Rapid isolation, culture method, and usefulness of truffle fungus (TRUFFLE FUNGI)

Abstract

A method for agricultural cultivation, domestication, or cultivation of truffle fungi, including a method for isolation and culture to produce pure truffle fungi or mini-truffles, typically species of the genus Tuber or Iamai. Examples of truffles include Tuber melanosporum, Tuber magnatum, Tuber aestivum, Tuber uncinatum, Tuber borchii, species of the genus Imaia, Tuber macrosporum, Tuber gibbosum, Tuber oregonense, and Tuberlyonii. The isolated pure truffles may then be cultured on a nutrient substrate, which may be, for example, fruits, nuts, grains, or parts thereof, to produce truffle-flavored foods. Non-limiting examples of such substrates include rye, barley, lentils, wheat, rice, soybeans, pecans, hazelnuts, pine nuts, walnuts, coffee beans, mustard, cocoa, sesame, sunflowers, grapes, blackberries, blueberries, cherries, kiwis, mangoes, raspberries, and huckleberries.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 455,304, filed Mar. 29, 2023, by Gary A. Strobel, entitled "Rapid Isolation, Cultivation and Novel Utilities of Truffle fungi (Tuber spp.)", the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials A computer-readable nucleotide / amino acid sequence list, submitted simultaneously with this specification and identified as follows, is incorporated herein by reference in its entirety: one XML file of 7,511 bytes, designated "22630.002US-PAT", created on Mar. 28, 2024.

[0003] The present disclosure relates to methods for agricultural cultivation or domestication of truffle fungi. In some embodiments, the present disclosure relates to isolation and culturing methods for producing pure truffle fungi.

Background Art

[0004] Truffle fruiting bodies have been highly sought after for thousands of years, their flavor prized and making them extremely expensive in any market. Most commonly, they are collected from the soil surrounding a particular tree species, and specific dogs and trained pigs are used to find the precious truffles. Once found, the truffles are dug up from the soil and transported for commercial distribution. The truffles themselves range in diameter from about 1 cm to over 6-8 cm, have a rough and uneven surface, and can be used to enhance the flavor of any food. Truffle cultivation has traditionally been carried out by assigning land to a particular type of tree (usually oak, but other types including pecan and hazelnut), and then inoculating the roots of the planted tree with harvested truffle material containing spores and mycelial fragments that invade the young roots. Ultimately, the infected roots begin to support the growth of the fungus attached to them, causing root blistering that includes truffle fungus, plant tissue, and numerous microorganisms associated with the soil. The fungal growth begins as a small sphere composed of enlarged root growths, eventually growing into a truffle of normal size. It takes 5 to 10 years for the truffle to fully develop.

[0005] In addition to the demand for truffles themselves, the long process required for truffle production and their short shelf life after being dug from the soil also contribute to the high price of this popular culinary delicacy. Because truffle production is costly and time-consuming, truffles can be expensive (for example, black truffles can cost $700-$1,000 or more per pound, and white truffles can cost $3,000 or more per pound).

[0006] To meet market demand, there is a need for methods of agriculturally cultivating truffles. [Overview of the project] [Problems that the invention aims to solve]

[0007] Truffle cultivation is a long and complex process, which contributes to the high price of this culinary delicacy. Methods for cultivating and farming truffles would help lower the costs associated with truffle production by increasing their availability on the market without relying on traditional harvesting methods. [Means for solving the problem]

[0008] This disclosure relates to methods for isolating and cultivating truffle species as an alternative to the domestication of major truffle species. This disclosure includes methods for cultivating commercially valuable truffles without the need for soil, thereby reducing the time required for truffle production. This disclosure includes methods for isolating pure cultures of ascomycetes that can be cultivated on plates, such methods providing an alternative to obtaining inoculum free from soil-derived contamination.

[0009] Some embodiments of the present disclosure include a method for cultivating truffles, the method comprising the steps of: obtaining a spore-producing fruiting body of an ascomycete from soil; scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample; placing the fruiting body sample on a first surface containing a vegetative substrate, wherein the vegetative substrate comprises monosaccharides, plant-derived isolates, and agar; incubating the fruiting body sample on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed, wherein the presence of mycelium indicates new fungal growth; and transferring the new fungal growth to a second surface containing a vegetative substrate to obtain a pure culture of the ascomycete.

[0010] Some embodiments of the present disclosure include a method for cultivating truffles without soil, the method comprising the steps of: obtaining a spore-producing fruiting body of an ascomycete from soil; collecting a fruiting body sample by scraping the inside of the spore-producing fruiting body of the ascomycete; placing the fruiting body sample on a first surface containing a vegetative substrate; incubating the fruiting body sample on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed; and transferring the new fungal growth to a second surface containing a vegetative substrate to obtain a pure culture of the ascomycete. The presence of mycelium indicates new fungal growth. In some embodiments of the present disclosure, the vegetative substrate includes monosaccharides, plant-derived isolates, and agar.

[0011] In some aspects of this disclosure, ascomycetes include species of the genera Tuber and Iamai. In another aspect of this disclosure, ascomycetes are selected from the group consisting of Tuber melanosporum, Tuber magnatum, Tuber aestivum, Tuber uncinatum, Tuber borchii, species of the genera Imaia, Tuber macrosporum, Tuber gibbosum, Tuber oregonense, and Tuber lyonii (also known as Tuber Texense). In yet another aspect of this disclosure, plant-derived isolates include fruits, nuts, grains, or parts thereof.

[0012] In some embodiments of the present disclosure, the antimicrobial volatile mixture is derived from volatile organic compounds produced by organisms of the genus Muscodor. In some embodiments, the antimicrobial volatile mixture comprises at least one of the following: methyl 2-methylpropanoate, ethanol, 2-methylpropyl acetate, 2-methylpropyl 2-methylpropanoate, 2-methyl-1-propanol, (E)-2-methyl-2-butenal, 3-methyl-1-butanol acetate, 2-methylbutyl 2-methylpropanoate, 3-methyl-1-butanol, 2-methylpropanoic acid or isobutyric acid, and 2-phenylethyl acetate.

[0013] In some embodiments, the nutrient substrate comprises potato modextrose agar. Other nutrient substrates may comprise pecan agar. In some embodiments of this disclosure, the second nutrient substrate may comprise a liquid. In some embodiments, the nutrient substrate may comprise potato modextrose broth.

[0014] Another embodiment of the present disclosure includes a method for cultivating miniature truffles. Some embodiments include obtaining a spore-producing fruiting body of an ascomycete from soil; scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample; placing the fruiting body sample on a first surface containing a vegetative substrate; incubating the fruiting body sample on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed; transferring new fungal growths to a second surface containing a vegetative substrate to obtain a pure culture of the ascomycete; incubating the pure culture with a fruit, nut, grain, or a portion thereof for at least one week to prepare an inoculated plant sample; and placing the inoculated plant sample on a third surface containing a vegetative substrate to produce miniature truffles. In some embodiments, the volatile organic compounds include compounds produced by organisms of the genus Muscodor, such as Muscodor crispans. The nutritional substrate may include, among several non-limiting examples, dextrose agar, potato dextrose broth, and pecan agar. In yet another embodiment of the present disclosure, the third surface may include pecan agar.

[0015] In yet another embodiment, a method for cultivating mini-truffles may further include: incubating a pure culture with fruits, nuts, grains, or portions thereof for at least two weeks to prepare an inoculated plant sample; immersing the inoculated plant sample in a sugar solution for at least one week to prepare an inoculated sugar solution; and incubating the inoculated sugar solution on a third surface containing a nutrient substrate to produce mini-truffles. In some non-limiting examples, the third surface nutrient substrate includes potato dextrose agar or pecan agar. In some non-limiting aspects of the present disclosure, the sugar solution includes honey or maple syrup. Some non-exclusive examples of fruits, nuts, grains, or parts thereof include rye, barley, lentils, wheat, rice, soybeans, pecans, hazelnuts, pine nuts, walnuts, coffee beans, mustard, cocoa, sesame, sunflowers, grapes, blackberries, blueberries, cherries, kiwis, mangoes, raspberries, and huckleberries.

[0016] In yet another embodiment, a method for producing truffle-flavored food is disclosed herein. This method comprises incubating a pure culture of ascomycetes with fruits, nuts, grains, or portions thereof for at least one week to produce an inoculated plant sample, and processing the inoculated plant sample into a food. In one embodiment of this embodiment, the processing step of this method comprises at least one of grinding the inoculated plant sample into a powder and making a mash using the inoculated plant sample. In another embodiment of this disclosure, the powder or mash is mixed with a food. In some non-limiting examples of food, the powder or mash is used to make salts, oils, dressings, spreads, and beverages.

[0017] Another embodiment disclosed herein includes a method for preserving pure cultures of ascomycetes. In some embodiments, the method includes providing sterile water, adding pure cultures of ascomycetes to the sterile water to prepare an inoculated solution, and preserving the inoculated solution under controlled temperature conditions of 1 to 5°C. In another embodiment, the method for preserving pure cultures of ascomycetes includes providing a sterile medium selected from the group consisting of nuts or portions thereof and grains or portions thereof, inoculating pure cultures of ascomycetes into the sterile medium to prepare a group of ascomycetes seeds, and preserving the group of ascomycetes seeds under controlled temperature conditions. In some embodiments of the disclosure, the controlled temperature conditions are -80°C to -20°C. In some other embodiments of the disclosure, the controlled temperature conditions are 20°C to 25°C, and the method further includes a step of drying the group of ascomycetes seeds.

[0018] In yet another embodiment, the Disclosure includes a method for cultivating truffles of known species. The method includes obtaining spore-producing fruiting bodies of ascomycetes from soil; collecting fruiting body samples by scraping the inside of the spore-producing fruiting bodies of ascomycetes; placing the fruiting body samples on a first surface containing a vegetative substrate, wherein the vegetative substrate comprises monosaccharides, plant-derived isolates, and agar; incubating the fruiting body samples on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed, the presence of mycelium indicates new fungal growth; transferring the new fungal growth to a second surface containing a vegetative substrate and a sterile medium selected from the group consisting of nuts or parts thereof and grains or parts thereof to obtain an inoculum; mixing sterile water and the inoculum in a volume ratio of 4 parts water to 1 part inoculum to prepare a suspension; mixing the suspension with a soil mixture to prepare a fungal soil mixture; and applying the fungal soil mixture to the roots or parts thereof of a plant. In some embodiments, the fungal soil mixture is air-dried and stored at 3°C. In other embodiments, the fungal soil mixture is stored at -20°C and -80°C. This method may further include mixing the fungal soil mixture with soil from an area where the roots of a host tree are growing and inoculating it with a pure culture of ascomycetes.

[0019] In additional embodiments, truffle cultivation compositions are disclosed herein. These compositions comprise grains and mycelium derived from truffle species. In some embodiments of this disclosure, the truffle species comprises at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and the grains are inoculated with the truffle species.

[0020] The foregoing and other aspects, features, and advantages will be apparent from the specification and drawings, and from the claims, if included.

[0021] This patent or patent application includes at least one color drawing. A copy of the published patent or patent application containing the color drawing(s) may be provided by the Japan Patent Office upon request and payment of the prescribed fee.

Brief Description of the Drawings

[0022] [Figure 1] An image of a freshly harvested white truffle Tuber magnatum, representing the typical appearance of an intact truffle, and also showing a newly cut portion of the truffle (the truffle in the upper right corner of the image; white arrow) for sampling and culturing. [Figure 2] An image display of the distribution of freshly cut truffle fragments on the surface of potato dextrose agar (PDA), showing the state where a plastic center well containing a B-23 mixture is placed on the plate. The plate is then sealed with parafilm (registered trademark) tape. [Figure 3A] An image showing the microscopic structure of black truffle fungus. It is a drawing of a conidiophore fascicle of the fungus Tuber melanosporum. The diameter of each spore is about 3 microns. [Figure 3B] An image showing the microscopic structure of black truffle fungus. It is an enlarged drawing of conidiospore production in the conidiophore fascicle of T. melanosporum. The diameter of the spores is 3 - 4 microns. [Figure 3C] An image showing the microscopic structure of black truffle fungus. It is an image of a scanning electron micrograph of a 6-week-old black truffle culture (Tuber melanosporum). This micrograph shows a conidiophore fascicle consisting of two or more conidiogenous conidiophores that are intertwined and mainly produce conidia along the entire length. The conidia germinate randomly from the surface of the conidiophores (see the inset photo showing the germ of the spore at the base of the large conidiophore). The average length of the spores is 2.5 - 3.0 μm, and the diameter of the germ is about 0.5 μm. [Figure 4]Image showing the production of mini-truffles on PDA by an isolated culture of T. melanosporum, which was stored in honey for 6 days and then a small part of the honey was plated 5 times on a new PDA plate and incubated at 22 °C for 10 months. The dissected truffle had the same structure as a large mature truffle harvested in the field. [Figure 5] Image showing a scanning electron micrograph of conidia (conidiospores) of T. magnatum, showing some single cells (3.0 μm) and some larger two-celled (6.7 μm). The end of the conidiophore with the detachment scar is shown in the inset photograph. [Figure 6] Image showing arthrospores of Tuber aestivum (summer truffle) photographed by a scanning electron microscope, showing spores with a width of 3.5 μm and a length of 5.2 μm. [Figure 7] Image showing the hyphae and conidia of Tuber borchii viewed by a scanning electron microscope. The spores have an average width of 4 μm and a length of 5.6 μm, and there are several longer ones. [Figure 8] Image showing a scanning electron micrograph of the spores and hyphae of Tuber uncinatum. The shape and size of the spores are diverse, with an average length of 9.2 μm and a diameter of 3.6 μm. A part of the matrix material of the thin film of the mini-truffle is also shown. [Figure 9] Image showing experimentally determining the optimal storage conditions of a rice fermentation product / soil mixture as an inoculum for T. melanosporum on trees. The wet mixtures kept at 3 °C and -20 °C contained neither obvious bacterial colony contamination nor fungal colony contamination, while both the wet preparation and the dry preparation kept at 22 °C showed significant contamination. [Figure 10] Image showing a cross-section of an oak root 2 weeks after inoculation with a T. melanosporum inoculum mixture. The formation of mini-truffles had started, as can be seen from the whiteish mycelial tufts forming on the cross-section of the oak root. [Figure 11]This image shows a more aged culture of the black truffle fungus (T. melanosporum). The grayish hue is the result of numerous conidiophorous bundles producing a large number of conidia. This fungus is growing on sterilized barley seeds. [Figure 12] This image shows rye truffle powder, produced from fermented black truffles on sterilized rye seeds. [Figure 13] This image shows that barley seeds that had been dried and stored for one year were inoculated with T. melanosporum and placed in moist soil for two days, and already showed the formation of new mycelium, conidiophores, and spores (arrows). [Modes for carrying out the invention]

[0023] Detailed aspects and applications of this disclosure will be described below in the following drawings and detailed description of the Technology. Unless otherwise indicated, the words and phrases in this specification and the claims are intended to have meanings that are plain, ordinary, and idiomatic to those skilled in the art in which they apply.

[0024] The following description includes numerous specific details to help you fully understand the various aspects of this disclosure for illustrative purposes. However, those skilled in the art will understand that embodiments of the technology disclosed herein can be implemented without these specific details. Note that there are many different alternative configurations, apparatuses, and techniques to which the disclosed technology may be applied. The full scope of the technology disclosed herein is not limited to the examples described below.

[0025] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates a different meaning. Therefore, for example, a reference to "step" can refer to one or more such steps.

[0026] The terms “exemplary,” “example,” and their various forms are used herein to mean examples, specific examples, or explanatory materials. Any aspect or design described herein as “exemplary” or “example” is not necessarily construed as being preferable or more advantageous than any other aspect or design. Furthermore, examples are provided solely for clarity and understanding and are not intended to limit or restrict in any way the subject matter or relevant portion of this disclosure. It should be understood that countless additional or alternative examples of various ranges could have been presented but have been omitted for the sake of brevity.

[0027] When a range of values ​​is expressed, another embodiment includes one specific value and / or another specific value. Similarly, when a value is expressed as an approximation by using the preceding “approximately,” it will be understood that the specific value forms another embodiment. All ranges are inclusive and combinable.

[0028] Throughout this specification and the claims, the words “comprise” and “contain,” and variations thereof, such as “comprising” and “comprises,” mean “contain, but not limited to,” and are not intended (and shall not) exclude other components.

[0029] Where necessary, detailed embodiments of this disclosure are included herein. It should be understood that the disclosed embodiments are merely examples of the invention and can be embodied in various forms. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but merely as a basis for instructing those skilled in the art on how to use the invention. The following specific examples may provide a deeper understanding of this disclosure; however, these are provided merely as guidance and do not imply any limitation.

[0030] This disclosure may be more readily understood by referring to the following detailed description provided in connection with the accompanying drawings and examples that form part of this disclosure. This disclosure is not limited to specific materials, apparatus, methods, uses, conditions, or parameters described and / or shown herein, and it should be understood that the terms used herein are intended to illustrate specific embodiments only as examples and are not intended to limit the claimed invention. The term “multiple” as used herein means more than one. Where a range of values ​​is expressed, another embodiment includes one specific value and / or other specific values. Similarly, where a value is expressed as an approximation by using the preceding “about,” it should be understood that that specific value forms another embodiment. All ranges are comprehensive and combinable.

[0031] More specifically, the present disclosure, its aspects and embodiments are not limited to the specific material types, components, methods, or other examples disclosed herein. Many additional material types, components, methods, and procedures known in the art are intended for use with the particular embodiments of the present disclosure. Thus, for example, while a particular embodiment is disclosed, such an embodiment and its components may include any parts, models, types, materials, versions, and / or quantities known in the art for such a system and its components, which are consistent with the intended operation.

[0032] This disclosure relates to methods for isolating and culturing truffle species. The truffle species described and utilized include ascomycetes of the genera Tuber and Imaia. The described methods are useful for the cultivation of truffle species by isolation and cultivation. The cultivation of major truffle species has great significance for commercially valuable truffles as it reduces the time required for truffle production. Therefore, a method for cultivating truffles that does not require the use of soil is disclosed. In some embodiments, the method establishes a pure culture of truffle fungi. In some embodiments, the method cultivates mini-truffles. In some embodiments, the method cultivates truffles of known species.

[0033] The disclosed methods provide effective and useful procedures for the successful isolation of truffle fungi. Furthermore, the disclosure also describes methods for successfully preserving pure isolated fungal cultures, reducing the need for constant harvesting from soil. In addition, the methods described herein include cultivating pure truffle fungal cultures on natural and artificial substrates, and using such cultures as root inoculants for propagating truffle fungi in the field. Moreover, the pure isolated cultures obtained by these methods can also be used to create novel food products, among many others, such as beverages, spreads, salad dressings, and flavorings.

[0034] Having each truffle fungus in a pure culture offers numerous advantages, making it possible to utilize them to produce a variety of novel food-related products that retain the truffle flavor after cultivation on specific foods such as grains, nuts, or fruits. It should be noted that, to the best of our knowledge, no organization in the world possesses any of these fungi in a pure culture and uses them to prepare any food product.

[0035] As mentioned earlier, the only truffle products currently available everywhere are those of natural origin, containing truffles themselves. Typically, truffle flakes or flaks are added to dishes as freshly harvested truffles or dried flakes of harvested truffles themselves. The range of flavor is remarkably limited, because this fungus is cultivated and obtained from only one tree source. Here, depending on the conditions and substrate in which the fungus is cultivated, the secondary products that the fungus produces depend on these nutrients available for growth. In other words, having a pure culture of a fungus, such as truffle fungus, allows them to be cultivated on a variety of substrates and under various conditions, which ultimately affects the flavor of the products produced by the fungus because the fungus alters the biochemical components of the host. Taking advantage of this unique characteristic of fungal fermentation, numerous novel and promising foods, as described herein, have been discovered.

[0036] One possible solution to the problems associated with obtaining truffles is the prospect of isolating the fungus and cultivating it simply in a laboratory facility using either liquid or solid fermentation. Such a method would dramatically reduce all costs, especially those associated with the period of field production. This solution is ideal because there is no reason to believe that truffles grown on the surface of tree roots from soil could have any superior taste or nutritional value to truffle fungi cultivated on sterile, known-composition substrates or natural substrates. Commercially, this feat has not been achieved anywhere in the world. One reason why cultivation has not progressed in this way is that these truffle fungal species are unavailable from state or national microbial strain preservation institutions as well as university microbial strain preservation institutions. This is because isolating these fungi in pure culture is extremely difficult. Therefore, if one wishes to use a particular regional strain for cultivation, it is important and necessary to obtain the truffles and then proceed with fungal isolation.

[0037] One of the difficulties in the fungal isolation process is that truffles themselves are surrounded by soil-dwelling bacteria, other fungi, and contaminated water, making isolation difficult, and in most cases impossible. Furthermore, all truffle species grow extremely slowly, and their growth is easily outpaced by other contaminating fungi and bacteria that coexist with the truffles living underground. Successful isolation methods would eliminate all the costs and time spent cultivating and harvesting truffles from inoculated trees in the wild. Truffle fungi, when cultivated on various substrates, can multiply and become available within weeks. These various substrates include natural ones, and each substrate has the potential to offer new products to the market. The potential market for these truffles and their products is enormous. Examples of such products include food and beverage items, among many other possible products.

[0038] A novel and unexpected solution to the problem of truffle fungus isolation is presented in this disclosure. A method demonstrating that the isolation of almost all major truffle species can be achieved relatively quickly is described below. This isolation can be achieved by a culture selection technique involving the use of a special mixture of volatile organic compounds (VOCs) that inhibit the growth of soil microorganisms that contaminate the truffle itself. The special mixture of VOCs is based on VOCs naturally produced by truffle fungi, including alcohols, esters, aldehydes, ketones, aromatic compounds, furans, alkanes, terpenoids, and, most importantly, sulfur-containing compounds. However, mastery of mycological techniques is required to fully and successfully execute this novel procedure. Ultimately, once obtained, individual truffle species can be cultivated on selected natural substrates, including grains, nuts, and berries, to obtain a whole new range of useful, attractive, and flavorful products, each providing a useful and healthy added value to the food market. Such possibilities are a result of having pure cultures of fungi obtained by the method disclosed herein.

[0039] A method for cultivating truffles includes obtaining spore-producing fruiting bodies of ascomycetes from soil, scraping the inside of the spore-producing fruiting bodies to collect fruiting body samples, placing the fruiting body samples on a first surface containing a vegetative substrate, incubating the fruiting body samples on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed, and transferring the new fungal growth to a second surface containing a vegetative substrate to obtain a pure culture of ascomycetes. The presence of mycelium indicates new fungal growth. In some embodiments of this disclosure, the vegetative substrate comprises monosaccharides, plant-derived isolates, and agar. In certain embodiments, the vegetative substrate further comprises at least one protein component, e.g., peptone and / or yeast extract. In some embodiments, the plant-derived isolate is a fruit, nut, grain, or a portion thereof. In certain embodiments, the vegetative substrate is potato dextrose agar. In other embodiments, the vegetative substrate is pecan agar. In some embodiments of the method, the second vegetative substrate comprises a liquid. In some embodiments, the nutritional substrate includes potato modextrose.

[0040] Another embodiment of the present disclosure includes a method for cultivating mini-truffles. In some non-limiting examples, mini-truffles include densely clustered small, round spheres ranging in diameter from 2 mm, which is smaller than the size typically expected for wild truffles. Some embodiments include obtaining a spore-producing fruiting body of an ascomycete from soil; scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample; placing the fruiting body sample on a first surface containing a vegetative substrate; incubating the fruiting body sample on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed; transferring the new fungal growth to a second surface containing a vegetative substrate to obtain a pure culture of the ascomycete; incubating the pure culture with a fruit, nut, grain, or a portion thereof for at least one week to prepare an inoculated plant sample; and placing the inoculated plant sample on a third surface containing a vegetative substrate to produce mini-truffles. The volatile organic compounds include compounds produced by organisms of the genus Muscodor. The nutritional substrates include, among several non-limiting examples, potato modextrose agar, potato modextrose broth, and pecan agar. In yet another embodiment of this disclosure, the third surface is pecan agar.

[0041] In yet another embodiment, a method for cultivating mini-truffles further comprises the steps of: incubating a pure culture with fruit, nuts, grains, or a portion thereof for at least one week to prepare an inoculated plant sample; immersing the inoculated plant sample in a sugar solution for at least one week to prepare an inoculated sugar solution; and incubating the inoculated sugar solution on a third surface containing a nutrient substrate to produce mini-truffles. In some non-limiting examples, the third surface nutrient substrate includes potato dextrose or pecan agar. In some non-limiting aspects of the present disclosure, the sugar solution includes honey or maple syrup. Some non-exclusive examples of fruits, nuts, grains, or parts thereof include rye, barley, lentils, wheat, rice, soybeans, pecans, hazelnuts, pine nuts, walnuts, coffee beans, mustard, cocoa, sesame, sunflowers, grapes, blackberries, blueberries, cherries, kiwis, mangoes, raspberries, and huckleberries.

[0042] In yet another embodiment, the Disclosure includes a method for cultivating truffles of known species. This method includes obtaining spore-producing fruiting bodies of ascomycetes from soil, collecting fruiting body samples by scraping the inside of the spore-producing fruiting bodies of ascomycetes, placing the fruiting body samples on a first surface containing a vegetative substrate, wherein the vegetative substrate comprises monosaccharides, plant-derived isolates, and agar, incubating the fruiting body samples on the vegetative substrate with an antimicrobial volatile mixture until mycelium is formed, the presence of mycelium indicates new fungal growth, incubating, transferring the new fungal growth to a second surface containing a vegetative substrate and a sterile medium selected from the group consisting of nuts or parts thereof and grains or parts thereof, to obtain an inoculum, mixing sterile water and the inoculum in a preferred volume ratio of 4 parts water to 1 part inoculum to prepare a suspension, mixing the suspension with a soil mixture to prepare a fungal soil mixture, and applying the fungal soil mixture to the roots or parts thereof of a plant. In some embodiments, the fungal soil mixture is air-dried and stored at 3°C. In other embodiments, the fungal soil mixture is stored at -20°C and -80°C. This method may further include the step of mixing the fungal soil mixture with soil from an area where the roots of a host tree are growing and inoculating it with a pure culture of ascomycetes. In additional embodiments, a truffle cultivation composition is disclosed herein. This composition comprises grain and mycelium derived from a truffle species. In some embodiments of this disclosure, the truffle species comprises at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and the grain is inoculated with the truffle species.

[0043] Another embodiment disclosed herein includes a method for preserving pure cultures of ascomycetes. In some embodiments, the method comprises providing sterile water, adding pure cultures of ascomycetes to the sterile water to prepare an inoculated solution, and preserving the inoculated solution under controlled temperature conditions of 1 to 5°C. In another embodiment, the method for preserving pure cultures of ascomycetes comprises providing a sterile medium selected from the group consisting of nuts or portions thereof and grains or portions thereof, inoculating pure cultures of ascomycetes into the sterile medium to prepare a group of ascomycete-inoculated seeds, and preserving the group of ascomycete-inoculated seeds under controlled temperature conditions. In some embodiments of the disclosure, the controlled temperature conditions are -80°C to -20°C. In some other embodiments of the disclosure, the controlled temperature conditions are 20°C to 25°C, and the method further comprises drying the group of ascomycete seeds.

[0044] In yet another embodiment, a method for producing truffle-flavored food is disclosed herein. This method comprises incubating a pure culture of ascomycetes with fruits, nuts, grains, or portions thereof for at least two weeks to produce an inoculated plant sample, and processing the inoculated plant sample into a food. In one embodiment of this embodiment, the processing step of this method comprises at least one of drying the product, grinding the inoculated plant sample into a powder, making a moist mash using the inoculated plant sample, and / or using the moist product in combination with several other products, such as honey or maple syrup. In another embodiment of this disclosure, the powder or mash is mixed with a food. In several non-limiting examples of food, the powder or mash is used to make salts, oils, dressings, spreads, and beverages. Cultured truffle fungi, when in a cultured state, can provide useful new food and flavors.

[0045] Mixture of volatile organic compounds In some embodiments of the present disclosure, the antimicrobial volatile mixture is derived from volatile organic compounds produced by organisms of the genus Muscodor.

[0046] Approximately 20 years ago, a new but closely related ascomycete (M. albus) was isolated, and its volatile organic compounds (VOCs) were not only inhibitory but also lethal to many other microorganisms. This fungus produces volatile antibiotics, some of which are identical to or related to those of truffle fungi. Therefore, the concept applied in this disclosure is understood to be that there are fungal species that do not kill themselves or closely related ascomycete (VOC-producing) fungal species, but which may kill or significantly inhibit contaminating microorganisms coexisting with truffles. It seemed likely that a technique could be developed that would easily allow for the substantial decontamination of truffles without killing the truffle-producing fungi, ultimately enabling truffles to grow on basal media. Subsequently, another species of the genus Muscodor was isolated that produced fewer VOCs and exhibited similar activity to M. albus. Thus, the truffle fungus sorting technique is based on the VOCs of a new Muscodor species named Muscodor crispans. This procedure is explained in more detail below.

[0047] The information behind this unique approach is based on the discovery of the novel endophytic fungus Muscodor albus and its related species. Each of these species is known to produce one or more biologically active volatile organic compounds against a variety of targets, from insects to microorganisms. Any artificially prepared mixture of these fungal-produced compounds has the ability to mimic the biological effects of the fungus itself. This organism is one of the most effective biological weapons ever discovered.

[0048] As of 2023, 22 species of the genus Muscodor are known, and several isolates have been reported from various geographical locations around the world. These are continuously utilized for the development of numerous industrial applications. Some areas of industrial application include health, environment, and food, following the agricultural sector. Members of the genus Muscodor are among the most well-studied fungi in terms of their gaseous chemicals (VOCs), which have a very wide range of direct and indirect applications. All isolates have been found as endophytes, and in most cases, each possesses antimicrobial properties at the VOC stage when using well-established bioassay systems. Furthermore, the chemical properties of VOC mixtures are unique to all individual species of the genus Muscodor described so far.

[0049] The first isolate of Muscodor, Muscodor albus, released up to 37 different VOCs. Artificial mixtures containing at least 18 of these VOCs were prepared through organic synthesis and commercial purchase, ultimately creating a mixture that faithfully mimicked the biological activity of the fungus itself. This mixture exhibited broad-spectrum activity against a wide range of fungi, although Trichoderma species were generally relatively unaffected. Therefore, collaborative efforts were undertaken to discover other species of this fungus that possessed fewer VOCs but still potent antimicrobial activity. The original fungus was used as a screening tool for these purposes. For this purpose, a novel species of the Muscodor genus was identified from wild pineapple plants collected in the upper Amazon region of Bolivia. This fungus, named Muscodor crispans, was shown to produce only 17 VOCs. At least five of these were considered non-essential for biological activity or posed questionable safety risks to humans and / or the environment. These five molecules include ethanol, ethyl acetate, N-(1-methylpropyl)-formamide, 1,2-dimethyl-3,5-bis(1-methylethenyl)cyclohexane, and 2,3-dimethyl-D-hexane. Subsequently, artificial mixtures of the remaining 12 compounds were prepared and tested, demonstrating their high activity against a wide range of pathogenic fungi and bacteria in plants and humans. This mixture of VOCs from Muscodor crispans was given the name B-23, after the original geographical name and the plant number assigned to the wild pineapple plant. Thus, the B-23 mixture was discovered and is now used as an important component for isolating various fungi of the genus Tuber (Table 3). This method has been successfully applied to the isolation of truffle fungi belonging to the genus Imaia.

[0050] Therefore, in some embodiments, the antimicrobial volatile mixture comprises methyl 2-methylpropanoate; ethanol, 2-methylpropyl acetate, 2-methylpropyl 2-methylpropanoate, 2-methyl-1-propanol, (e)-2-methyl-2-butenal, 3-methyl-1-butanol acetate, 2-methylbutyl 2-methylpropanoate, 3-methyl-1-butanol, 2-methylpropanoic acid or isobutyric acid, and 2-phenylethyl acetate.

[0051] Truffle seeds The truffle species to which the disclosed method may be applied include species of the genera Tuber and Iamai. For example, the disclosed method cultivates ascomycete species selected from the group consisting of Tuber melanosporum, Tuber magnatum, Tuber aestivum, Tuber uncinatum, Tuber borchii, species of the genera Imaia, Tuber macrosporum, Tuber gibbosum, Tuber oregonense, and Tuber lyonii syn. Texense.

[0052] In short, some of the characteristics of isolated Tuber melanosporum, or black truffle, are as follows: 1. Growth in PDA medium is relatively slow, at 1 mm per day. 2. Slightly yellowish mycelium develops and becomes extremely entangled. 3. The fused hyphae immediately form conidiophorous bundles (structures that form fruiting bodies). 4. It produces abundant conidia (conidia) with a diameter of approximately 2.5 μm on conidiophorous bundles (Figures 3 and 4). Initially, spores are produced at the ends of branched conidiophores. Subsequently, they bud from the entire length of the structure on older conidiophorous bundles (Figures 3 and 4). 5. The initial colony is yellowish-white in color, which eventually changes to gray, and then to black. 6. Older colonies become grayish due to numerous conidiophorous bundles, as well as conidiophores and conidia (Figure 5). 7. A distinct truffle odor develops on the colony. This odor is a result of the fungus producing volatile organic compounds (VOCs). GC / MS analysis results have been published for black truffles, as well as many other truffle fungi. Standard SPME fiber analysis has identified compounds, revealing the presence of sulfur compounds, organic alcohols, acids, esters, aldehydes, ketones, and aromatic compounds, furans, terpenoids, etc. It should be noted that all analyses published in the literature to date have been performed on intact truffle material, not pure fungal cultures, and that all truffles are composed of a large and complex mixture of microorganisms, including bacteria, other fungal species, Streptomyces, and sometimes even insects, not to mention the plant tissue material on which truffles form. Therefore, it is expected that the GC / MS analysis results of pure truffle cultures will not be identical to the analysis results of each truffle type that the culture may produce, because fungal products are likely to be easily modified by other microorganisms within the truffle tissue. This is likely to result in a mixture of products mainly derived from bacteria and other fungi. Nevertheless, GC / MS analysis of the VOCs of black truffle cultures revealed the following compounds common to the published analysis results of black truffles themselves, such as acetic acid, ethylbenzene, nonanal, decanal, ethylbenzene, 1,24-trimethylbenzene, and benzaldehyde. Most importantly, methyl 3-thiophenecarboxylate was detected. Thiophene is a known product of truffle fungi. These products were found in samples obtained from fungal cultures cultivated on white rice for 4-5 weeks, as described later. Gases were sampled using SPME fibers, and soluble gaseous compounds were analyzed using a C8 solid-phase extraction cartridge. The GC / MS data supports the claim that the black truffle fungus was successfully isolated. Needless to say, not all of the volatile compounds published about black truffles were recovered here.This is because truffles are a microbiome composed of many microorganisms and other organisms, and these undoubtedly contribute to the complexity of the VOC composition, regardless of the species of the genus Tuber. 8. Mini truffles are ultimately formed on the PDA plate. The fungus was cultivated on sterile pecan fruit for 6 weeks, and then placed in honey for 1 week. After that, it was pressed onto a PDA plate and cultivated for 1 year, where mini truffles were produced (Figure 4). Furthermore, when the culture was transferred from PDA to pecan agar, mini truffle formation began in about 6 weeks. To the best of our knowledge, this is the first reported discovery of its kind. This has great significance as a new product, namely artificially obtained truffles. 9. The ITS sequence of T. melanosporum is provided in Sequence ID No. 1. T. melanosporum shares 99.8% identity with species of the genus Cosmospora, but is by no means identical to this fungus. Interestingly, almost all ITS sequences reported in the literature for truffle fungi have been derived from DNA extracted from truffles themselves, which are composed of contaminants derived from plants, other fungi, and bacteria (11). Therefore, this method cannot effectively determine the actual ITS sequence. Accurate ITS sequence information can only be obtained when a pure fungal culture is the DNA source, as described above.

[0053] Some of the characteristics of white truffles (Tuber magnatum) are as follows: 1. Growth in PDA medium is relatively slow, at 1.1 mm per day. 2. Slightly transparent mycelium develops, and a whitish mycelial body grows. 3. The fused hyphae immediately form conidiophorous bundles (structures that form fruiting bodies). 4. Conidia are abundantly produced on conidiophores and multiple conidiophores (bundles of conidiophores), which are dimorphic, with some in the range of approximately 3.0 μm in length and others being two-celled and longer, approximately 6.7 μm (Figure 5). Initially, spores are produced at the ends of branched conidiophores, and during the fixation process, the conidia detach from the conidiophores, leaving detachment marks at the ends of these structures (inset in Figure 5). 5. The initial colony color is white, and eventually changes to a partially yellowish-brown to brownish color. 6. Older colonies will be dark brown in color. 7. A distinct truffle scent is detected on the colony. 8. After 1-2 weeks, the mini truffles will have formed on the pecan agar plate. The ITS sequence for 9.T.magnatum is provided in Sequence ID No. 2. This is not identical to that of any other truffle species.

[0054] Furthermore, some of the characteristics of summer truffles (Tuber aestivum) are as follows: 1. Growth in PDA medium is relatively fast, at 2.8 mm per day. 2. Transparent hyphae develop, followed by white mycelial bodies, which form intertwined, chain-like strands. 3. Conidiophores, followed by conidiophorous bundles and numerous segmented spores (conidia), develop. The spores are barrel-shaped with their tips truncated, and have an average length of 5.2 μm and a width of 3.5 μm (Figure 6). 4. Older colonies remain whitish. 5. An initial fruity smell develops in the culture plate. 6. Mini truffles will form on the pecan agar in 2-3 weeks. 7. The ITS sequence of T. aestivum is provided in Sequence ID 3. This shows 98% sequence identity with the genus Geotrichum, which also produces segmented spores, but is not a Geotrichum species based on all other indicators.

[0055] The characteristics of Burgundy truffles (Tuber uncinatum) are as follows: 1. Growth is relatively slow, at 1.0 mm per day. 2. Transparent hyphae develop, which eventually form a white mycelium that creates a chain-like, intertwined string. 3. A bundle of conidiophores is formed on the conidiophores, containing a large number of conidia. The conidia have an unusual shape, but on average they are 3.6 × 9.2 μm in size (Figure 8). 4. After several weeks on the PDA, the culture medium begins to change to a burgundy color, which is fitting for this intriguing truffle. 5. After 4-5 weeks on pecan agar, the fungus begins to form mini-truffles that are whitish, small, and have a protruding surface similar to naturally harvested mature truffles, i.e., 0.3 mm in size. Scanning electron micrographs show that a clean thin film layer has formed on the surface of the mini-truffles. 6. Cultures of this fungus generally have an oily appearance due to the formation of a vast number of conidia. The mycelium remains whitish for several months. 7. The ITS sequence of T. uncinatum is provided in SEQ ID NO: 4. This is not related to any other truffle fungus.

[0056] Some of the characteristics of the whitish truffle fungus (Tuber borchii) are as follows: 1. Growth in PDA medium is relatively fast, at 3.0 mm per day. 2. Transparent hyphae develop, which eventually form a white mycelium that creates a chain-like, intertwined string. 3. At the end of the branch, a conidiophore containing conidia develops, followed by a conidiophorous bundle and numerous segmental spores (conidia). The spores are barrel-shaped with their tips truncated, and have an average length of 5.6 μm and a width of 4.0 μm (Figure 7). 4. Older colonies remain whitish. 5. An initial fruity smell develops in the culture plate. 6. Mini truffles will form in 2-3 weeks using pecan agar. 7. The ITS sequence of T. borchii is provided in Sequence ID No. 5. This sequence is very closely related to the sequence of T. aestivum, but not closely related to other truffle fungi such as T. melanosporum (mentioned above).

[0057] The characteristics of North American truffle fungi (species of the genus Imai) are as follows:

[0058] The genus Imaia is a fungal genus belonging to the family Morchellaceae, found in Japan and the Appalachian Mountains of the United States. The monotypic genus Imaia was defined in 2008 by James Martin Trappe and Gabor M. Kovacsto and includes the truffle-like species formerly known as Terfezia gigantea. This is because molecular analysis has demonstrated that its DNA sequence is significantly different from that of the genus Terfezia. The fruiting body of Imaia gigantea is irregularly shaped, ranging from spherical to nearly elliptical, brown in color, and usually cracks as it ages. The internal gleba contains a brown, sac-like compartment of asci separated by white, vein-like tissue. The spores are spherical or nearly spherical, up to 70 μm in length, and enclosed in a thick outer membrane.

[0059] After isolation and investigation, the following characteristics were observed. 1. Transparent mycelium grows on a PDA at a rate of 2.5 mm per day at 22°C. 2. When kept on a PDA, it produces a completely whitish mycelium that does not show any coloration for several months. After incubation at 3.22°C for two weeks, whitish mini-truffles, 3-5 mm in diameter, formed on pecan agar. 4. After cultivating on a PDA for one week, a wonderfully fragrant mixture of volatile organic compounds was produced. 5. Numerous conidiophorous bundles were formed on the PDA, and the conidia (segmented spores) associated with these bundles were similar (in size and shape) to those of both T. aestivum and T. borchii (Figures 6 and 7). 6. This fungus was fermented on Thomson seedless grapes and crushed pecans at 22°C for two weeks. Decanted and filtered grape juice was given a score of 8.5, while unfermented juice was given a score of 6.0 (see Table 7 below). In the case of pecans, the fungal product was dried and crushed. This yielded a delicious truffle-flavored powder with a pecan flavor in the background (see Table 5 below). 7. This truffle mycelium isolate can be stored by culturing it on sterile white rice grains for several weeks and then placing it in a cryovial at -80°C, or by storing the conidia in a vial with sterile water at 2-3°C (see the storage section below).

[0060] Table 1 provides a brief description of commercially available truffle species. Table 2 lists the ITS sequences of truffle pure cultures. Each truffle fungus listed in Tables 1 and 2 was originally obtained from their natural geographical sources as freshly harvested truffles, and subsequently used as a biological source for pure cultures after successful isolation of these organisms.

[0061] Table 1: Brief description of truffle species [Table 1]

[0062] Table 2 ITS sequences of isolated truffle fungi. [Table 2-1] [Table 2-2]

[0063] Basically, many of the truffle fungi mentioned above are now available as pure cultures. These truffle fungi can now be cultivated on a variety of natural or artificial media, which makes available countless new product candidates, each with different sets or combinations of flavors. This is because, by providing the fungi with various substrates for growth, the substances contained in the growth source are converted into other flavor-like compounds and their combinations. Similarly, this isolation procedure makes it easy to collect numerous regional strains of the fungus, which can be useful in finding unique and distinctive natural foods. In this case, novel flavors can be created by actually cultivating truffle fungi on grains, nuts, powders, flower parts, or literally natural food sources or mixtures of such products.

[0064] Currently, truffle fungi are inoculated into trees by immersing their roots in a puree of the desired truffle fungus. This is a costly, unsatisfactory, and unscientific approach because truffles themselves do not contain countless infectious conidia, but are contaminated with numerous other fungi and bacteria, all of which suggest unexpectedly bad and undesirable results. However, pure cultures of many truffle fungi can now be easily cultivated on sterile grains such as barley, rye, wheat, or rice with the outer husk removed, in which case an abundance of infectious conidia is produced (Figure 11). These grains can be dried at room temperature under sterile conditions after cultivating the fungus for 2-3 weeks and stored for up to one year under refrigeration (freezing or 40°F) or room temperature (22°F) without significantly reducing their viability (Figure 13). These inoculated grains can then be used as inoculations for plant roots by placing them near the roots of the desired plant species that will act as the host. [Examples]

[0065] This disclosure is further illustrated by the following embodiments, which should not be construed as limiting. All references, patents, and published patent applications, as well as drawings, cited herein are incorporated herein by reference in their entirety for all purposes.

[0066] Example 1: Isolation procedure for cultivating major truffle species The isolation of the target truffle species began by sourcing fresh truffles from a commercially available, intact truffle supply. The truffles were cleaned, rinsed, dried, and sent to the inventors' Montana address via a rapid delivery service. The procedure described below is best suited for intact truffles. The truffles were preserved, wrapped in paper towels, and stored at refrigerator temperature. After each truffle was cut, it was split open. Small pieces of approximately 0.5–2 mm were scraped directly from the freshly exposed surface into a petri dish containing potato dextrose agar (PDA) (Figures 1 and 2). The lid, removed from a small microcentrifuge vial, was placed in the center of the plate. Ten microliters of B-23 solution were then added to the lid. The composition of B-23 is listed in Table 3. The B-23 patent product mixture is available from Jeneil Biotech Co. (Saukville, WI). Truffle fragments were scattered generously around the plate (Figure 2). This plate was incubated at 22°C for 3–4 days. At this point, some microbial growth was observed quite far from the central well, but not immediately adjacent to it. After 5 days of incubation, we were able to carefully pick mycelial fragments of fungal colonies, presumably of the genus Tuber. These fungal colonies, particularly those with conidiophorous bundles, were targeted for transplantation. Colony transplantation was performed using the tip of a sterilized, sharp knife. The lid of the petri dish was removed (to degas), and then replaced. For several more days, particles adjacent to the wells were examined for fungal growth. Once growth occurred, agar fragments containing the tips of the fungal hyphae were carefully picked and transplanted into a new PDA petri dish. Any remaining bacterial contaminants were removed by placing a small amount of powdered tetracycline HCl on top of the culture. Next, fungi suspected to be species of the genus Tuber were examined for conidiophorous bundles, asexual spores, pigments, and the ability to produce the typical truffle odor. Throughout this process, it is necessary for those skilled in mycology, particularly the biology of truffle fungi, to be able to accurately identify these fungi. The first species of the genus Tuber isolated in this manner was Tuber melanosporum.The isolated samples met all of the aforementioned culticultural and morphological characteristics of the specific fungus.

[0067] Table 3 shows the GC / MS analysis of volatile compounds produced by M. crispans. Several minor and breakthrough peaks, which accounted for only 1% of the total area, were omitted from the overall analysis results. Compounds found in the control PDA plate are not included in this table. [Table 3]

[0068] * The symbol indicates that the compound shown had the same retention time (RT) and mass spectrum (MS) as the genuine standard material. It also indicates that the compound was used in the artificial mixture of VOCs. All other compounds in the list match the most likely compounds in the NIST database, but have not been confirmed by retention time or MS using appropriate identical standard materials. The symbol # indicates that the genuine standard material does not have the same retention time as the compound shown. The symbol + indicates that it was not used in the B-23 compound mixture. The B-23 compound is made by adding each component shown in this figure as a volume derived from the calculation of the total area of ​​this table. Isobutyric acid at 13.37 min was replaced with propanoic acid because this acid has malodorous properties.

[0069] To isolate a pure culture of the genus Imaia, approximately 1.5 kg of freshly harvested truffles were obtained from a producer in North Carolina. One truffle was rinsed with a spray of 70% ethanol and air-dried. A sterilized, sharp blade was then inserted into the fleshy part of the truffle, and the truffle was split along the line of the blade to expose the undamaged inner surface. Using a sterilized blade, a fragment of the truffle flesh was transferred to a Petri dish (PDA). A small plastic well containing 10 μl of B-23 mixture was placed in the center of the plate, and the plate was sealed by covering it twice with Parafilm. This plate was set to incubate at 22°C for 4 days. At the end of this period, only one fungal colony had grown from the truffle flesh fragment. This colony was labeled NC-1 and pinned to another PDA plate to check the culture purity. This culture had promising quality to become a new pure culture isolate of truffle fungus. Further investigation revealed the aforementioned characteristics.

[0070] Materials and methods 1. Mini truffles and pecan agar We cultivated T. melanosporum on crushed pecans for six weeks, then placed them in honey for one week to kill the fungus. Afterward, we prepared agar by spreading it thinly on a PDA plate to determine if the fungus had died. Quite unexpectedly, the fungus appeared to curl up and form small spheres of mycelium within a week, and after one year, these small spheres emerged as mini-truffles. These were densely clustered small, round spheres, each about 2 mm in diameter, visible to the naked eye, but smaller than those typically expected in wild truffles. In comparison, on ordinary agar PDA, the fungus only grew as flat mycelium, and mini-truffles had never appeared before. Thus, it seemed that components in pecans could induce mini-truffle formation in this fungus. Therefore, we created a special pecan agar and used it as a substrate to investigate whether truffle formation could be induced in various fungi suspected to be truffle-producing. If so, this medium can serve as a test medium to determine whether any suspected fungus could be a truffle fungus, and as a result, can provide additional supporting information for identifying the organism.

[0071] The ingredients for pecan agar jelly are as follows: 10g finely crushed pecans 2.5g glucose 0.5g of peptone 1g of yeast extract 3g of agar

[0072] Add water until the total volume reaches 200ml, then autoclave for 20 minutes.

[0073] Pour 35 ml of liquid onto the plate.

[0074] Next, pure cultures of each truffle fungus are inoculated onto plates.

[0075] The plates were incubated at 22°C for 2-3 weeks, and then photographs were taken when the mini truffles began to appear.

[0076] 2. Scanning electron microscopy (SEM) SEM was performed on truffle isolates cultivated on pecan agar (see below) for various periods depending on the given isolate. The agar pieces supporting fungal growth were placed in filter paper bags, then placed in 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2-7.4) with the addition of the wetting agent TritonX 100, aspirated for 5 minutes, and left overnight. The next day, they were washed 6 times with a 1:1 mixture of water and buffer, changing every 15 minutes, then washed 5 times with 10% ethanol for 15 minutes, 30% ethanol for 15 minutes, 50% ethanol for 15 minutes, and 70% ethanol, changing every 15 minutes, and then left overnight or longer in 70% ethanol. After that, they were rinsed 6 times with 95% ethanol for 15 minutes each, and then 3 times with 100% ethanol, changing every 15 minutes. Microbial materials were critically dried using a Tousimis Autosamdri® 931 GL critical point dryer, sputter-coated with AuPd (80:20) using a Quorum Q 150T ES sputter coater, and images were recorded using a ThermoFisher Apreo C SEM. Mycelial hyphae were measured using ImageJ software (available online: http: / / rsb.info.nih.gov / ij / ).

[0077] 3. Determination of the DNA sequence of ITS-truffle fungus. DNA Extraction - Genomic DNA was obtained from fungal isolates using a rapid DNA extraction method. Mycelium was picked using a sterile pipette tip and transferred to a 0.2 ml Eppendorf tube containing 15 μl of Kawasaki lysis buffer (10 mM Tris HCl pH 8, 1 mM EDTA, 0.5% Tween 20, 10 μg / ml proteinase K) and 15 μl of ultrapure water, followed by incubation at 85°C for 15 minutes. After the incubation period, the tube was centrifuged at 10,000 rpm for 1 minute. DNA extraction was repeated twice for each fungal isolate.

[0078] Fungal DNA was amplified using PCR-rRNA gene primers ITS1F and ITS4R (9). A PCR reaction (20 μl) was performed in a 0.2 ml Eppendorf tube containing 2 μl of template DNA, 10 μl of 2× PCR Hot Start Green Master Mix, and 500 nM primers. The PCR thermocycler conditions consisted of an initial denaturation step of 5 minutes at 95°C, followed by 35 amplification cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 60 seconds at 72°C, and a final extension step of 10 minutes at 72°C. All reactions were performed in duplicate. The amplification was visualized at 10,000x magnification in a 1% agarose gel stained with GelRed® nucleic acid gel.

[0079] The PCR products were purified and sequenced using the Sanger method at Genewiz / Azenta in New Jersey. The sequencing results were searched for using BLAST against the NCBI database. These analyses were performed by Erica Consoli of MSU.

[0080] 4. Gas chromatography / mass spectrometry of volatile organic compounds (VOCs) from species of the genus Tuber Using a solid-phase microextraction syringe, volatile substances (in the gas phase) from the fungal truffle were collected following the procedure used for VOC analysis of Muscodor albus. The fiber material (Supelco) consisted of polydimethylsiloxane on a stableflex fiber topped with 50 / 30 divinylbenzene / carburen. The syringe was inserted through a small hole in the side of a petri dish and exposed to the gas phase for 45 minutes. The syringe was then inserted into a gas chromatograph (Agilent 8890) equipped with a mass-selective detector. A 30m × 0.25mm (inner diameter) 5% phenylmethylpolysiloxane (HP-5ms) Agilent J&W capillary column (film thickness 0.25μm) was used for the separation of volatile substances. The column temperature was programmed as follows: 25°C for 2 minutes, then increased to 220°C at 5°C / min. The carrier gas was helium (ultra-high purity (UHP); local distributor), and the initial column head pressure was 50 kPa. The helium pressure was varied in accordance with the rise in oven temperature to maintain a constant carrier gas flow rate during the separation process. Before collecting volatile substances, the fiber was pre-conditioned by flowing helium gas through it at 240°C for 20 minutes. The sample fiber was introduced into the gas chromatograph using an injection time of 30 seconds. The chromatograph was connected to an Agilent 7000D triple quadrupole mass spectrometer operating at a mass resolution of 1500. The mass spectrum was scanned over the mass range of 35–360 Da at a rate of 0.50 seconds per decade. Data acquisition and processing were performed using the Agilent Mass Hunter software package. Initial identification of unknown substances produced by fungi was performed by library comparison using the NIST database.

[0081] Analysis of compounds at low concentrations in the gas phase was achieved by cultivating fungi on a rice and water slurry. A water extract (25 mL) obtained from this mixture was filtered through Whatman No. 2 filter paper, and this solution was then applied to a C8 solid-phase extraction cartridge (Silicycle® brand, 500 mg). The C8 cartridge was prepared before use by passing 15 mL of HPLC-grade methanol, followed by 20 mL of HPLC-grade water, through the cartridge. A color change indicated that the fungal extract had adhered to the stationary phase. The cartridge was then washed under vacuum with 20 mL of HPLC-grade water in a solid-phase extraction manifold. Air was passed through the cartridge for 5 minutes after loading to remove excess moisture. The compounds retained in the cartridge were eluted by adding 5 mL of HPLC-grade methanol and collecting it in a 3-drum vial as the eluent. 1 mL of this eluent was placed in a GC vial, capped, and analyzed using the aforementioned temperature program and process. These analyses were performed by Dr. James Harper of BYU.

[0082] Example 2: Preservation of isolated truffle species One crucial requirement for cultivating these truffle fungi is the ability to successfully preserve them for future use by harvesting the truffles. Many techniques are available for this purpose, and we validated several standard procedures. Testing of isolated truffle species showed that we successfully preserved these isolated cultures using the techniques described below.

[0083] 1. Storage in sterile water at refrigerated temperatures. The surface of agar plates containing 2-3 week cultures of each truffle fungus, grown on PDA potato dextrose agar, was scratched with a bacterial loop. The spores and hyphae fragments picked up with the loop were then transferred to a small vial containing approximately 1 ml of sterile distilled water, shaken as needed. The vial was then capped and stored at 3°C ​​for various periods. Subsequently, the loop was reinserted into the spore suspension and thinly spread onto the surface of a newly prepared PDA plate. Isolates of T. melanosporum and T. borchii showed growth on the plate within 2 days, even after storage for over a year. Isolates of T. aestivum, T. magnatum, T. uncinatum, and species of the genus Imaia all showed clear growth even after storage for 3 months, and even up to 1 year. This technique is a promising method for maintaining viable cultures of truffle fungi.

[0084] 2. Storage of inoculated grains at -80°C After cultivating truffle mycelium on PDA for several weeks, groups of 20-30 sterile (autoclaved) barley or rice seeds were placed on top of the growing fungal culture on an agar surface to allow the fungus to inoculate the seeds. The seeds were pre-treated by autoclaving them after adding 1 volume of seeds to 1 volume of water, then cooling them, and autoclaving them again after adding 0.5 volumes of water to 1 volume of grain. The two autoclaves were necessary to kill any spore-forming bacteria that normally accompany them. The sterile grains were incubated on plates inoculated with truffle mycelium for 2-3 weeks, and then placed in appropriately labeled cryovials at -80°C for an extended period. The tested truffle mycelium could be easily recovered from this storage state for at least one year after being stored at this temperature.

[0085] 3. Storage of inoculated dried grains at 22°C Instead of storing the inoculated seeds at -80°C as outlined above, they were simply removed from the PDA plates, placed in empty plastic petri dishes, and dried at 22°C. These plates were then wrapped in Parafilm and stored at 22°C.

[0086] Seeds were intermittently extracted and placed on a standard PDA to examine the viability of the fungi. Each truffle fungus tested was able to withstand drying and storage at room temperature for at least one year.

[0087] Other observations regarding the preservation and durability of truffle fungi at various temperatures have revealed the following: a. Inoculated seeds can be stored at -18 to -20°C, and the fungi maintain their viability for extended periods. Furthermore, they maintain their viability even after being removed from storage, thawed, and refrozen. b. Inoculated seeds retain their viability even after being stored at -80°C, removed, thawed, and refrozen. This is not a typical observation, as most fungi die when frozen, thawed, and refrozen. c. Fungi can withstand heat exposure of up to 35-50°C for several minutes. d. Truffle fungus is usually killed when exposed to 65-70°C for 2 hours.

[0088] Finally, each truffle fungus was cultivated on sterilized barley seeds for several weeks, and then these colonized seeds were transferred to cryovials and stored at -80°C. All of these can be successfully preserved for extended periods under these conditions. They are all successfully preserved and maintained at the Montana State University Microbial Strain Preservation Center and the Black Boar Truffle Co. Preservation Center.

[0089] Example 3: Use of isolated truffle fungus species Many pure cultures of the aforementioned truffle fungi are now available. These can now be cultivated on artificial and natural media, as well as on various natural products, such as grains, nuts, and fruits, making countless new product candidates available, each with different sets or combinations of flavors. Truffle fungi have the potential to convert natural products in grains or fruits into other compounds with interesting and useful flavor substances, adding novelty to fermentation processes and new product development.

[0090] Furthermore, the novel truffle fungal isolation procedure disclosed above facilitates the recovery of numerous regional strains of the fungus, which may be useful in discovering unique and distinctive natural foods. In this case, novel flavors can be created by cultivating truffle regional strains of the fungus on grains, nuts, powders, flower parts, or mixtures of such natural plant products.

[0091] The techniques for cultivating these fungi on seeds and grains are primarily carried out by solid-state fermentation, as described below. Use 1.1 liters of product, such as rye, rice, or barley seed water. 2. Add 0.6 to 3.0 liters of water (depending on the seeds used). 3. Next, add 5g of glucose, 1g of peptone, and 2g of yeast extract, and mix thoroughly.

[0092] Various sizes of Erlenmeyer flasks or other specially designed glass flasks can be used to carry out the fermentation process. Truffle fungi do not thrive in water, and therefore the amount of water present in the final mixture is crucial for fungal growth and development. However, for mass production in a solid state, the mixture is usually placed in a plastic bag with an opening 1.5-2 inches wide, sealed with a foam cylinder, and covered with aluminum foil after inoculation. Autoclaving is performed for 30-40 minutes to achieve complete sterilization of the contents. After cooling, the bag is then inoculated by introducing spores and mycelium of one species of truffle fungus into the bag through the opening. One method of inoculating the bag with spores is, for example, to inoculate 10-20 barley seeds inoculated with a Tuber species that have been incubated on a PDA plate for several weeks. The spores and mycelium then grow throughout the contents of the plastic bag. Incubate the bag in a dark, undisturbed place at 19-20°C for 4-6 weeks, stirring the contents manually from time to time. A temperature lower than normal room temperature, i.e., 19-20°C, is preferred.

[0093] Depending on what is being fermented, the contents of the bag may undergo different processing. Initially, various grains / seeds, including wheat, rice, rye, lentils, and barley, were fermented using the truffle fungus (Tuber melanosporum), which had been successfully isolated for the first time. After fermentation, the seeds were removed, placed on a flat metal pan, and heated at 60°C to effectively dry the product. The seeds were then ground into a fine powder, thus preparing the product for testing. The dark powdered rye truffle product (Figure 12) is now widely sold as an additive to salt (truffle salt) and olive oil (truffle oil), as well as in packaged finished products such as truffle potato chips (Table 4). Development of other powders obtained by fermenting this truffle fungus on various other seeds has been underway, and the products have been tested and are currently being prepared for the sales process (Table 4). This includes novel black truffle products made using lentils, brown rice, wheat, and barley as substrates for the fungi. Finally, when soybeans were used as a substrate for the black truffle fungus, both liquid (filtrate from the fermentation process) and dried powder yielded a basic, conventional truffle-flavored product. This liquid could be called truffle soy sauce, after the famous Japanese soy sauce, but with added truffle flavor characteristics (Table 4). The various powders produced from these grain fermentations can be used as condiments for hot beverages, salt, oil, and vinegar, as well as flavorings for meat, baked goods, eggs, and soups. Appropriate testing was conducted to demonstrate the practical applications of each of these plant-derived fermented powders.

[0094] Table 4 shows the fermentation of black truffle (T. melanosporum) on various substrates, as well as the resulting products and uses. Note that each of these products has its own unique aroma and flavor. There are currently and potentially a wide range of applications. [Table 4]

[0095] Besides grains, there are numerous other seeds and nuts that can serve as potential fermentation sources for truffle fungi. For this purpose, a representative list of commonly available natural products was tested for their ability to support the growth of various truffle fungi and create novel, usable, and desirable foods. In the case of nuts and seeds, fermentation was carried out as described above in the experiments testing grains. The first test was performed using pecans incubated with black truffle fungus. The final product was a viscous, liquid, mash-like product that could be used in various ways. White truffle fungus was also fermented in this manner. The final fermentation product can also be dried, ground, and used as a powder (Table 5). However, the viscous liquid product obtained directly from fermentation can ideally be used as a sauce for bread, biscuits, toast, waffles, or crackers, and various other foods, and in particular, when mixed with honey or maple syrup, it can be used as a sweetened truffle / pecan mixture.

[0096] One example of the usefulness of black truffle and pecan mash is the creation of a delicious and versatile mixture. This product is made by creating a 50:50 v / v mixture with honey and can be used as a novel spread for biscuits, toast, or waffles. The mixture can be modified by changing the ratio of honey to pecan / truffle ferment, and an acceptable taste can still be obtained even when the ratio is reduced to 5%. When the 50 / 50 mixture was taste-tested on a local adult panel, the overwhelming response indicated that they found the product delicious and novel (Table 6). Two respondents disliked the smell and taste of truffle, but this is in complete agreement with the general population, meaning that approximately 20% of people feel the same way about truffle products. When a similar mixture was made using walnuts, dried products with completely different tastes were obtained, regardless of whether black truffle or white truffle ferment was used. Products with different ratios of honey to nuts / truffles were also created and tested, yielding similar results. Other candidates include hazelnuts, peanuts, almonds, Brazil nuts, cashews, pistachios, macadamia nuts, chestnuts, and cocoa. These results have encouraged further research into other nuts and seeds, as shown in Table 5.

[0097] As mentioned above, many other nuts and seeds were also fermented using the exact same fermentation formulations and conditions as those described for pecans and walnuts, as shown in Table 5. For example, coffee beans were fermented for 6 weeks on a sterilized coffee bean and sugar / peptone mixture, and the (previously) final dried product was ground and mixed with standard coffee bean powder at varying levels to produce a delicious beverage. The final product was made by mixing either 10% or 20% truffle coffee bean powder with 80-90% regular coffee beans and pouring hot water over it to produce the best truffle / coffee flavor. Adding cream to the coffee bean mixture also resulted in a wonderfully flavored truffle / coffee / latte (Table 5). The same method was also applied when making truffle cocoa mix using one or more truffle powders or mashed products to mix / add to standard cocoa powder or powder mixture to make a beverage (Table 5). In the case of nuts / beans, novel products can be made as confectionery mixed with chocolate, white chocolate, or other sweet substances to obtain products with novel flavors, and each product can be labeled with the truffle seeds used to make the product. The same applies to hazelnuts, whose flavor was distinctly different from other nut products (Table 5).

[0098] In each of these examples using black truffles, the method can be replaced with any other truffle fungus isolated by the described method, and then the fermentation schedule outlined for black truffles can be applied (Table 4). Essentially, since each truffle has a different flavor, it is possible to create entirely new products. Almost all major truffle species have been isolated in this manner, and tests have shown that they can grow on natural products such as seeds and beans, as in T. melanosporum (black truffle). Furthermore, individual truffle isolates are very likely to have their own important and subtle flavor characteristics, thus further increasing the importance of isolating and obtaining individual truffle strains.

[0099] [Table 5] * As an example of how these new products were evaluated, please refer to the table showing the participant evaluation results for this product (Table 6). This fungus formed large, usable truffle-like structures in this culture. These are very promising and very unexpected new products.

[0100] When the fermentation product of white truffle fungus was cultivated on sunflower seeds, completely unexpected results were obtained (Table 5). The dried fermentation product yielded a product with a unique, rich umami / aroma, making it a versatile all-purpose flavoring for roasted meats, steaks, and seafood. This was evaluated by well-trained chefs, and its use in tapenade was also suggested (Table 5). Interestingly, the product from pine nuts yielded a very spicy, almost peppery taste that could be used in salad dressings and other condiments (Table 5). Similarly, the fermentation filtrate (white truffle) and powders obtained from soybean tests also yielded good umami-like products (Table 5).

[0101] When long-grain white rice and long-grain brown rice were used with black truffle fungus, the products yielded black powder products that could be used as common condiments. The advantage of using rice as a substrate is that truffle fungus ferments more easily and quickly due to its relatively low contamination and the ease and short time it takes for the fungus to grow and develop on these substrates. Both Burgundy truffle fungus and whitish truffle fungus yielded mild-flavored product candidates on white long-grain white rice, but no product could be obtained from summer truffles (Table 5). Finally, mustard sauces made from the fermented products of white and black truffles yielded a completely new flavor of mustard with nuances of white and black truffle taste, possessing a completely unique, highly distinctive, and appetizing flavor (Table 5).

[0102] Table 6. Taste panel evaluation of black truffle pecan honey syrup products mixed with honey at a ratio of 10-20% by weight.* [Table 6]

[0103] All participants were adults residing in Bozeman, many of whom were professionals or student volunteers. They offered numerous suggestions for product uses, including as a spread for toast, waffles, and fine crackers. Two outliers reflected the average population, with approximately 20% disliking the smell or taste of truffles or truffle products.

[0104] In addition to cultivating truffle fungus on seeds (grains) and nuts, we fermented a wide variety of berries and fruits to make juices / beverages, resulting in novel flavors (Table 7). In all cases, the following preparations were made to carry out the fermentation and evaluation processes. 1. Use 150g of thoroughly crushed fruit or berry product. 2.0.25g of peptone was added. 3. Add 100ml of water.

[0105] After incubation at 19-20°C for two weeks, the contents were filtered through four layers of coffee filter paper to obtain a nearly clear product. Taste tests were conducted with at least three participants, who ranked the products on a scale of 1 to 10 based on flavor, sweetness, uniqueness, desirability, and overall potential as a new beverage, possibly for use as an aperitif or digestif. Data were averaged, and the standard deviation of the mean was calculated (Table 7). Note that with Thomson seedless grapes, each truffle fungus produced a better-tasting product than the liquid product (control) that did not support fungal growth (Table 7). Similar results were obtained with all products containing grape additives, including red seedless grapes alone, black seedless grapes alone, and / or blackberries alone or with grape additives (Table 7). Dry cherry beverages using either black or white truffle fungus also performed well. However, golden kiwi generally did not yield products with particularly outstanding potential (Table 7). Interestingly, commercially viable yellow mango beverages were those produced using black truffle fungus, whitish truffle fungus, and summer truffle fungus (Table 7). Similarly, red raspberries and huckleberries also yielded excellent beverage products, particularly with black truffle fungus, while Burgundy truffles produced superior results when used with Thompson seedless grapes, dried cherries, and huckleberries (Table 7).

[0106] Table 7. Evaluation of various berry / fruit truffle fermentation juices collected by filtration through a four-layer coffee filter after incubation at 22°C for two weeks. Evaluations were conducted by at least five individuals, ranking the juices based on flavor, sweetness, uniqueness, desirability, and overall potential as a novel beverage. The scale was 1 to 10, with 1 being the lowest. Table 7 Truffle fungi used in fermentation [Table 7]

[0107] *Dried Montmorency dried cherries—In the initial tests, the yeast extract somewhat negatively affected the flavor, so fermentation was carried out without using the yeast extract.

[0108] E. Cultivation of Tuber species in a special culture medium to produce only mycelium. If it is desired that the fermentation product be relatively pure, consisting only of mycelium and not mixed with any major seeds or nuts, or with any other natural plant products such as seed coats or other unfermented natural seed products, the following procedure is used. The fermentation process is carried out using only liquid medium in a static culture to produce complete mycelium. 1.0.5g glucose 2.0.2g of peptone 3.0.4g of yeast extract 4.10 grams of finely ground white rice flour 5,100 ml of water (this amount may vary depending on water loss during autoclaving). Since fungi do not grow well under liquid culture conditions, only a thin layer of water is needed at the bottom of the flask.

[0109] Place the mixture in a 2-liter flask and add the truffle mycelium. During the incubation process, do not agitate or mix the contents of the flask, and incubate at 19-20°C. In either case, very good solid mycelium will develop. This can be dried and ground into a powder, or sold as complete truffle mycelium itself. For example, the mycelium may be harvested by filtration and dried at 60°C. The dried, dark mycelium can then be ground into a powder and used as needed. Alternatively, the complete mycelium can be rolled out into tortillas and / or dried to make the final product.

[0110] This process may require fermentation for 4 to 8 weeks in media with several variations in the broth formulation, including the addition of methionine to obtain different sets of sulfur-containing flavoring products, particularly using black truffle and white truffle fungi. The mycelium is then harvested by simply sliding it out of the flask and processed by drying or rolling it into a cylindrical shape.

[0111] F. Truffle inoculation for trees planted in the wild for the purpose of wild truffle production. To test the use of inoculated grains for inoculation into trees in the field, one-year-old dried barley seeds inoculated with T. melanosporum were placed in 50 grams of loamy soil in a petri dish at 22°C. The soil was then moistened with sterile water, and the seeds were observed as a function of time. After 3 days of incubation, visual and microscopic observations revealed abundant conidiophores and infectious conidia in each inoculated seed (Figure 13). Artificial infection of tree roots should be carried out under field conditions by placing moistened roots with a large quantity of inoculated barley seeds for several days before planting.

[0112] Experimentally, the optimal truffle fungal inoculum was determined by cultivating T. melanosporum in long-grain white rice for 6-8 weeks with frequent stirring and mixing in an aqueous suspension of 1 volume of rice per 4 volumes of water (the same formulation as described above on page 26). This fungal suspension (3 volumes, i.e., 300 ml) was added, while mixing, to 5 volumes (500 ml) of pre-autoclaved MiracleGro® brand planting soil mixture or other organic soil mixtures. The fungal soil mixture was then placed in plastic bags and stored under various conditions. Essentially, this allowed for the determination of which conditions were optimal for storage and for the shipment of viable inoculated products. 1. A portion of the fungal soil mixture was air-dried in a fume hood at 22°C for two days, and then stored in a plastic bag at 22°C. 2. A portion of the mixture was placed in a plastic bag and stored at room temperature of 22°C. 3. A portion of the mixture was placed in a plastic bag and stored in the refrigerator at 3°C. 4. A portion was placed in a plastic bag and stored at -20°C, which is the standard freezer temperature of a refrigerator. 5. Subsequently, a portion was stored at -80°C in a special laboratory freezer.

[0113] After one week under each of these conditions, several milligrams of the contents of each bag were aseptically taken and placed in separate locations on a PDA plate to measure the viability of the truffle fungus and the degree of microbial contamination. After three days of incubation, both the dried preparations and wet samples kept at room temperature showed contamination of the black truffle fungus by other fungi, which is unacceptable (Figure 9). However, both the preparations kept at a freezing temperature of -20°C and the normal refrigeration temperature of 3°C showed little contamination, and the truffle fungus grew well and remained viable. This was also true for the preparation kept at -80°C. Therefore, any of these methods for truffle inoculum would likely be useful as products developed for sale to those interested in field truffle production, although the -80°C method would likely prove too costly to be feasible for implementation and transportation. The dried product would also be useful as a product, but the drying process would need to be carried out under sterile conditions due to the presence of contaminants in addition to the fungus (Figure 9).

[0114] Finally, to investigate whether the T. melanosporum / soil mixture (-20°C and -3°C) is effective in producing truffle-like bodies in nature, experiments were conducted by placing the mixture on oak roots. Roots of 1-2 year old white oak trees, recently felled from a Virginia forest, were obtained. The roots were sawn to a cross-section approximately 2-3 mm thick. They were then sterilized in an autoclave for 20 minutes, and further sterilized only the surface by treating with 70% ethanol and then briefly burning with a flame for a few seconds until the alcohol disappeared from the tissue. This latter process was repeated three times. The root sections were then placed on agar water, and 1-2 mg of the fungus / soil mixture was carefully inoculated onto the surface of the root sections, which had been stored at either -3°C or -20°C. After two weeks, small mini-truffles (0.5-1.0 mm) appeared on the surface of all root sections in both the autoclaved and surface-treated oak root sections (Figure 10). These observations strongly suggest that the fungal / soil mixture (in any of these temperature schemes) could serve as a viable and potentially superior alternative to field inoculation of host trees for wild truffle production. Furthermore, when this mixture was added to water and placed in several 3-4 cm holes made with iron rods in the root systems of white oak trees growing in the Bozeman (Montana) region, the application method was fully functional, although it may take several years to see results.

[0115] References incorporated by citation and reference 1. Nowak, Z. 2015. Truffle: A Global History. Reaktion Books. 128P. 2.De Aza,C.,et al.,2022.Fungal and bacterial communities in Tuber melanosporum plantations from northern Spain.Forests 13:385- 3.Cullere,L.et al.,2009.Characterization of aroma active compounds in black truffles (Tuber melansporum) and summer truffles (Tuber aestivum) by gas chromatography.olfactometry.Food Chemistry 122- 300-306. 4.Strobel,G.A.,Dirksie,E.,Sears,J.,and Markworth,C.2001.Volatile antimicrobials from a Novel Endophytic Fungus.Microbiol.147:2943-2950. 5.Saxena,S and Strobel,G.A.2021.Marvelous Muscodor spp.:update on their biology and applications.Microbial Ecology 82:5-20. 6.Mitchell,A.M.Strobel,G.A.,Moore,E.,Robison,R.,and Sears,J.2010 Volatile antimicrobials from Muscodor crispans.Microbiology 156:270-277. 7.Urban,A.,et al.,2004.Molecular studies on terricolous microfungi reveal novel anamorph of two Tuber species.Mycol.Rev 108:749-758. 8.Iotti,M.et al.,2002.Morphological and molecular characterization of mycelia of some Tuber species in pure culture.New Phytologist 155:499-505. 9.White,T.J.(1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics.In:PCR Protocols,a Guide to Methods and Applications,315-322. 10.Mustafa,A.M.et al.,2020.An overview of truffle aroma and main volatile compounds.Molecules.25:5948;https: / / doi.org / 10.3390 / molecules25245948 11.Vahdatzaheh,M.,et al.,2015.The Role of the Microbiome of Truffles in Aroma Formation:A Meta-Analysis Approach.Appl.Environ.Microbiol.81:6946-6952.

Claims

1. A method for cultivating truffles, The steps involve obtaining the spore-producing fruiting bodies of ascomycetes from the soil, The steps include: scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample; A step of placing the fruiting body sample on a first surface containing a nutrient substrate, wherein the nutrient substrate is monosaccharide, Plant-derived isolates, and Agar Steps including, The step of incubating the fruiting body sample on the nutrient substrate with an antimicrobial volatile mixture until mycelium is formed, wherein the presence of mycelium indicates new fungal growth. The steps include transferring the new fungal growth to a second surface containing the nutrient substrate to obtain a pure culture of the ascomycetes, Methods that include...

2. The method according to claim 1, wherein the ascomycetes include species of the genus Tuber and species of the genus Iamai.

3. The method according to claim 2, wherein the ascomycetes are selected from the group consisting of Tuber melanosporum, Tuber magnatum, Tuber aestivum, Tuber uncinatum, Tuber borchii, species of the genus Imaia, Tuber macrosporum, Tuber gibbosum, Tuber oregonense, and Tuber lyonii syn. texense.

4. The method according to claim 1, wherein the antimicrobial volatile mixture is derived from volatile organic compounds produced by organisms of the genus Muscodor.

5. The method according to claim 4, wherein the antimicrobial volatile mixture comprises at least one of methyl 2-methylpropanoate, ethanol, 2-methylpropyl acetate, 2-methylpropyl 2-methylpropanoate, 2-methyl-1-propanol, (E)-2-methyl-2-butenal, 3-methyl-1-butanol acetate, 2-methylbutyl 2-methylpropanoate, 3-methyl-1-butanol, 2-methylpropanoic acid or isobutyric acid, or 2-phenylethyl acetate.

6. The method according to claim 4, wherein the antimicrobial volatile mixture comprises methyl 2-methylpropanoate, ethanol, 2-methylpropyl acetate, 2-methylpropyl 2-methylpropanoate, 2-methyl-1-propanol, (E)-2-methyl-2-butenal, 3-methyl-1-butanol acetate, 2-methylbutyl 2-methylpropanoate, 3-methyl-1-butanol, 2-methylpropanoic acid or isobutyric acid, and 2-phenylethyl acetate.

7. The method according to claim 1, wherein the second nutrient substrate is a liquid.

8. The method according to claim 1, wherein the nutrient substrate is potato modextrose agar.

9. The method according to claim 7, wherein the nutrient substrate is potato dextrose broth.

10. The method according to claim 8, wherein the nutrient substrate is pecan agar.

11. A method for cultivating mini truffles, Obtaining spore-producing fruiting bodies of ascomycetes from soil, The process involves scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample, The process involves placing the fruiting body sample on a first surface containing a nutrient substrate, wherein the nutrient substrate is monosaccharide, Plant-derived isolates, and Agar Including, posting, The incubation involves incubating the fruiting body sample on the nutrient substrate with an antimicrobial volatile mixture until mycelium is formed, wherein the presence of mycelium indicates new fungal growth. The process involves transferring the new fungal growth to a second surface containing the nutrient substrate to obtain a pure culture of the ascomycetes, The aforementioned pure culture is incubated with fruits, nuts, grains, or parts thereof for at least one week to prepare an inoculated plant sample. Mini truffles are produced by placing the inoculated plant sample on a third surface containing the aforementioned nutrient substrate, Methods that include...

12. The method according to claim 11, wherein the third surface contains pecan agar.

13. A method for cultivating mini truffles, Obtaining spore-producing fruiting bodies of ascomycetes from soil, The process involves scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample, The process involves placing the fruiting body sample on a first surface containing a nutrient substrate, wherein the nutrient substrate is monosaccharide, Plant-derived isolates, and Agar Including, posting, To provide the fruiting body sample on the aforementioned nutrient substrate, The incubation involves incubating the fruiting body sample on the nutrient substrate with an antimicrobial volatile mixture until mycelium is formed, wherein the presence of mycelium indicates new fungal growth. The process involves transferring the new fungal growth to a second surface containing the nutrient substrate to obtain a pure culture of the ascomycetes, The aforementioned pure culture is incubated with fruits, nuts, grains, or parts thereof for at least one week to prepare an inoculated plant sample. The inoculated plant sample is placed in the sugar solution for at least one week to prepare the inoculated sugar solution. Mini truffles are produced by incubating the inoculated sugar solution on a third surface containing the aforementioned nutrient substrate. Methods that include...

14. The method according to claim 13, wherein the third surface comprises potato dextrose agar or pecan agar.

15. The method according to claim 14, wherein the sugar solution is honey or maple syrup.

16. A method for producing truffle-flavored food, To prepare an inoculated plant sample by incubating a pure culture of ascomycetes with fruit, nuts, grains, or a portion thereof for at least one week, The aforementioned inoculated plant sample is processed into food, Methods that include...

17. The method according to claim 16, wherein the pure culture is obtained according to the method according to any one of claims 1 to 15.

18. The aforementioned processing step is The aforementioned inoculated plant sample, moistened, is used in combination with food. The inoculated plant sample is dried. The inoculated plant sample is crushed into a powder. Making a mash using the aforementioned inoculated plant sample, and Mix the aforementioned powder or mash with the aforementioned food product. The method according to claim 16, comprising at least one of the following.

19. A method for preserving pure cultures of ascomycetes, The step of providing sterile water, The steps include adding the pure culture of the ascomycetes to the sterile water to prepare an inoculated solution, The steps include storing the inoculated solution under controlled temperature conditions of 1°C to 5°C, Methods that include...

20. A method for preserving pure cultures of ascomycetes, A step of providing a sterile culture medium selected from the group consisting of nuts or parts thereof and grains or parts thereof, The steps include: inoculating the pure culture of the ascomycetes into the sterile medium to produce a seed group inoculated with ascomycetes; The steps include storing the seed group inoculated with the aforementioned ascomycetes under controlled temperature conditions, Methods that include...

21. The method according to claim 20, wherein the controlled temperature conditions are -80°C to -20°C.

22. The method according to claim 20, wherein the controlled temperature conditions are 20°C to 25°C, and the method further comprises drying the ascomycete seed group.

23. A method for cultivating known truffle species, Obtaining spore-producing fruiting bodies of ascomycetes from soil, The process involves scraping the inside of the spore-producing fruiting body of the ascomycete to collect a fruiting body sample, The process involves placing the fruiting body sample on a first surface containing a nutrient substrate, wherein the nutrient substrate is monosaccharide, Plant-derived isolates, and Agar Including, posting, To provide the fruiting body sample on the aforementioned nutrient substrate, The incubation involves incubating the fruiting body sample on the nutrient substrate with an antimicrobial volatile mixture until mycelium is formed, wherein the presence of mycelium indicates new fungal growth. The process involves transferring the new fungal growth to a second surface containing the aforementioned nutrient substrate and a sterile medium selected from the group consisting of nuts or a portion thereof and grains or a portion thereof, in order to obtain an inoculum. A suspension is prepared by mixing sterile water and the inoculant in a volume ratio of 4 parts water to 1 part inoculant. The suspension is mixed with a soil mixture to produce a fungal soil mixture, Applying the aforementioned fungal soil mixture to the roots of a plant or a part thereof, Methods that include...

24. The method according to claim 23, wherein the fungal soil mixture is air-dried and stored at 3°C.

25. The method according to claim 23, wherein the fungal soil mixture is stored at -20°C.

26. The method according to claim 23, wherein the fungal soil mixture is stored at -80°C.

27. The method according to claim 23, further comprising the step of mixing the fungal soil mixture with soil in an area where the roots of a desired host tree are growing.

28. A truffle cultivation composition, grains, and Mycelium derived from truffle species The truffle species includes at least one of Sequence ID No. 1, Sequence ID No. 2, Sequence ID No. 3, Sequence ID No. 4, or Sequence ID No. 5, and the grain is inoculated with the truffle species. Truffle cultivation composition.

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