Electron beam radiation for sterilization of media prior to inoculation of fungi and related methods

Abstract

The present invention provides a continuous method for sterilizing a substrate. The method comprises the step of preparing a matrix mixture having a moisture content. Further steps include loading the substrate mixture into a carrier, while the next step includes exposing the substrate mixture to electron beam ionizing radiation in an electron beam blindage housing to produce a sterilised substrate having the same moisture content as the substrate mixture. The last step includes unloading the sterilized substrate from the carrier such that the carrier can be reused in a continuous process.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 399,425, filed on August 19, 2022, the subject matter of which is incorporated herein by reference. Technical Field

[0003] The present invention generally relates to the use of ionizing radiation (including electron beam radiation or X-ray radiation) to sterilize substrates or other culture media prior to inoculation of various fungi. The present invention also relates to the uniform mixing, uniform hydration and sterilization of various culture media or substrates by ionizing (i.e., electron beam or X-ray) radiation in a continuous process, without heat or steam during the sterilization process, prior to inoculation of various fungi. The methods of the present invention provide more accurate moisture content and better simulate the natural particles comprising the substrate or culture media, while creating a more efficient process for the cultivation of mushrooms or other fungi. Background Art

[0004] It is generally accepted that mushroom and other fungal cultures are very similar to cultures of other microorganisms in that they are prepared, maintained, propagated, and stored on sterile media using microbiological laboratory methods. Cells of pure cultures are manipulated using sterile tools and aseptic techniques within a cleanroom or sterile transfer hood or carrier for a variety of purposes, including clonal propagation, development of new strains, and generally for the production or cultivation of such cultures. A variety of different techniques may be used, including spore germination on sterile growth media and controlled hybridization on sterile growth media. Commercial culture inoculants and production materials include mushroom spawn and sleeve inoculants. Synthetic "logs" are also prepared using large-scale microbial production methods, such as by aseptically introducing an inoculum of a pure culture of a fungal strain into 1 to 14,000 liters of sterile growth media under sterile conditions. Typically, the inoculated culture is provided to the end user as a pure culture on sterile growth media contained within sterile packaging.

[0005] The mushroom or other fungal product is then cultivated commercially in a dedicated structure in a dedicated facility. While there are many variations in the method, the following description is typical. A substrate prepared with a lignocellulosic material (e.g., straw, sawdust, or wood chips), to which a nitrogen-containing material is added, is finished in a suitable facility and pasteurized or sterilized. Mushroom spawn or other suitable inoculant is mixed with the prepared substrate and incubated at a controlled temperature for about 10 to about 90 days, during which time the mycelium of the mushroom culture colonizes the entire substrate and begins to digest it. The mushroom spawn or other suitable inoculant includes a sterile, friable "carrier matrix" or liquid medium to which a pure culture of one or more fungal strains is aseptically added through the inoculum and then propagated. For certain mushroom-forming fungi, including Agaricus bisporus, a non-nutritious "cover" material (e.g., peat) may then be placed over the substrate to a thickness of about 1.5 to about 2 inches. Additional "mulch inoculum" comprising the same mushroom culture may be added to the mulch layer to accelerate the formation and harvest of fungal biomass (e.g., mushrooms) and to improve the uniformity of mycelium distribution within and on the mulch surface. Environmental conditions in the production facility, including temperature and humidity, are then carefully managed to promote and control the transition of the culture from vegetative to reproductive growth at the mulch / air interface. For Agaricus bisporus, the mushrooms will develop to the correct stage for harvesting and marketing within approximately 13 to 18 days after mulching. A batch of mushrooms including the original culture will be picked within a period of 3 to 4 days. Additional batches of mushrooms will appear at intervals of about every other week. Commercially, two to three batches of mushrooms are produced and harvested before the compost is removed and placed back into the cultivation facility.

[0006] In the cultivation of mushroom mycelium or other fungus-based products, the substrate must be sterilized before inoculation with the appropriate fungal culture to avoid the growth of other bacteria and molds, which inhibit the growth of desired organisms. In traditional methods of cultivating such materials, the sterilization of the substrate is labor-intensive and is carried out by "batch" processing, which means that only a certain amount can be sterilized each time, depending on the capacity of the heating equipment. That is, the sterilization process is usually carried out in a kiln or autoclave or other heating equipment at a high temperature far above 35°C (more likely above 50°C, even more likely in the range of 100-125°C) for a considerable period of time, i.e. at least 12 to 24 hours, which reduces the moisture content of the substrate and may change the texture and nutritional value of the substrate, which is an undesirable feature of traditional methods. Another disadvantage of having to sterilize the substrate at high temperature is that since the heated substrate needs time to cool, the process must be carried out in a batch process rather than a more efficient continuous process. Therefore, there is a need in the art for a method of sterilizing the substrate before inoculation to avoid the defects of previous methods, such as unnecessarily reducing the moisture content of the substrate. There is also a need for a more efficient method of cultivating mushrooms or other fungi. Summary of the invention

[0007] At least one aspect of the present invention provides a continuous method for sterilizing a substrate, the method comprising the steps of: preparing a substrate mixture having a moisture content; loading the substrate mixture onto a carrier; exposing the substrate mixture to electron beam or X-ray ionizing radiation in an electron beam or X-ray shelter housing to produce a sterilized substrate having the same moisture content as the substrate mixture; and unloading the sterilized substrate from the carrier so that the carrier can be reused in the continuous method. In one or more embodiments, the method steps can be sequential.

[0008] Another embodiment of the present invention provides a continuous method as described in the above embodiment, further comprising: packaging the sterilized matrix into a package for transportation. In some embodiments, this will be a step after the unloading step.

[0009] Another embodiment of the present invention provides a continuous method as described above, further comprising: a step of inoculating the sterilized matrix with an inoculum to produce an inoculated matrix. In some embodiments, this will be a step after the unloading step.

[0010] Another embodiment of the present invention provides a continuous method as described in the above embodiment, further comprising, after the inoculation step: packaging the inoculation matrix into a package for transportation.

[0011] Another embodiment of the present invention provides a continuous process as described above, wherein the inoculum is an organism selected from, but not limited to, the following group: Agaricus, Morchella, Trichoderma, Beauveria, Lentinus, Ganoderma, or Pseudomonas. It should be understood that the present invention is not necessarily limited to these specific spore-forming organisms. These happen to be the most commercially acceptable organisms currently.

[0012] Another aspect of the present invention provides a continuous method as described in any of the above embodiments, wherein in the step of unloading the sterilized matrix, the carrier is brought to a clean room directly adjacent to the electron beam or X-ray shelter housing after being exposed to the electron beam or X-ray ionizing radiation, and the unloading step is performed in the clean room.

[0013] Another embodiment of the present invention provides a continuous method as described in any of the above embodiments, wherein in the step of unloading the sterilized matrix, the carrier is transported to a clean room not directly adjacent to the electron beam or X-ray shelter housing after exposure to the electron beam ionizing radiation.

[0014] Another embodiment of the present invention provides a continuous method as described in any of the above embodiments, wherein the substrate mixture includes nutrient particles and optionally added water.

[0015] Another embodiment of the present invention provides a continuous method as in any of the above embodiments, wherein the nutrient particles are selected from sawdust, fermented sawdust, millet, sorghum, grains, agricultural waste, oilseed products, grains, prepared compost, fermented compost, peat moss, gypsum, chalk, vermiculite, perlite, clay, waste paper materials and combinations thereof.

[0016] Another embodiment of the present invention provides the continuous method as described in any one of the above embodiments, wherein the moisture content of the substrate mixture is between 30% and 70%.

[0017] Another embodiment of the present invention provides a continuous method as in any of the above embodiments, wherein the exposing step comprises subjecting the matrix mixture to an ionizing radiation dose between about 5 kGy and about 40 kGy.

[0018] Another embodiment of the present invention provides a continuous method as described in any embodiment, wherein the exposing step is performed at a temperature between about 10°C and about 35°C.

[0019] As will be appreciated, these and other aspects of the invention provide advantages in sterilizing culture media and other substrates that were not previously available by heating the substrate. The amount of time required to conduct the continuous process is seconds to minutes (up to an hour or less, but typically only 1 to 10 minutes) compared to prior art batch processes, where bulk substrates must be heated and cooled over a considerable period of time (12-24 hours or more) and where the substrate loses at least a portion of its moisture content.

[0020] It should also be understood that while electron beam and / or X-ray radiation are known in the art of sterilization, such sterilization has never been performed in a continuous process for producing a substrate culture medium for mushrooms or other fungi prior to inoculation. In fact, if it has been used before, electron beam radiation has only been used for the final product, which in the case of fungi is also meant to kill the inoculum material that is trying to grow on the substrate described in this specification. In the present invention, electron beam and X-ray radiation are used to sterilize the culture medium so that other things can be grown without further sterilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention and to the accompanying drawings, in which:

[0022] Figure 1 shows a simplified representation of a first embodiment of a continuous method for sterilizing a substrate;

[0023] Figure 2 shows a simplified representation of a second embodiment of a continuous method for sterilizing a substrate;

[0024] Figure 3 shows a simplified representation of a third embodiment of a continuous method for sterilizing a substrate;

[0025] Figure 4 A simplified representation of a fourth embodiment of a continuous method for sterilizing a substrate is shown. DETAILED DESCRIPTION

[0026] The present invention provides a continuous method that utilizes electron beam radiation to sterilize substrates or other culture media prior to inoculation with various fungi. Figure 1A simplified representation of a continuous method 10 for sterilizing a matrix mixture 12 is shown. In the first step of the method 10, a matrix mixture 12 is prepared in a mixer 14. The matrix mixture 12 will include nutrient particles and optionally added water to provide a matrix mixture 12 with a moisture content. Once the matrix mixture 12 is prepared, the next step is to load the matrix mixture 12 onto a carrier 16. The carrier 16 is then fed into an electron beam shelter housing 20 (or alternatively, an X-ray shelter housing) along a conveyor 18 to expose the matrix mixture 12 to electron beam ionizing radiation, thereby producing a sterilized matrix 22. It should be understood that throughout this application, X-ray radiation can replace electron beam radiation, and "X-ray shelter housing" can replace the "electron beam shelter housing" used in this application. In one or more embodiments, the carrier 16 is a covered carrier that contains the matrix mixture 12 in the carrier 16 using a lid or similar object. In other embodiments, the carrier 16 is an uncovered carrier that exposes the matrix mixture 12 to the environment.

[0027] It is worth noting that the method 10 of the present invention is a continuous method, meaning that the carrier 16 can continuously travel around the conveyor 18 and can be used in the method 10 of the present invention multiple times.

[0028] It is noteworthy that the continuous method 10 of the present invention also does not heat the matrix mixture 12 to sterilize the matrix mixture. This is more advantageous than previous matrix mixture sterilization methods because no water is lost in the matrix mixture 12 during sterilization.

[0029] Notably, in the continuous method 10 of the present invention, the sterilized matrix 22 exposed to electron beam ionizing radiation in the electron beam shelter housing 20 will have the same moisture content as the matrix mixture 12 before exposure of the matrix mixture 12 to the electron beam ionizing radiation in the electron beam shelter housing 20.

[0030] The next step in the continuous process 10 is to unload the sterilized substrate 22 from the carrier 16 so that the carrier 16 can be retrieved along the conveyor 18 and reused in the continuous process 10. In some embodiments, the action of unloading the sterilized substrate 22 from the carrier 16 is accomplished by a pneumatic carrier turner 24, such as Figure 1 shown. Figure 1 It is also shown that unloading of the sterilized matrix 22 from the carrier 16 is performed in a clean room 26a, which is directly adjacent to the electron beam shelter housing 20. Unloading directly in the clean room 26a will allow additional steps discussed below to be performed directly after the sterilized matrix 22 is unloaded from the carrier 16. Figure 1As shown, clean room 26a includes HEPA walls 27a and 27b. HEPA walls 27a and 27b provide clean, filtered, laminar air flow throughout the enclosure, allowing workers to operate in the clean room while minimizing the possibility of contaminating any exposed sterile substrates 22.

[0031] In another embodiment, Figure 2 As shown, the action of unloading the sterilized substrate 22 from the carrier 16 is not completed in the clean room. In this embodiment, the substrate is covered or placed in a sealed carrier and sterilized. After unloading, the sterilized substrate 22 is transported to a clean room 26b that is not directly adjacent to the electron beam shelter housing 20. Figure 1 Examples and Figure 2 The differences between the embodiments will be based on the design parameters of the facility performing the method 10. If the facility has the ability to place the clean room 26a directly adjacent to the electron beam shelter housing 20, then they may choose to do so. If the facility does not have the space to place the clean room 26b directly adjacent to the electron beam shelter housing 20, then they may choose to place the clean room 26b in a location that is not directly adjacent to the electron beam shelter housing 20. The location of the clean rooms 26a / 26b does not change the scope of the present invention.

[0032] In another embodiment, Figure 3 As shown, the continuous method 10 includes an additional step, which occurs after the unloading step, of packaging the sterilized matrix 22 into a package 28 for shipping. The packaging step is shown in FIG. Figure 3 As shown, it is completed by a bagging machine or an automatic bagging machine 30. Once the sterilized matrix 22 is packaged into the packaging 28, it can be transported to different facilities for inoculation. Figure 3 As shown, the packages 28 are initially removed from a bagging machine or automatic bagging machine along a conveyor 32. Figure 3 The bagging machine or automated bagging machine 30 is shown located within the confines of the clean room 26a, but it is contemplated that the bagging machine or automated bagging machine 30 may be located at a location outside of the confines of the clean room 26a.

[0033] In another embodiment, Figure 4 As shown, the continuous process 10 includes an additional step, which occurs after the unloading step, of inoculating the sterilized matrix 22 with an inoculum to produce an inoculated matrix 34 . Figure 4 The inoculation step shown in FIG. 1 is completed by an inoculation mixing screw 36, but the present invention is not limited to such an inoculation device. It is important to note that the inoculation mixing screw 36 needs to be located in the clean room 26a. Figure 2 Also shown is an inoculated mixing screw 36 located within the clean room 26b.

[0034] Figure 4Also shown is the additional step of packaging the seeding matrix 34 into packaging 28 for shipping, which is performed after the seeding step. Figure 4 , the packaging step is shown, which is completed by a bagging machine or an automatic bagging machine 30. Once the inoculated substrate 34 is packaged into the packaging material 28, it can be transported to the customer to harvest the crops planted by the inoculated substrate 34. Figure 4 The bagging machine or automated bagging machine 30 is shown located within the confines of the clean room 26a, but it is contemplated that the bagging machine or automated bagging machine 30 may be located at a location outside of the confines of the clean room 26a.

[0035] In the whole continuous process 10, as Figures 1 to 4 As shown in any embodiment of the present invention, the substrate mixture 12 includes nutrient particles and optionally added water. The nutrient particles are selected from the group consisting of sawdust, fermented sawdust, millet, sorghum, grains, oilseed products, agricultural waste, grains, prepared compost, fermented compost, peat moss, gypsum, chalk, vermiculite, perlite, clay, waste paper materials and combinations thereof. The present invention contemplates that the moisture content of the substrate mixture 12 should be between 30% and 70%. Depending on the moisture content of the nutrient particles themselves and the desired moisture content of the substrate mixture 12, water can be added to the substrate mixture.

[0036] In the whole continuous process 10, as Figures 1 to 4 As shown in any of the embodiments, during the step of exposing the matrix mixture 12 to electron beam ionizing radiation in the electron beam shelter housing 20 to produce a sterilized matrix 22 having the same moisture content as the matrix mixture 12, the matrix mixture 12 is exposed to an ionizing radiation dose between about 5 kGy and about 40 kGy. The dose of ionizing radiation is determined based on the thickness of the matrix mixture 12 being treated, and the effective penetration of the ionizing radiation depends on the thickness of the matrix mixture 12. In addition, the exposure step occurs at a temperature between about 10° C. and about 35° C.

[0037] In the entire continuous method 10, if Figures 1 to 4As shown in any of the embodiments of the present invention, it should be noted that the electron beam ionizing radiation applied to the matrix mixture 12 within the electron beam shelter housing 20 can only penetrate 9 cm into the matrix mixture 12. Therefore, if the thickness of the matrix mixture 12 is greater than 9 cm, it is expected that the matrix mixture can pass through the electron beam shelter housing 20 more than once, or pass through multiple beams (i.e., above and below). In some embodiments, the matrix mixture 12 passes through the electron beam shelter housing 20 for the first time, then the matrix mixture 12 is rotated 180 degrees, and then passes through the electron beam shelter housing 20 for the second time. In some embodiments, the matrix mixture 12 passes through the electron beam shelter housing 20 for the first time, then the matrix mixture 12 is rotated 90 degrees, then passes through the electron beam shelter housing 20 for the second time, then the matrix mixture 12 is rotated 90 degrees again, then passes through the electron beam shelter housing 20 for the third time, then the matrix mixture 12 is rotated 90 degrees again, and then passes through the electron beam shelter housing 20 for the fourth and final time.

[0038] exist Figure 4 In the illustrated embodiment of the continuous process 10 of the present invention, the inoculant used to inoculate the sterilized substrate 22 to produce the inoculated substrate 32 is a spore-forming organism selected from the group consisting of Agaricus, Morchella, Trichoderma, Beauveria, Lentinus, Ganoderma, or Pseudomonas. This list of inoculants is only a limited list of potential inocula that can be used in the continuous process 10 of the present invention, and other different types of fungi not listed here can also be used in the continuous process 10 of the present invention.

[0039] In view of the foregoing, it should be appreciated that the present invention has significantly promoted the development of this field by providing a continuous method for sterilizing a substrate that is improved in various ways in structure and function. Although specific embodiments of the present invention are disclosed in detail herein, it should be appreciated that the present invention is not limited thereto or thereby limited, because those of ordinary skill in the art will readily appreciate the variations of the present invention herein. The scope of the present invention should be understood from the following claims.

[0040] Example

[0041] In the first example, cooked millet, milo (sorghum), and rye grains with moisture contents between 43% and 46% were sampled to determine their initial bioburden (as shown in Table 1 below). 2.5 lbs of the sample were then placed in a suitable sealed plastic container and then exposed to varying levels of electron beam ionizing radiation between 5.96 kGy and 15.8 kGy by adjusting the speed at which the substrate mixture passed through the exposure field generated in the electron beam shelter enclosure.

[0042] Table 1 – Bioburden in untreated nutrient pellets

[0043]

[0044]

[0045] 1 colony forming unit

[0046] 2Below the minimum detection level, equivalent to <200 CFU / g

[0047] After exposure, the contents of each container were aseptically sampled to determine the degree of sterility achieved (results are shown in Tables 2, 3, and 4 below). The remainder of the treated material was inoculated into an appropriate commercial Agaricus bisporus inoculum and the extent of colonization was observed over the next two to three weeks and compared to a commercial control. The extent of colonization was observed on three different media: potato dextrose agar, tryptic soy agar, and tryptic soy agar. Potato dextrose agar is a microbial growth medium for fungi, while tryptic soy agar is a microbial growth medium for bacteria. Tryptic soy agar is a common growth medium for low-level detection of bacteria. Enrichment of the broth involved a 5-day incubation of the broth boxes followed by a sterility assay by streaking the broth on tryptic soy agar plates.

[0048] Table 2 – Sterility test of millet treated with electron beam ionizing radiation

[0049]

[0050] 3Potato Dextrose Agar

[0051] 4Tryptic Soy Agar

[0052] 5 Tryptic Soy Broth

[0053] Table 3 - Sterility Test of Milo Treated with Electron Beam Ionizing Radiation

[0054]

[0055] Table 4 – Sterility tests for rye grain treated with electron beam ionizing radiation

[0056]

[0057] In a second example, a mixed granular substrate containing an inert solid carrier (vermiculite, perlite, Biodac blend) and a representative nutrient mixture of corn meal, feather meal, wheat bran, soybean meal, yeast, chalk, gypsum, lime, and composted wheat straw was combined and mixed with water to obtain a uniform substrate mixture with a moisture content between 43% and 46% and a pH suitable for fungal cell growth. The material was sampled to determine the initial bioburden (Table 5). A 2.5 pound sample was then placed in a suitable sealed plastic container and then exposed to different levels of electron beam ionizing radiation between 5.9 kGy and 16.1 kGy by adjusting the speed at which the substrate mixture passed through the exposure field generated in the electron beam shelter enclosure.

[0058] Table 5 – Bioburden of untreated mixed granular matrix

[0059] Bacterial concentration (CFU / g) Fungal concentration (CFU / g) 1.34E+08 BMDL

[0060] After exposure, the contents of each container were aseptically sampled to determine the degree of sterility achieved (Table 6). The remainder of the treated material was inoculated with an appropriate commercial Agaricus bisporus inoculum and the extent of colonization was observed over the next two to three weeks and compared to a commercial control.

[0061] Table 6 - Sterility test of mixed granular matrix treated with electron beam ionizing radiation

[0062]

[0063] In a third example, a mixed matrix containing sawdust, wheat bran, and gypsum was mixed with water to obtain a homogenous matrix material with a moisture content between 45% and 65% and a pH suitable for fungal cell growth. The material was sampled to determine the initial bioburden (Table 7). Then, 2.5Kg of the sample was placed in a suitable sealed cylindrical plastic tube, compacted to a density of 0.9g / cm3, and then exposed to a fixed level of 10kGy of ionizing radiation at a rate of 4.92FPM through an exposure field created in an electron beam shelter enclosure. The bag was rotated 180 degrees and passed through the field a second time, or the cylinder was rotated in 90 degree increments for a total of four passes through the field.

[0064] Table 7 - Bioburden of untreated mixed sawdust substrate

[0065] Bacterial concentration (CFU / g) Fungal concentration (CFU / g) 7.5E+04 5.6E+06

[0066] After exposure, the sterilized substrate in each bag was aseptically sampled to determine the degree of sterility achieved (Table 8). The remainder of the sterilized substrate material was inoculated with a commercial shiitake inoculum from a suitable source and observed for degree of colonization over a period of 15 to 60 days compared to a commercial control.

[0067] Table 8 - Sterility test of mixed sawdust matrix treated with electron beam ionizing radiation

[0068]

[0069] In another example, further research was conducted by irradiating Shiitake mushroom growing substrates, comparing the effects of ionizing radiation using 10 MeV electron beam irradiation and 7 MV (megavolt) X-ray irradiation to compare the levels of bioburden reduction achieved by the two techniques.

[0070] A shiitake mushroom growth substrate (hardwood sawdust amended with wheat bran, corn, chalk and gypsum, adjusted to a moisture content between 60% and 65%) was prepared and samples (between 250 and 500 grams of substrate) were placed in plastic bags, tightly closed, and stored at room temperature as a control. 1.4 kg bags were then treated with different radiation treatments to determine the exposure required to achieve a bioburden reduction of 10e6 or better. The results are shown in Table 9. The prepared samples were exposed to doses of 10 kGy, 15 kGy, 20 kGy, 25 kGy (electron beam, 10 bags per dose) and 10 kGy, 15 kGy, 20 kGy, 25 kGy (X-ray, 9 bags per dose). In addition, a group of ten bags were steam sterilized as an additional control.

[0071] Table 9 – Bioburden Reduction Test Levels

[0072]

[0073] Note: Shiitake mushroom refers to the growth medium of shiitake mushrooms

[0074] The results showed that 10 kGy of X-ray treatment was not sufficient to reduce the bioburden below the target level. X-ray irradiation exposure, another form of ionizing radiation, can be used to produce a sterile matrix for fungal biomass growth at exposure levels of 15 kGy or higher.

[0075] Although the present invention has been described in this application with specific embodiments, those skilled in the art may generate additional embodiments and modifications based on the teachings herein without departing from the spirit of the claimed invention or exceeding its scope. Therefore, it should be understood that the description herein is only used to help understand the present invention and should not be interpreted as limiting its scope.

Claims

1. A continuous method for substrate sterilization, the method comprising the following steps: a. preparing a substrate mixture having a moisture content; b. loading the matrix mixture onto a carrier; c. exposing the matrix mixture to electron beam or X-ray ionizing radiation in an electron beam or X-ray shelter housing to produce a sterilized matrix having the same moisture content as the matrix mixture; as well as d. Unloading the sterilized matrix from the carrier so that the carrier can be reused in the continuous process.

2. The continuous method according to claim 1, further comprising the step of packaging the sterilized matrix into packages for transportation.

3. The continuous process of claim 1, further comprising the step of inoculating the sterilized matrix with an inoculum to produce an inoculated matrix.

4. The continuous method according to claim 3, further comprising the step of packaging the inoculated matrix into packages for transportation.

5. The method of claim 4, wherein the inoculant is an organism selected from the group consisting of Agaricus, Morchella, Trichoderma, Beauveria, Lentinus, Ganoderma, and Pseudomonas.

6. A continuous method according to claim 1, wherein in the step of unloading the sterilized matrix, the carrier is brought to a clean room directly adjacent to the electron beam or X-ray shelter housing after being exposed to the electron beam or X-ray ionizing radiation, respectively, and the unloading step is performed in the clean room.

7. The continuous method according to claim 1, wherein in the step of unloading the sterilized matrix, the carrier is transported to a clean room not directly adjacent to the electron beam or X-ray shelter housing after being exposed to the electron beam or X-ray ionizing radiation, respectively.

8. The continuous process of claim 1, wherein the substrate mixture comprises nutrient particles and optionally added water.

9. The continuous process of claim 8, wherein the nutrient particles are selected from the group consisting of sawdust, fermented sawdust, millet, sorghum, grains, oilseed products, agricultural waste, cereals, prepared compost, fermented compost, peat moss, gypsum, chalk, vermiculite, perlite, clay, waste paper material, and combinations thereof.

10. The continuous process of claim 1, wherein the moisture content of the substrate mixture is between 30% and 70%.

11. The continuous method of claim 1, wherein the exposing step comprises subjecting the matrix mixture to an ionizing radiation dose of between about 5 kGy and about 40 kGy.

12. The continuous process of claim 1, wherein the exposing step is performed at a temperature between about 10°C and about 35°C.

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