A submerged liquid state biomass fermentation process

Abstract

The present technology generally relates to a process for the development of mushroom mycelium using byproducts of protein isolate production and fractionation or milling.

Description

[0001] A SUBMERGED LIQUID STATE BIOMASS FERMENTATION PROCESS

[0002] FIELD OF TECHNOLOGY

[0003]

[0001] The present technology relates to methods and compositions for producing mushroom mycelium using pea starch via a biomass fermentation process.

[0004] BACKGROUND

[0005]

[0002] In an era where sustainability is increasingly becoming a priority, industries are seeking innovative, cost-effective alternatives that align with environmental goals. One such solution is the use of agricultural byproducts as growth media for mycelium. Mycelium, the root structure of fungi, has vast applications in food production, bio-based materials, and waste management.

[0006]

[0003] Agricultural byproducts are underutilized waste products in farming and food production. By repurposing these materials, manufacturers can not only reduce waste but also contribute to a circular economy. This transformation of waste into a valuable resource is both cost-effective and environmentally responsible, creating a closed-loop system that benefits industries and ecosystems alike (Kamthan & Tiwari, 2017). Many agricultural byproducts are biodegradable, making them an ideal substrate for mycelium, which can decompose and transform these materials into useful products. This process also aids in organic waste bioremediation. Certain fungi are highly effective in breaking down complex substances like cellulose, hemicellulose, and lignin (Geethanjali et al., 2020).

[0007]

[0004] The composition of agricultural byproducts is generally consistent, making them reliable for large-scale, standardized mycelium production. This consistency ensures high-quality mycelium, which is crucial for various applications such as food products (like mycoproteins) and innovative biomaterials, such as mycelium-based packaging (Barta et al., 2024).

[0008]

[0005] Mushroom mycelium production through fermentation is a growing area of interest due to its applications in food, pharmaceuticals, and biotechnology. Traditional methods often rely on substrates that may not be sustainable or economically viable. The present technology utilizes pea- based ingredients, which offer a renewable and cost-effective alternative, enhancing the overall efficiency and yield of mushroom mycelium production.

[0009]

[0006] Mushrooms have been used in a variety of new ways such as incorporating into food products such as bread and meat, adding unique flavours and characteristics (De Cianni et al., 2023; Du et al., 2021; Guinard et al., 2016; all incorporated herein by reference).

[0010]

[0007] Mushrooms have been grown using a variety of methods such as solid state or submerged state liquid culture while also using a variety of substrates such as pea protein and starches (A. Martin et al., 1992; Dulay et al., 2015; Souza Filho et al., 2018; all incorporated herein by reference).

[0011]

[0008] Mushrooms, specifically the Pleurotus ostreatus mushroom which has been characterized in depth, has been developed using liquid fermentation in a variety of bioprocesses (Hadar & Cohen-Arazi, 1986; Hesham Ali El-Enshasy et al., 2010; Raman et al., 2021; W. Manu-Tawiah et al., 1987; all incorporated herein by reference).

[0012]

[0009] Other methods of producing mycelium biomass have been previously developed using yeast and molds grown on a variety of substrates such as starch rich material (Avniel et al., 2024; SONI et al., 2020; Thanh, 2020; T et al., 2024; all incorporated herein by reference).

[0013]

[0010] There thus remains a need in the art for improved methods of producing mycelium biomass that alleviate at least some of the drawbacks observed in the art.

[0014] SUMMARY OF TECHNOLOGY

[0015]

[0011] In one aspect, the present technology relates to a process for production of mushroom mycelium, the process comprising: a) fermenting a fermentation composition, the fermentation composition comprising: i) at least one carbohydrate source; and ii) at least one protein source, to obtain a fermentation media; b) sterilizing the fermentation media to obtain a sterilized medium; c) inoculating the sterilized medium with a pure culture of mushroom species to obtain an inoculated medium; d) fermenting the inoculated medium under controlled conditions; and e) harvesting the mushroom mycelium from the fermented inoculated medium. In one aspect, the is a submerged liquid state fermentation process.

[0016]

[0012] In one aspect, the carbohydrate source is selected from legume-derived starches. In one aspect, the at least one carbohydrate source is pea starch. In one aspect, the carbohydrate source is bean starch. In one aspect, the carbohydrate source is pea flour. In one aspect, the carbohydrate source is bean flour.

[0017]

[0013] In one aspect the protein source is selected from plant-derived proteins. In one aspect the protein source is a legume protein. In one aspect the protein source is pea protein. In one aspect the protein source is bean protein.

[0018]

[0014] In one aspect, the carbohydrate source is present at a concentration of between about 1% and about 30% w / v of the total weight of the fermentation composition. In one aspect, the carbohydrate source is present at a concentration of between about 1% and about 15% w / v of the total weight of the fermentation composition.

[0019]

[0015] In one aspect, the protein source is present at a concentration of between about 1% and about 30% w / v of the total weight of the fermentation composition. In one aspect, the protein source is present at a concentration of between about 1% and about 15% w / v of the total weight of the fermentation composition. In one aspect, the protein source is present at a concentration of between about 1% and about 10% w / v of the total weight of the fermentation composition. In one aspect, the protein source is present at a concentration of between about 1% and about 3% w / v of the total weight of the fermentation composition.

[0020]

[0016] In one aspect, the fermentation composition further comprises a yeast extract, vitamins, minerals or a combination thereof.

[0021]

[0017] In one aspect, the process of the present technology further comprises adjusting the pH. In one aspect, the pH is adjusted with NaOH, H2SO4, organic acids, or buffering agents.

[0018] In one aspect, the process of the present technology further comprises adjusting brix through enzyme saccharification. In one aspect, the brix is adjusted through physical, enzymatic, or chemical means. In one aspect, the brix is measured at a temperature of between about 20°C and about 121°C. In one aspect, the brix is measured at a pressure of between about atmospheric and about 15 psi.

[0022]

[0019] In one aspect, the inoculation step of the process of the present technology is performed using liquid culture, solid culture, or spore suspension.

[0023]

[0020] In one aspect, the fermentation step of the process of the present technology is carried out in a bioreactor, a fermenter, or a continuous flow system.

[0024]

[0021] In one aspect, the sterilization step is performed at a temperature between about 80°C and about 121°C.

[0025]

[0022] In one aspect, the harvesting step is performed using disk stack centrifugation or screw press or decanter. In one aspect, the process further comprises drying the harvested mushroom mycelium using infrared microwave drying.

[0026]

[0023] In one aspect, the process of the present technology further comprises adjusting fermentation conditions throughout the process. In one aspect, the fermentation conditions are selected from pH, temperature, and aeration. In one aspect, fermentation is carried out for between about 5 and about 14 days or for between about 5 and about 10 days.

[0027]

[0024] In one aspect, the fermentation step is carried out at scales of between about 1, about 10, about 100, about 1000, about 10 000, about 100 000, about 1 000 000, and about 1 000 000 L of production capacity. In one aspect, the fermentation is carried out at scales of between about 1 and about 1 000 000 L of production capacity. BRIEF DESCRIPTION OF FIGURES

[0028]

[0025] For a better understanding of the present technology, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0029]

[0026] Figure 1 is a schematic representation of a fermentation and extrusion process for the creation of fresh mycelium and extruded texturized vegetable protein, CanPro according to one embodiment of the present technology.

[0030]

[0027] Figure 2 is a block flow diagram of the fermentation process according to one embodiment of the present technology. Other embodiments can include blending of material, pressing, or vacuum sealing.

[0031]

[0028] Figure 3 is a graph showing growth of strains on pea flour only.

[0032]

[0029] Figure 4 are pictures showing growth of strains on mixed carbon source (50% sugar, 50% flour).

[0033]

[0030] Figure 5 are pictures showing representative growth and material morphology for three different strains on pea flour only.

[0034]

[0031] Figure 6 is a graph showing the wet weight for mycelium grown on potato starch and Faba bean starch at varying concentration levels.

[0035]

[0032] Figure 7 are microscopic observations of a high-density inoculum growth on faba starch substrates at 100 mL over nine days at 100X magnification.

[0036]

[0033] Figure 8 shows microscopic observations of faba, wheat and pea after Day 1 culture at 100X magnification.

[0034] Figure 9 are pictures of saccharified faba starch agar plate (left) and molasses agar (right) plate showing colony growth after nine days.

[0037]

[0035] Figure 10 are pictures showing representative samples of potato and faba bean starches from 1%, 3%, and 5% w / v of starch in media fermented with P. ostreatus.

[0038]

[0036] Figure 11 shows Agarose gel electrophoresis of ITS1 / ITS4 PCR products (-750 bp expected size) from mycelium DNA extracts, visualized alongside FroggaBio 1 kb DNA ladder (1.5% agarose in 1 * TAE; 90-110 V, 30-45 min). (A) Gradient PCR optimization using samples G1 and Cl at annealing temperatures 61°C, 55.2 °C, 51.7°C, and 49.1°C. (B) PCR amplification at optimized 60 °C annealing temperature for samples Al-El. (C) PCR amplification at optimized 60 °C annealing temperature for samples E2, Fl, F2, G2, and a negative control.

[0039] DETAILED DESCRIPTION OF TECHNOLOGY

[0040]

[0037] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having", "containing", "involving" and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items. In the following description, the same numerical references refer to similar elements.

[0041]

[0038] In the context of the present specification, unless expressly provided otherwise, the words “first”, “second”, “third”, etc. have been used as adjectives only for the purpose of allowing for distinction between the nouns that they modify from one another, and not for the purpose of describing any particular relationship between those nouns.

[0042]

[0039] It must be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0040] As used herein, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.

[0043]

[0041] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0044]

[0042] As used herein, the term “isolated” means the separation from and the removal of other materials. For example, a protein isolated from a legume is separated from the legume as whole and removed from the remainder of the legume.

[0045]

[0043] As used herein, the term “submerged” means to be suspended in solution fully in the liquid phase; below the gas phase. For example, surface fermentation uses the surface of the media but not the volume of; submerged uses the volume of the space.

[0046]

[0044] In one embodiment, the present technology provides a process for producing mushroom mycelium through a biomass fermentation process. The process comprises using pea starch, flour, and protein ingredients as represented in Figure 1. This process leverages and focuses on the unique properties of pea-based substrates to optimize the growth conditions and yield of mushroom mycelium. Although the present technology is being disclosed herein using pea starch, it is to be understood that the present technology is readily adaptable to use substrates derived from grains such as wheat, corn, and rice.

[0047]

[0045] In one embodiment, the fungus is selected from the genus Pleurotus, including but not limited to, Oyster (P. ostreatus) Phoenix Oyster (P. pulminarius), Black King Oyster (P. eryngii), Pink Oyster (P. dejamour), P. citrinopileatus, P. luber-regium. and P. salmoneostramineus. The fungus may also be selected from the genus Agaricus (e.g., Agaricus bisporus, Agaricus blazei), Coprinus (e.g., Coprinus comatus), Lentinula (e.g., Lentinula edodes, also known as Shiitake), Trametes (e.g., Trametes versicolor), Ganoderma (e.g., Ganoderma lucidum, known as Reishi). The fungus types mentioned are examples and should not limit the scope of the present technology. The fungi can be utilized at various lifecycle stages, including spores, mycelium, and mature fruiting bodies.

[0048]

[0046] Non-limiting examples of isolated forms of Pleurotus that may be suitable for the present technology include: Common Oyster Mushroom (Pleurotus ostreatus), King Oyster Mushroom (Pleurotus eryngii), Golden Oyster Mushroom (Pleurotus cilrinopHealus), Pink Oyster Mushroom (Pleurotus djamor), Phoenix Oyster Mushroom (Pleurotus pulmonarius), Branched Oyster Mushroom (Pleurotus cornucopiae), Veiled Oyster Mushroom (Pleurotus dryinus), Abalone Mushroom (Pleurotus cyslidiosus). Ferula Oyster Mushroom (Pleurotus ferulae), Blue Oyster Mushroom (Pleurotus Columbians). Salmon Oyster Mushroom (Pleurotus salmoneoslramineus). Indian Oyster Mushroom (Pleurotus sajor-caju), Cinnamon Cap Mushroom (Pleurotus calyptratus), Florida Oyster Mushroom (Pleurotus florida), Tasty Oyster Mushroom (Pleurotus sapidus), Aspen Oyster Mushroom (Pleurotus populinus), Fragrant Oyster Mushroom (Pleurotus euosmus). Golden Hue Branched Oyster Mushroom (Pleurotus cornucopiae var. cilrinopHealus). and King Tuber Mushroom (Pleurotus tuber-regium).

[0049]

[0047] Non-limiting examples of isolated Hericium species include: Lion's Mane Mushroom (Hericium erinaceus). Bear's Head Tooth Mushroom (Hericium americanum). Bearded Tooth Mushroom (Hericium coraHoides). Comb Tooth Mushroom (Hericium ramosum). Monkey's Head Mushroom (Hericium alpeslre). Pom Pom Mushroom (Hericium abielis), Eastern Coral Tooth Mushroom (Hericium flagellum), Pine-Spike Mushroom (Hericium abielis). Snow Coral Mushroom (Hericium dalhroides), and Hazel Bracket (Hericium circinatum).

[0050]

[0048] Non-limiting examples of isolated Trametes species include: Turkey Tail Mushroom (Trametes versicolor), Violet Toothed Polypore (Trametes pubescens), Cinnamon Bracket (Trametes cinnabarina), Multicolor Gill Polypore (Trametes hirsuta), Trametes suaveolens, Trametes ochracea, Trametes gibbosa, Trametes trogii, Trametes villosa, Trametes meyenii, Trametes lactinea, Trametes elegans, Trametes ljubarskyi, and Trametes menziesii.

[0051]

[0049] Non-limiting examples of isolated Agaricus species include: Button Mushroom (Agaricus bisporus), Horse Mushroom (Agaricus arvensis), Field Mushroom (Agaricus campestris), Prince Mushroom (Agaricus augustus), Almond Mushroom (Agaricus subrufescens), Yellow-staining Mushroom (Agaricus xanthodermas). Wood Mushroom Agaricus silvicola). Scaly Wood Mushroom (Agaricus langei). Garden Mushroom (Agaricus bitorquis), Blewit-Like Agaricus (Agaricus p / acomyces)(iox'c), Blushing Wood Mushroom (Agaricus sHvalicus). Agaricus devoniensis. Snowball Mushroom (Agaricus niveus), Sweet Almond Mushroom (Agaricus dulcidulus), Agaricus bernardii, Agaricus impudicus, Agaricus moelleri, Agaricus phaeolepidotus, Agaricus cupreobrunneus, and Agaricus pseudopratensis.

[0052]

[0050] In one aspect, isolated Agaricus species include edible mushrooms.

[0053]

[0051] In one embodiment, the present technology relates to a fermentation system for the production of mycelium material that is compatible with current legume or grain protein isolate processing that uses fractionation or milling techniques. The system uses waste flour or starch material from the milling.

[0054]

[0052] In one embodiment the fresh mycelium is dried and used as an ingredient in the production of textured vegetable proteins and textured protein products, and other food products as a mushroom ingredient additive in which the protein isolate used in the blend is also sourced from the isolation process.

[0055]

[0053] In one embodiment the fermentation media is supplemented with glucose or dextrose to reach an optimal brix level of between about 2 and about 10, or between about 3 and about 10, or between about 4 and about 10, or between about 5 and about 10.

[0056]

[0054] In one embodiment the fermentation media is supplemented with protein isolate as a nitrogen source.

[0057]

[0055] In one embodiment the fresh mycelium is used as a wet flavour enhancing agent without drying.

[0058]

[0056] In one embodiment the fresh mycelium is used in a meat-mushroom blended product.

[0057] In one embodiment the fresh mycelium is used as a binding agent.

[0059]

[0058] In one embodiment the fresh mycelium is frozen and used in non-animal dairy products.

[0060]

[0059] In one embodiment the fresh mycelium is used as in confectionery and baking applications.

[0061]

[0060] In one embodiment the fresh mycelium is used alone as an alternative protein product.

[0062]

[0061] In one embodiment the fresh mycelium undergoes further extraction to isolate specific bioactive compounds.

[0063]

[0062] In one embodiment the fresh mycelium is used in non-food applications as a gelling agent and bioactive ingredient.

[0064]

[0063] In one embodiment the fresh mycelium is dried and used as a flavour modifying agent.

[0065]

[0064] In one embodiment the fresh mycelium is used as a material for extrusion and creation of textured protein products.

[0066]

[0065] Figure 2 outlines one embodiment of the process of the present technology wherein the steps of the process are disclosed below.

[0067] Media preparation

[0068]

[0066] The media components (1-4) necessary for mycelium growth, such as carbon (e.g., sugars and / or feedstocks) and nitrogen (e.g., ammonia or ammonium salts) are sourced and stored. Media additives such as micronutrients and pH adjustment chemicals to optimize growth conditions (5,6) may also be added as media components. The media components are transported to a tank using conveyors or pumps and are then mixed with water in a tank. Media preparation is completed once the mixture is homogeneous and the pH is adjusted. The media is then pumped out of the tank and into the bioreactor.

[0067] The pea-based ingredients are mixed with water and sterilized at 121°C and 15 psi to create a nutrient-rich fermentation medium. In some embodiments the pH is adjusted to about 5, about 6, about 7, or about 8. In some embodiments the fermentation media is pasteurized at 80°C for 60 minutes. In some embodiments the fermentation media undergoes a cold alkali wash to pasteurize the material before inoculation.

[0069]

[0068] In some embodiments the pea starch is present in an amount of between 2 and 30% w / v. In some embodiments, the pea flour is present in an amount of between 1 and 50% w / v. In some embodiments, the pea protein is present in an amount of between 1 and 50% w / v. In some embodiments, additional nutrients may be present in the composition, such as, but not limited to: Yeast extract, vitamins, carbon, and minerals are used minimally to ensure C:N ratio is only a limiting agent. Table 1 provides an example of a pea flour composition.

[0070] Table 1 : Pea flour composition

[0071] Sterilization

[0072]

[0069] Sterilization of the reactor and media (7-9) is important to ensure optimal mycelium growth and ensure product quality. Steam is produced in a boiler and used to sterilize the media and bioreactor. Heating occurs at 121°C for 15 to 90 minutes. The heat transfer between steam and bioreactor happens by moving the steam to the bioreactor jacket, a secondary wall outside the bioreactor, allowing for heat exchange without direct contact with the inside of the bioreactor. This technique is known as indirect steam injection. The steam condensate is collected and recycled to the boiler.

[0073] Fermentation

[0074]

[0070] Once the system has cooled after sterilization, mycelium is added to the bioreactor, where it is inoculated with mycelium (12,13). A pure culture of the desired mushroom species (e.g., Agaricus bisporus, Pleurotus ostreatus) is prepared. The prepared inoculum is introduced into the sterilized medium under aseptic conditions. During fermentation, the bioreactor is maintained at specific conditions (temperature, aeration, and agitation) to facilitate optimal mycelium growth. The mycelium consumes the nutrients in the media and proliferates, forming biomass.

[0075]

[0071] Throughout the fermentation process (10), air is continuously supplied to the bioreactor to ensure the mycelium receives enough oxygen. The air is supplied using a compressor. After a predetermined period, the fermentation is completed and the mycelium is ready for downstream processing. The fermentation vessel may comprise a sparger and continuous stirrer apparatus and impeller to reduce shear force and increase oxygen and material transfer. The fermentation is carried out under controlled conditions (temperature: 25-30°C, pH: 6-7, aeration: 1-2 vvm, or 30%) for 1-10 days, depending on the mushroom species.

[0076] RNA reduction

[0077]

[0072] The biomass then undergoes RNA reduction (14-17), lowering the amount of RNA to less than 2 w / w%, as per health and safety guidelines. This process enhances the nutritional profile and ensures safety of the product. This is achieved by heating the reactor using steam in a process similar to sterilization. Heating occurs at 60, 70, 80, or 90 degrees, for 30, 40, 50, 60, 70, or 80 minutes.

[0078] Dewatering

[0079]

[0073] At this point the biomass is safe for consumption but comprises too much water for the desired application. Excess water is removed (18) from the mycelium using centrifugation, an efficient, scalable and controllable method of removing moisture from the mycelium, resulting in a concentrated, solid product. The mushroom mycelium is separated from the fermentation broth (19). The separated water, now considered wastewater, is collected and treated or disposed of according to environmental regulations (20). The dewatered mycelium is now ready for packaging, further processing, or direct use in various applications (FIGs. 3 and 4). Centrifuged, washed, and dried or frozen for further use. Table 2 outlines the dry biomass for three different mushroom species on different media. Table 2. Dry biomass of three different mushroom species on different media.

[0080]

[0074] The process disclosed herein offers a sustainable and efficient approach to producing mushroom mycelium, suitable for applications in food production, pharmaceuticals, and other biotechnological industries. The use of pea-based ingredients as a fermentation substrate provides an economically viable alternative to traditional methods.

[0081] EXAMPLES

[0082]

[0075] The examples below are given so as to illustrate the practice of various embodiments of the present disclosure. They are not intended to limit or define the entire scope of this disclosure. It should be appreciated that the disclosure is not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the disclosure as defined in the appended embodiments.

[0083] Example 1 - Production of mushroom mycelium

[0084]

[0076] Starch and protein from the Fabaceae family and the media were tested using varying levels of pea starch and supplemented dextrose (Table 3). Ranges for media included A. 30 g / L pea starch + 10 g / L dextrose; B. 20 g / L pea starch + 20 g / L dextrose; or C. 30 g / L pea starch + 0 g / L dextrose. The media was sterilized using steam and inoculated with P. Ostreatus mushroom strain. The media and mushroom were agitated using an impeller and aerated. The fermentation was allowed to run for 5 to 7 days before material was harvested. Table 3 indicates the wet weight for control. Pea starch at varying levels of inclusion as described above Media A, Media B, Media C. group was higher than that of the treatment groups. Table 3: Pea starch submerged liquid fermentation

[0085]

[0077] Potato starch and faba bean (Vicia) starch were compared at different concentrations (FIG 6). The data analysis was conducted using Minitab software. Comparison of different media concentrations revealed that the mycelial wet weight was highest at 5% concentration compared to 1% and 3%. A comparison of different Faba bean concentrations in the control treatment showed that mycelial wet weight was highest at 5% and lowest at 1%. An increasing trend was observed as the concentration increased from 1% to 5%. We found that increasing the concentration of both Faba bean and potato starch leads to an increase in Brix. The Brix value of the media was highest at the 5% concentration for both potato starch and Faba bean compared to the lower concentration.

[0086]

[0078] By increasing the concentration of both potato starch and faba bean, the mycelial dry weight increased, indicating an overall upward trend across all treatments and the control. This increase was statistically significant within each treatment. This implies that the dry weight of mycelium grown on Faba bean was significantly higher than that on potato starch at all tested concentrations. The potato starch concentration at 5% resulted in a significantly higher mycelium dry weight compared to the other concentrations.

[0079] Comparing different control (Faba bean) concentration shows that the dry weight of 5% Faba bean is significantly higher than other treatments. The pH of the Faba group is significantly higher than that of each experimental group across all concentrations. Additionally, the pH of the Faba bean remains around 6 at all concentrations, while the pH of the potato starch is consistently around 5 across all concentrations. There is no significant difference between the pH of the 3% and 5% potato starch concentrations, but the 1% potato starch concentration exhibits the lowest pH. A comparison of the control treatments indicates no significant difference in pH between the 1% and 5% Faba bean concentrations, both of which exhibited the highest values. In contrast, the 3% concentration showed the lowest pH.

[0087]

[0080] Growth was assessed using 100 ml shake flasks on saccharified faba bean starch inoculated with P. ostreatus. Microscopic observations indicated the presence of mycelia. Fewer starch granules were observed following saccharification, an indication that saccharification was effective and the sugars released were being used as substrates for growth. While mycelial growth was still visible under the microscope, visually dense clusters of mycelia could not be observed in the flask, showing that high-density inoculum is needed for effective colonization. To determine if high-density inoculum and non-saccharified starch would encourage effective colonization, faba starch was used as a substrate. The texture of the culture appeared denser than with low-density inoculum. Microscopic observations indicated extensive mycelia growth and fewer starch granules as growth proceeded (FIG 7). The starch substrates supported better mycelial growth upon saccharification. Therefore, for the 1 L working volumes for faba, wheat and pea starches saccharification was conducted and microscopic assessment showed variations in composition (FIG 8). After saccharification, flasks were inoculated with high-density inoculum. As suspected, the pea starch behaves similarly to the faba bean starch.

[0088]

[0081] Growth colony diameter was used to monitor growth dynamics in saccharified faba starch agar plates compared to molasses agar plates showed that mycelium grew much slower on saccharified faba starch (FIG 9).

[0089]

[0082] The color of the mycelium for each treatment and replication was matched using the RHS Color Chart, and the results are presented in the table below and visibly in (FIG 10). The faba bean starch becomes noticeably darker following inoculation. Table 4 shows the results of a colour analysis of mycelium grown on representative starch material.

[0090] Table 4: Results of Colour analysis

[0091] Example 2 - Extraction of Genomic DNA and amplification of fungal barcode loci

[0092]

[0083] Extraction of genomic DNA from the mycelium produced in Example 1 and the amplification of fungal barcode loci (ITS, EFla, RPB2) to support species confirmation were performed. Using the ITS1 / ITS4 primer pair, a gradient PCR established an optimal annealing temperature of 60°C, and -750 bp amplicons were obtained across all samples, confirming successful fungal DNA extraction; gradient optimisations for EFla and RPB2 were completed on representative extracts (Cl, Gl). Next, ITS will be re-amplified with a high-fidelity polymerase at 60 °C, products will be cleaned and submitted for Sanger sequencing, and EFla / RPB2 will be extended across the sample set to provide multilocus confirmation.

[0093]

[0084] The DNA extraction process was adapted from the Qiagen DNeasy Plant Mini Kit, which in its standard protocol employs a TissueLyser II to mechanically disrupt samples via beadbeating. As this instrument was unavailable, a modified approach was implemented using 0.5 mm glass beads and a standard laboratory vortex mixer. Multiple variations of this adapted lysis step were trialled, including the incorporation of a lyticase pretreatment to enzymatically weaken fungal cell walls, adjustments to vortexing duration and intensity, and modifications to the timing of lysis buffer addition. Based on these preliminary trials, a consolidated experimental design was developed to evaluate multiple protocol variants in parallel, to identify the most effective conditions for producing high-quality DNA suitable for PCR amplification. DNA yield and purity for each extract were quantified using NanoDrop spectrophotometry.

[0094]

[0085] For mechanical disruption, 0.10 mL of 0.5 mm glass beads were added to each 1.7 mL microcentrifuge tube, followed by the wet biomass amount specified in the sample matrix (30 mg or 75 mg). In enzyme-assisted variants, samples were pelleted by centrifugation and resuspended in sorbitol-EDTA buffer containing lyticase at the concentration assigned for that sample (5 U / mg or 10 U / mg biomass). Samples undergoing enzymatic pretreatment were incubated at 30°C for 60 minutes to promote spheroplast formation, after which the lyticase solution was removed by centrifugation.

[0095]

[0086] Lysis buffer and RNase A were then added following the kit protocol, and samples were vortexed for either 10 or 15 minutes to achieve bead-bashing. Subsequent steps: including incubation, DNA binding to the silica column, washing, and elution — were performed according to the manufacturer’s instructions. DNA yield and purity were quantified using a NanoDrop spectrophotometer, with A260 / 280 and A260 / 230 ratios recorded. The complete sample matrix for these extractions is presented in Table 5.

[0096] Table 5: RNA extraction protocol DNA Extraction protocol and sample parameters used for mycelium DNA isolation

[0097]

[0087] Polymerase chain reaction (PCR) was employed to amplify fungal barcode regions from the extracted DNA for species identification. Three primer sets were selected to target distinct loci: ITS1 / ITS4 for the internal transcribed spacer (ITS) region, EF595F / EF1160R for the EFla region, and two RPB2 primer pairs (fRPB2-5F / bRPB2-7.1R and bRPB2-6.9F / bRPB2-l 1R1). Gradient PCRs were first performed on representative DNA extracts (samples Cl and Gl) to determine the optimal annealing temperature for each primer set. Once these optimal temperatures are established, full-scale PCRs will be conducted across all extracted DNA samples. To date, a full- scale PCR using the ITS1 / ITS4 primer pair has been completed on all extracted samples.

[0098]

[0088] PCR products were analyzed by agarose gel electrophoresis to assess amplification success and confirm amplicon sizes. The presence of a single, distinct band at the expected size was taken as evidence of specific amplification, whereas smears or additional bands were interpreted as indicators of non-specific amplification or template degradation.

[0099]

[0089] In one embodiment the methods of PCR amplifications were performed (FIG. 11) in 25 pL reaction volumes containing 12.5 pL of 2* Taq FroggaMix, 0.5 pL of each primer (10 pM), 1 pL of template DNA (5-20 ng), and nuclease-free water to volume. For optimization experiments, annealing temperatures were varied in a gradient (e.g., 49-61 °C for ITS primers) to determine the condition yielding the highest specificity and product yield. The cycling program for Taq polymerase comprised an initial denaturation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at the designated gradient temperature for 30 seconds, and extension at 72°C for 45-120 seconds depending on the expected amplicon size. A final extension was carried out at 72°C for 2-10 minutes.

[0100]

[0090] Following amplification, 5 pL of each PCR product was combined with loading dye and resolved on 1.5% (w / v) agarose gels prepared in l x TAE buffer. A 1000 bp DNA ladder was included in each gel for size calibration. Electrophoresis was conducted at 90-110 V for 30-45 minutes, after which gels were stained with a DNA-safe dye and visualized under blue light or UV illumination. Gel images were captured and archived for subsequent analysis and documentation.

[0101]

[0091] Gradient amplification of the internal transcribed spacer using the ITS1 / ITS4 primer pair produced strong, well-resolved bands across the temperature range tested on samples Cl and G1 (Figure 1). An annealing temperature of 60 °C was selected as optimal based on band intensity and specificity. Using this condition, amplification of the ITS region was successful for all extracted samples, yielding discrete products at the expected size range. Because ITS1 / ITS4 targets the fungal ribosomal internal transcribed spacer, the consistent recovery of amplicons from all extracts supports that the extractions contained fungal genomic DNA suitable for downstream identification.

[0102]

[0092] The system is designed for > 1 000 000 L of fermentation and downstream processing capacity. In this example, the facility is co-located with a legume fractionation facility.

[0103]

[0093] All references cited in this specification, and their references, are incorporated by reference herein in their entirety where appropriate for teachings of additional or alternative details, features, and / or technical background.

[0104]

[0094] While the disclosure has been particularly shown and described with reference to particular embodiments, it will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

[0105] REFERENCES

[0106] A. Martin, Antonio M. Martin, & Antonio M. Martin. (1992). Study of the Growth and Biomass Composition of the Edible Mushroom Pleurotus Ostreatus. Developments in Food Science. https: / / doi.org / 10.1016 / b978-Q-444-88834-l.50024-7

[0107] Avniel, Y. C., Kozubal, M. A., & MACUR, R. E. (2024). Filamentous fungal biomats, methods of their production and methods of their use (Patent AU2024200784A1). https: / / patents. google. com / patent / AU2024200784Al / en?q=(fermentation+AND+mycoprotein+

[0108] AND+Mushroom+AND+protein+AND+2024)&oq=fermentation+AND+mycoprotein+AND+M ushroom+AND+protein+AND+2024&sort=new&clustered=true

[0109] Barta, D.-G., Simion, I., Tiuc, A.-E., & Vasile, O. (2024). Mycelium-based composites as a sustainable solution for waste management and circular economy. Materials (Basel), 17(2), 404.

[0110] De Cianni, R., Pippinato, L., & Mancuso, T. (2023). A systematic review on drivers influencing consumption of edible mushrooms and innovative mushroom-containing products. Appetite, 182, 106454. https: / / doi.Org / 10.1016 / i .appet.2023.106454

[0111] Du, X., Muniz, A., Sissons, J., Wang, W., & Juma, S. (2021). Consumer acceptance of egg white partially substituted with mushrooms and mushroom-egg white flavor pairing. Food Science & Nutrition, 9(3), 1410-1421. https: / / doi.org / 10.1002 / fsn3.21Q5

[0112] Dulay, R. M. R., Dulay, R. M. R., Ray, K., Ray, K., Hou, C. T., & Hou, C. T. (2015).

[0113] Optimization of liquid culture conditions of Philippine wild edible mushrooms as potential source of bioactive lipids. Biocatalysis and Agricultural Biotechnology. https: / / doi.Org / 10.1016 / j.bcab.2015.04.003

[0114] Geethanjali, P. A., Gowtham, H. G., & Jayashankar, M. (2020). Biodegradation potential of indigenous litter dwelling ligninolytic fungi on agricultural wastes. Bulletin of the National Research Centre, 44, 173.

[0115] Guinard, J.-X., Myrdal Miller, A., Mills, K., Wong, T., Lee, S. M., Sirimuangmoon, C., Schaefer, S. E., & Drescher, G. (2016). Consumer acceptance of dishes in which beef has been partially substituted with mushrooms and sodium has been reduced. Appetite, 105, 449-459. https: / / doi.Org / 10.1016 / i.appet.2016.06.018

[0116] Hadar, Y., & Cohen-Arazi, E. (1986). Chemical Composition of the Edible Mushroom Pleurotus ostreatus Produced by Fermentation. Applied and Environmental Microbiology, 51(6), 1352— 1354. https: / / doi.Org / 10.1128 / aem.51.6.1352-1354.1986

[0117] Hashem, M., & Darwish, S. M. I. (2010). Production of bioethanol and associated by-products from potato starch residue stream by Saccharomyces cerevisiae. Biomass and Bioenergy, 34(7), 953-959.

[0118] Hesham Ali El-Enshasy, Hesham A. El-Enshasy, Ayman S. Daba, A. Daba, M. El-Demella, M.

[0119] El-Demella, M. El-Demellawy, A. Ibrahim, A. Ibrahim, Ahmed S. Ibrahim, Ahmed B. Ibrahim, Ahmed Ibrahim, Salah El Sayed, S. El-Sayed, S. El Sayed, I. El-Badry, & I. El-Badry. (2010). Bioprocess development for large scale production of anticancer exo-polysaccharide by Pleurotus ostreatus in submerged culture. Journal of Applied Sciences. https: / / doi.org / 10.3923 / jas.201Q.2523.2529

[0120] Kamthan, R., & Tiwari, I. (2017). Agricultural wastes. Potential substrates for mushroom cultivation. European Experimental Biology, 7(5), 31.

[0121] Raman, J., Jang, K.-Y., Oh, Y.-L., Oh, M., Im, J.-H., Lakshmanan, H., & Sabaratnam, V. (2021). Cultivation and Nutritional Value of Prominent Pleurotus spp.: An Overview. Mycobiology, 49(f), 1-14. https: / / doi.org / 10.1080 / 12298093.202Q.1835142

[0122] Singh, N., Isono, N., Srichuwong, S., Noda, T., & Nishinari, K. (2008). Structural, thermal and viscoelastic properties of potato starches. Food Hydrocolloids, 22(6), 979-988.

[0123] SONI, B. K., Kelly, B. J., Langan, J. P., DAVIS, H., & Hahn, A. D. (2020). Methods for the production and use of myceliated high protein food compositions (United States Patent US10806101B2). https: / / patents.google.com / patent / US 10806101B2 / en?q=(production+of+mushroom+protein+fer mentation+liquid+state)&scholar&oq=production+of+mushroom+protein+using+fermentation+l iquid+state

[0124] Souza Filho, P. F., Nair, R. B., Andersson, D., Lennartsson, P. R., & Taherzadeh, M. J. (2018). Vegan-mycoprotein concentrate from pea-processing industry byproduct using edible filamentous fungi. Fungal Biology and Biotechnology, 5(1), 5. https: / / doi.Org / 10.l 186 / s40694- 018-0050-9

[0125] Thanh, L. Q. (2020). Process for producing high protein biomass from starch-containing cereal materials by yeast strains (United States Patent US20200109428A1). https: / / patents.google.com / patent / US20200109428Al / en?q=(mycoprotein+AND+process+AND +fermentation+AND+mycelium)&country=US

[0126] W. Manu-Tawiah, W. Manu-Tawiah, A. Martin, & A.M. Martin. (1987). Chemical composition of Pleurotus ostreatus mycelial biomass. Food Microbiology. https: / / doi.org / 10.1016 / sQ740- 0020(87)80004-7 )+(Food)+Beyond+Meat&after=publication:20240101&oq=(submerged+fermentation)+(Mushro om)+(Food)+after:publication:20240101+Beyond+Meat

Claims

CLAIMS:

1. A process for production of mushroom mycelium, the process comprising: a) fermenting a fermentation composition, the fermentation composition comprising: i) at least one carbohydrate source; and ii) at least one protein source, to obtain a fermentation media; b) sterilizing the fermentation media to obtain a sterilized medium; c) inoculating the sterilized medium with a pure culture of mushroom species to obtain an inoculated medium; d) fermenting the inoculated medium under controlled conditions; and e) harvesting the mushroom mycelium from the fermented inoculated medium.

2. The process of claim 1, wherein the process is a submerged liquid state fermentation process.

3. The process of claim 1 or 2, wherein at least one carbohydrate source is selected from legume-derived starches.

4. The process of claim 3, wherein at least one carbohydrate source is pea starch.

5. The process of claim 3, wherein the at least one carbohydrate source is bean starch6. The process of claim 3, wherein the at least one carbohydrate source is pea flour.

7. The process of claim 3, wherein the at least on carbohydrate source is bean flour.

8. The process of any one of claimsl to 7, wherein at least one protein source is selected from plant-derived proteins.

9. The process of claim 8, wherein at least one protein source is a legume protein.

10. The process of claim 9, wherein at least one protein source is pea protein.

11. The process of claim 9, wherein the at least one protein source is bean protein.

12. The process of any one of claims 1 to 11, further comprising adjusting the pH.

13. The process of claim 12, wherein the pH is adjusted with NaOH, H2SO4, organic acids, or buffering agents.

14. The process of any one of claims 1 to 13, further comprising adjusting brix through enzyme saccharification.

15. The process of claim 14, wherein the brix is adjusted through physical, enzymatic, or chemical means,16. The process of claim 15, wherein the brix is measured at a temperature of between about 20°C and about 121°C.

17. The process of claim 15, wherein the brix is measured at a pressure of between about atmospheric and about 15 psi.

18. The process of any one of claims 1 to 17, wherein step c) is performed using liquid culture, solid culture, or spore suspension.

19. The process of any one of claims 1 to 18, wherein the fermentation is carried out in a bioreactor, a fermenter, or a continuous flow system.

20. The process of any one of claims 1 to 19, wherein step b) is performed at a temperature between about 80°C and about 121°C.

21. The process of any one of claims 1 to 20, wherein step e) is performed using disk stack centrifugation or screw press or decanter.

22. The process of any one of claims 1 to 21, further comprising drying the harvested mushroom mycelium using infrared microwave drying.

23. The process of any one of claims 1 to 22, wherein the at least one carbohydrate source is present at a concentration of between about 1% and about 30% w / v of the total weight of the fermentation composition.

24. The process of any one of claims 1 to 22, wherein the at least one carbohydrate source is present at a concentration of between about 1% and about 15% w / v of the total weight of the fermentation composition.

25. The process of any one of claims 1 to 24, wherein the at least one protein source is present at a concentration of between about 1% and about 30% w / v of the total weight of the fermentation composition.

26. The process of any one of claims 1 to 24, wherein the at least one protein source is present at a concentration of between about 1% and about 15% w / v of the total weight of the fermentation composition.

27. The process of any one of claims 1 to 24, wherein the at least one protein source is present at a concentration of between about 1% and about 10% w / v of the total weight of the fermentation composition.

28. The process of any one of claims 1 to 24, wherein the at least one protein source is present at a concentration of between about 1% and about 3% w / v of the total weight of the fermentation composition.

29. The process of any one of claims 1 to 28, wherein the fermentation composition further comprises a yeast extract, vitamins, minerals or a combination thereof.

30. The process of any one of claims 1 to 29, further comprising adjusting fermentation conditions throughout the process.

31. The process of claim 30, wherein the fermentation conditions are selected from pH, temperature, and aeration.

32. The process of any one of claims 1 to 31, wherein the fermentation is carried out for between about 5 and about 14 days.

33. The process of any one of claims 1 to 32, wherein the fermentation is carried out for between about 5 and about 10 days.

34. The process of any one of claims 1 to 33, wherein the fermentation is carried out at scales of between about 1 and about 1 000 000 L of production capacity.

Images