 24 hours after inoculation, as compared to much lower germination rates in other traditional and leachate media. The specific components of this leachate, determined through mass spectrometry, are listed below:
Butanoic acid 3 methyl
butyrolactone
cyclohexanone
glycerin
2-propanamine
2 5 dimethyl 4 hydroxy 3(2H) furanone
Formic acid ethenyl ester
maltol
pentanoic acid 4 oxo
4H pyran 4 one 2 3 dihydro 3 5 dihydroxy 6 methyl
isosorbide
propanedioic acid phenyl
isosorbide
4H 1 3 oxazine 5 6 dihydro 2 4 4 tetramethyl
Phenol 2 6 dimethoxy
d mannitol 1 4 anhydro
D erythro penose 2 deoxy
guanosine
heptanamine 5 methyl
heptane 3 methylene
The following are examples of the method of the invention.
Over the course of experimentation, mass spectrometry was performed on leachate samples derived from feedstocks including 5 different species of basidiomycetes grown on multiple substrates, both before and after heat sterilization. The exact components are variable depending on the substrate, species, and whether or not it has been heat processed, but the following general components are constant:
Most, but not all varieties of leachate also contain the following (depending on substrate and species composing the feedstock)
Below is an example of mass spectrometry results for a leachate sample derived from feedstock composed of hemp substrate colonized by a higher basidiomycete, both pre and post-heat sterilization in a pressure sterilizer at 15 psi for 1 hour. The substrate was dehydrated prior to sporocarp development, and leachate was produced.
Sample mass spectrometry result, leachate sample pre-heat sterilization
Sample mass spectrometry result, leachate sample post-heat sterilization
Below are other compounds frequently found in common between leachates produced from different feedstocks:
Many of these components, especially sugar alcohols, phenolics, and phenyls, are known to be by-products of fungal lignin decomposition. It has been demonstrated that leachate derived from virgin substrate (not colonized by a fungus) does not function as a fungal culture medium. Therefore, the fungal decomposition of the lignocellulose substrate prior to soaking to produce leachate is necessary to produce the components outlined above that are responsible for the functionality of the leachate.
The invention thus provides a process that employs leachate as a liquid culture medium for cultivating fungi and, particularly, a fungal leachate that does not require additional nutrient components.
The invention further provides a process for producing leachate in a less energy intensive manner than prior art processes.
This application claims the benefit of U.S. Provisional Patent Application 61/642,491 filed May 5, 2012.
| Number | Date | Country | |
|---|---|---|---|
| 61642491 | May 2012 | US |
