Preparation of a meaty flavor based on biotechnology

Abstract

This invention relates to a method for preparing one or more seasonings, the seasonings being selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds. The method includes: providing a culture medium containing one or more components, the culture medium supporting the growth of fungi of the Basidiomycota phylum and convertible into one or more seasonings selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur flavor compounds; culturing the fungi of the Basidiomycota phylum in or on the culture medium under conditions supporting fungal growth and the formation of one or more seasonings; and optionally, recovering one or more seasonings.

Description

Technical Field

[0001] This invention primarily relates to a method for preparing one or more seasonings, wherein the seasonings are selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds. This invention also relates to a method for preparing a natural seasoning with a meaty flavor, various uses of culture media, and uses of fungi belonging to the phylum Sulfonobacter. This invention further relates to a composition and its use as a nutrient preparation or in a nutrient preparation. Background Technology

[0002] Meat aromas are produced through complex interactions between aroma and flavor molecules. Significant differences in the composition of aroma molecules exist between different types of meat, thus forming their unique flavor properties. By measuring flavor dilution factors, some key aroma compounds have been identified as characteristic of chicken and beef.

[0003] The complex aroma molecular composition of meat includes, among other things, unsaturated aldehydes such as the decadienal isomer; and organosulfur compounds such as 3-methyl-2-furanthiol, bis(2-methyl-3-furanyl)disulfide, 2,5-dimethyl-3-furanthiol, and 2-furfurylthiol. These organosulfur compounds, with their extremely low odor thresholds, are associated with different “meat” aromas. The molecules typically form during heating or cooking, rather than being inherent in raw meat. The formation of organosulfur compounds begins with the Maillard reaction between reducing sugars and sulfur-containing amino acids. Conversely, the formation of unsaturated aldehydes begins with the autoxidation of unsaturated fatty acids. In industrial production, these target compounds are generally generated by performing heat treatment to carry out the aforementioned reactions.

[0004] Basidiomycetes are a phylum of fungi with a wide range of unique enzymatic activities, enabling them to play important roles in ecosystems. Most edible fungi belong to the Basidiomycetes phylum. These enzymatic activities are likely to be used in the biotechnology-based preparation of ingredients for food, flavorings, and pharmaceutical purposes.

[0005] An interesting example is that basidiomycetes are sulfur bacteria (LSU) (also known as: sulfur-colored bacteria). *L. suis*, commonly known as "chicken mushroom of the forest," is a fungus whose edible fruiting bodies are found throughout forests worldwide and are highly prized for their distinctive "chicken" flavor. The aroma molecules of the wild fruiting bodies have been studied. Multiple studies involving extracellular polysaccharides, pigments, lectins, and aroma molecules have been conducted by studying *L. suis* hyphae. In deep culture, *L. suis* hyphae have been described as imparting a "seasoning" aroma. This sensory impression is primarily associated with a specific conversion of the amino acid L-isoleucine to 3-hydroxy-4,5-dimethyl-2(5H)-furanone (cucurbitacin) and the formation of further furanones. Despite the distinctive name of this fungus, no volatile organosulfur compounds with a "meaty" aroma, such as 2-methyl-3-furanthiol, were detected in any of the aforementioned studies.

[0006] Unlike the well-known LSU, the associated sulfur bacteria, such as Dark Brown Stripe Polyporus portentosus (LPO), dark-colored LPER or alpine Fomitopsis (LPM), whether in its fruiting body or hyphae, has been almost entirely unstudied for biotechnological purposes to date. Furthermore, LPOR, LPO, and LPM have not yet been described in the area of ​​aroma or flavor.

[0007] The past few years have witnessed a growing trend towards vegetarian or vegan diets. Conversely, many consumers enjoy the taste and smell of meat, leading to the emergence of meat alternatives on the market. Additionally, a rising demand for clean-label natural seasonings has been observed. In this regard, it has been demonstrated that consumers prefer natural seasonings over those that do not mention their source ingredients. <x>Seasonings (FTNS, 95 / 5), wherein 95% of the flavoring components in the seasoning must be derived from the sources specified in the instructions for use (FTNS). However, to date, no biotechnology-based natural preparation method has been found for volatile organic sulfur compounds with a "meaty" flavor, such as 2-methyl-3-furanthiol, 2-methyl-(3-methylthio)-furan, 2-methyl-(3-methyldithio)-furan, and 2-methyl-(3-methyltrithio)-furan.

[0008] In addition, thousands of tons of food sideflows (such as onion residue and chicken fat) accumulate each year, and these sideflows are mostly disposed of. Better utilization of these sideflows would be highly beneficial. Summary of the Invention

[0009] Therefore, one object of the present invention is to overcome at least some of the above-mentioned disadvantages in the prior art, and in particular to develop a natural seasoning, especially a natural seasoning with a rich "meat" aroma. <x>A "green" preparation method for seasonings (FTNS, 95 / 5). Furthermore, this method should preferably be low-cost, resource-efficient, and integrateable into existing food-related methods.

[0010] In the first aspect, this objective is achieved by a method as defined in appended claim 1.

[0011] The method for preparing one or more seasonings, wherein the seasonings are selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds, includes the following steps:

[0012] A culture medium containing one or more components is provided, which supports the growth of fungi of the Basidiomycota phylum and can be converted into one or more seasonings selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and .

[0013] Basidiomycetes fungi are cultured in or on a culture medium under conditions that support fungal growth and the formation of one or more seasonings; and optionally,

[0014] Recycle one or more seasonings,

[0015] Fungi are preferably hyphae, and / or

[0016] One or more seasonings preferably contain at least two compounds from two groups, more preferably at least one compound from the unsaturated lactone group and at least one compound from the other group, and most preferably at least one compound from each group.

[0017] This invention is primarily based on the discovery that fungi of the phylum Basidiomycota (formerly classified as class Basidiomycota), particularly in their hyphal form, can be used to prepare novel natural seasonings with a rich meaty flavor using biotechnology. Therefore, a universal platform is provided, eliminating the need for the harsh manufacturing conditions, organic solvents, or heavy metal catalysts unavoidable in chemical synthesis. Thus, this invention provides an environmentally friendly alternative to chemical synthesis, while also satisfying the requirements for natural... <x>The growing demand for seasonings (FTNS, 95 / 5).

[0018] The term "flavoring" is used herein to refer to compounds that impart a perceptible taste and / or odor in an amount of aroma activity. In this document, the term "aroma activity" refers to the amount of a compound in a formulation sufficient to cause a sensory effect on odor, trigeminal nerve, and / or taste receptors. This effect can also be manifested by reducing or masking unpleasant taste- and / or odor-based sensory perceptions.

[0019] Furthermore, the method according to the invention is characterized in that it provides the possibility of using food sidestreams (which would otherwise be disposed of, as described further below) as a source of high-quality flavoring substances, and thus can advantageously integrate food sidestreams into the value chain of existing food-related preparation methods.

[0020] Of particular interest in this invention are the perceived pleasant taste and / or odor impressions. Whether a taste and / or odor impression is pleasant or rather unpleasant can be assessed through sensory analysis performed by a trained group based on ratings of sensory impressions between negative (pleasant) and positive (unpleasant). Additional criteria such as very negative, neutral, and very positive can be provided for more precise classification. The aroma profile of the seasoning to be evaluated, which may be in a mixture with other compounds and possibly co-occurs with other seasonings, can be determined, for example, by gas chromatography-olfactography. In the case of this invention, the seasoning imparts a meaty aroma.

[0021] Preferred seasonings prepared by the method of the present invention are selected from 2,4-decadienal, particularly (E,E)-2,4-decadienal, 5-butyl-2(5H)-furanone, and 2-methyl-3-(methylthio)-furan. Other preferred seasonings obtainable by the method of the present invention are selected from those compounds mentioned in the Examples section. The exact composition, in terms of chemical substances and the amount of seasoning prepared, can be influenced not only by the culture medium and specific fungi, but also by the harvest time, i.e., the time at which cultivation ceases. Therefore, among other things, harvesting can be carried out when the particularly pleasant aroma characteristics and / or the desired aroma notes are stronger than rather undesirable ones.

[0022] Both main parts of basidiomycetes—the fruiting body and the hyphae—can be used for cultivation. The main technical advantage of hyphae is the variety of cultivation possibilities, such as effluent cultivation or submerged (or liquid) cultivation. In particular, through submerged cultivation, hyphae can grow in a resource-efficient manner within bioreactor systems.

[0023] The process of recovering seasonings may involve separating the seasonings from fungi, preferably fungal hyphae. For example, fungi can be removed by liquid-solid separation, such as filtration, gravity, or centrifugation. Separation can also be achieved by thermal separation techniques such as distillation, whereby the fungi are retained in the residue, while the seasonings can be recovered from the distillate. Seasonings can also be separated from fungi by extraction. For example, seasonings can be selectively adsorbed onto a solid phase (solid-state extraction, e.g., […]). After fungal removal, the seasoning can be further processed as needed and provided in liquid or solid form.

[0024] Prior to culturing, seed culture can be performed. The purpose of seed culture is to generate a sufficient number of fungal cells from the total amount used for cell thawing or cell line maintenance to inoculate the bioreactor (master culture) used for preparation. Seed culture may include pre-culture, which is carried out on one or more agar plates (preferably on malt extract agar) and / or in one or more culture flasks (preferably in a sterile liquid medium such as malt extract or yeast extract, preferably with the pH adjusted to <2.5, and in the dark, with stirring or agitation, for 5 to 14 days). Depending on the final scale used for the (master) culture, seed culture may also include pre-culture in one or more (smaller) bioreactors.

[0025] For the (primary) culture, the culture medium or its components, such as a carbon source and / or nitrogen source, can be pasteurized or sterilized. After pasteurization or sterilization, temperature-sensitive culture medium components and fungi are added. After pasteurization or sterilization, the culture medium is cooled. Then, (optionally pre-cultured) fungal cultures (inoculum) and temperature-sensitive components and / or other components (e.g., thiamine and / or ascorbic acid in some embodiments) can be added to the pasteurized or sterilized (partial) culture medium or components.

[0026] Following a brief description of the accompanying drawings, other aspects and embodiments of the invention will be described in detail. Attached Figure Description

[0027] Figure 1 Sensory analysis of hyphae of different basidiomycetes after deep culture in MEP medium (supernatant, n=10 trained team members, intensity level 0-3).

[0028] Figure 2 Comparison of hyphae of different basidiomycetes after different depths of culture in MEP medium. Aroma analysis of the supernatant was performed by headspace-solid phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS) of selected analytes.

[0029] Figure 3 Comparison of different sulfur bacteria strains cultured in ME or MEP media. Aroma analysis of selected analytes was performed by headspace-SPME-GC-MS.

[0030] Figure 4 Comparison of the effects of different acidifiers on biomass formation: Submerged culture of sulfur bacteria (LSU) in MEYD medium adjusted to different initial pH values. (na = unadjusted pH).

[0031] Figure 5 LSU was cultured in deep layers in MEP medium adjusted to different initial pH values ​​with / without thiamine hydrochloride added. Dry biomass was measured twice by weight after 13 days of culture.

[0032] Figure 6 LSU was cultured in deep layers in MEP medium adjusted to different initial pH values ​​with / without thiamine hydrochloride. Oxalic acid content was determined based on ion-exchange chromatography.

[0033] Figure 7 Dry biomass (DBM) of LSU master cultures supplemented with thiamine hydrochloride and L-ascorbic acid; error indication related to biological repeatability assays.

[0034] Figure 8 Kinetic analysis of mycelia of a subspecies of *Sulphurella* after deep culture in MEPM medium: determination of dry biomass and pH. Error indications are correlated with biological repeatability.

[0035] Figure 9 Kinetic analysis of thiamine during LSU submerged culture: determination of thiamine levels in media with and without thiamine hydrochloride. Chemical control medium without LSU inoculation.

[0036] Figure 10 Optimization of biological processes in LSU deep culture: the impact of biological process parameters on fungal growth. A model developed based on experimental design.

[0037] Figure 11 Optimization of culture medium for LSU submerged culture: the effect of culture medium composition on fungal growth. Development of a model based on experimental design.

[0038] Figure 12 Kinetic analysis of 2,4-decadienal formation during LSU submerged culture: estimation of 2,4-decadienal grades using SPME-GC-MS with internal standard ((E,Z)-(2-6)-nonadienal). Comparison with and without thiamine hydrochloride. Chemical control medium without LSU inoculation.

[0039] Figure 13 Optimization of biological processes in LSU deep culture: the impact of biological process parameters on selected analytes. A development model based on experimental design.

[0040] Figure 14 Optimization of culture medium for LSU submerged culture: The effect of medium composition on the formation of (E,E)-2,4-decadienal. Comparison of two development models based on experimental design.

[0041] Figure 15 LSU deep culture in MED medium supplemented with various types of yeast extracts (15 g / L each): Effect on 2,4-decadienal formation. Concentration estimation was performed by SPME-GC-MS using the internal standard ((E,Z)-(2-6)-nonadienal).

[0042] Figure 16 Optimization of culture medium for LSU submerged culture: the effect of culture medium composition on the formation of 5-butyl-2(5H)-furanone. A model based on experimental design was developed.

[0043] Figure 17 Optimization of culture medium for LSU submerged culture: the effect of culture medium composition on the formation of 2-methyl-3-(meththio)-furan. A model based on experimental design was developed.

[0044] Figure 18 Effects of adding cysteine ​​(CYS), glutathione (GSH), and thiamine hydrochloride to LSU submerged cultures in MED on the formation of 2-methyl-3-(meththio)-furan.

[0045] Figure 19 Effects of adding cysteine ​​(CYS), glutathione (GSH), and thiamine hydrochloride to LSU submerged cultures in MED on the formation of 2-methyl-5-(meththio)-furan.

[0046] Figure 20 Effects of adding L-ascorbic acid (asc.) and thiamine hydrochloride to LSU submerged cultures in ME on the formation of 2-methyl-3-(methylthio)-furan and 2-methyl-3-furanthiol. Measurements were repeated twice. Samples 1a-1c: uninoculated culture medium (chemical control group).

[0047] Figure 21 Effects of adding L-ascorbic acid (asc.) and thiamine hydrochloride to LPER submerged cultures in ME on the formation of 2-methyl-3-(methylthio)-furan and 2-methyl-3-furanthiol. The assays were repeated twice.

[0048] Figure 22 Kinetic analysis of 2-methyl-3-(methylthio)-furan formation during 10-day deep culture of LSU in MEPM medium: estimation of 2-methyl-3-(methylthio)-furan concentration using an internal standard (3-heptanone) by SPME-GC-MS. Comparison with and without thiamine hydrochloride addition. Chemical control medium without LSU inoculation.

[0049] Figure 23 Kinetic analysis of 2-methyl-3-(methylthio)-furan formation during the first 30 hours of deep culture of LSU in ME medium supplemented with thiamine hydrochloride (2.7 g / L) and L-ascorbic acid (1.4 g / L): estimation of 2-methyl-3-(methylthio)-furan concentration by SPME-GC-MS using the internal standard (3-heptanone).

[0050] Figure 24 Aroma characteristics of sulfur bacteria (LSU) after deep culture in MEPM with or without thiamine hydrochloride for 14 days. Descriptive sensory analysis was performed by a trained panel of 11 members, with intensity ratings from 0 ("no attribute detected") to 5 ("very strong").

[0051] Figure 25 dark Aroma characteristics of LPER (Large Periostracum) after deep culturing in MEPM with or without thiamine hydrochloride for 14 days. Descriptive sensory analysis was performed by a trained panel of 11 members, with intensity ratings from 0 ("No attribute detected") to 5 ("Very strong").

[0052] Figure 26 Dark brown streaks Aroma characteristics of LPO fungi after deep culturing in MEPM with or without thiamine hydrochloride for 14 days. Descriptive sensory analysis was performed by a trained panel of 11 members, with intensity ratings from 0 ("No attribute detected") to 5 ("Very strong").

[0053] Figure 27 Research on onion residue as a biotransformation culture medium: amino acid composition analysis in mg / 100g dry matter, repeated three times.

[0054] Figure 28 Biotransformation of onion residue based on LSU deep culture: SPME-GC-MS chromatograms of the LSU-transformed (A) and the uninoculated chemical control group (B) were compared. Culture medium composition: fresh onion residue, 30 g / L; L-ascorbic acid, 2 g / L; thiamine hydrochloride, 5 g / L.

[0055] Figure 29 Biotransformation of culture media containing different plant parts: Selected aroma analytes were screened from an uninoculated chemical control group (left) and those after LSU biotransformation (right) for different substrates. Corresponding concentrations in the culture media: celery powder 20 g / L; leek powder, 20 g / L; onion extract, 15 g / L–55 g / L; fresh onion pomace, 30 g / L–35 g / L. Other technical aids: L-ascorbic acid, 1 g / L–2 g / L; thiamine hydrochloride, 3 g / L–5 g / L. Samples were analyzed by SPME-GC-MS, with total area count used for evaluation. Cumulative isomers of dimethylthiophene and dimethylmethylthiothiophene. Graphs were designed using the statistical tool JMP15.1.

[0056] Figure 30 Biotransformation of onion oil from LSU-based submerged culture: SPME-GC-MS chromatograms comparing the LSU-based biotransformation (top) with the uninoculated chemical control group (bottom). MYD medium was supplemented with onion oil (7.5 μl / L). Detailed Implementation

[0057] As described above, one or more seasonings prepared by the method of the present invention have a meaty aroma. The meaty aroma can be more specifically described as an aroma characteristic characterized by (further distinguished) sub-aromas, such as mushroom, sulfur-containing vegetables, fried, oily, roasted, meaty, fried bacon, broth ("Maggi-like"), and chicken skin. The present invention provides a platform method that is generally capable of providing aroma characteristics including many or substantially all of the above-described sub-aromas, as shown in the Examples section, for example. For example, the present invention can provide a seasoning (or, as described above, a seasoning mixture) characterized by 1, 2, 3, 4, 5, 6, 7, 8, or 9 aromas from mushroom, sulfur-containing vegetables, fried, oily, roasted, meaty, fried bacon, broth ("Maggi-like"), and chicken skin. Furthermore, specific sub-aromas can be enhanced by adjusting precise method conditions, such as the fungi used for cultivation / preparation.

[0058] In a preferred embodiment of the present invention, the fungus is selected from the genera *Sulphurella*, *Microphyte*, *Leptochloa*, *Lentinula*, and *Lentinula*, with *Sulphurella* being the most preferred. Preferred species include: *Sulphurella*, *Microphyte* (garlic-shaped), *Leptochloa* (garlic-leaf variety), *Lentinula* (shiitake), *Lentinula tamariscina*, and *Lentinula datura*. Fungi, dark brown streaks Fungi and Alpine Polyporus. Sulfur-producing fungi, especially sulfur fungi and dark-colored fungi are preferred. Pore ​​fungus, mountain Fungi, dark brown streaks Porcini. Because of its chicken-like aroma, the fruiting body of sulfur fungus is also known as sulfur rack, forest chicken, chicken mushroom, or chicken fungus.

[0059] As described above, the culture medium is designed to support fungal growth. Therefore, it may include carbon and nitrogen sources. Additionally, in a preferred embodiment, the culture medium comprises or is substantially composed of food ingredients and / or food side streams. The food side streams are preferably used as carbon and / or nitrogen sources. The phrase "substantially composed of" means that at least 50% of the total dry weight of carbon and / or nitrogen sources contained in the culture medium, preferably at least 60% of the dry weight, more preferably at least 70% of the dry weight, and most preferably at least 80% of the dry weight, are derived from food ingredients and / or food side streams.

[0060] In addition to food sidestream or food ingredients, the culture medium may optionally contain one or more other components. Preferably, the optional component is a sulfur source, preferably a natural sulfur source. Preferred (preferably natural) sulfur sources comprise or consist of thiamine, cysteine, glutathione, or methionine, especially thiamine. Natural sulfur sources containing thiamine include, for example, egg yolks, cashew nuts, or fermentation products as described in patents WO 2004 106,557 or WO 2019 012,058. Thiamine may also be a salt, preferably hydrochloride, or used as a monophosphate, diphosphate, or triphosphate, or adenosyl thiamine triphosphate, or a mixture of these derivatives. By adding a sulfur source, preferably thiamine, to the culture medium, a particularly rich meaty aroma is formed. Biomass production can be increased by containing the sulfur source, preferably thiamine or consisting of thiamine. Furthermore, the preparation of organosulfur compounds, particularly 2-methyl-3-(methylthio)-furan and 2-methyl-3-furanthiol, can be greatly improved. The sulfur source may also be a combination of sulfur-containing compounds. Particularly preferred combinations include cysteine ​​and / or glutathione in addition to thiamine. Unless the context otherwise requires, acids and bases mentioned herein include the corresponding salts. Specifically, the term "thiamine" specifically includes hydrochloride, phosphates containing adenosylated forms such as adenosyl thiamine triphosphate (e.g., monophosphate, diphosphate, and triphosphate), and mixtures thereof.

[0061] The amount of sulfur source (preferably thiamine) relative to the total volume of the culture medium is preferably in the range of 0.1 g / L to 10 g / L, more preferably 0.2 g / L to 10 g / L, and even more preferably 0.5 g / L to 5 g / L. The given amounts apply to thiamine in hydrochloride form.

[0062] Another preferred component of the culture medium is an antioxidant and / or organic acid, particularly ascorbic acid, preferably in its L-form. The amount of the antioxidant and / or organic acid, particularly ascorbic acid, relative to the total volume of the culture medium is preferably 0.1 g / L to 10 g / L, more preferably 0.2 g / L to 5 g / L, more preferably 1 g / L to 4 g / L, and most preferably 2 g / L to 3 g / L. The presence of the antioxidant and / or organic acid can improve biomass production. When ascorbic acid is used, its presence in the culture medium, preferably in conjunction with thiamine, can also improve the preparation of organosulfur compounds, particularly 2-methyl-3-(methylthio)-furan and 2-methyl-3-furanthiol.

[0063] The pH value during the cultivation process is preferably acidic and can be buffered within the range of 1.5 to 6, preferably 1.5 to 5, and more preferably 1.5 to 4.5.

[0064] The optimal cultivation period is at least 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or at least 7 days and / or no more than 20 days, 19 days, 18 days, 17 days, 16 days, 15 days or no more than 14 days.

[0065] The temperature during the cultivation process is in the range of 15℃ to 35℃, preferably 20℃ to 28℃.

[0066] In the context of this invention, all cultivation methods, including submerged culture, aqueous culture, and solid-state culture, are primarily envisioned, even if a particular method may affect the resulting seasoning, i.e., its aroma characteristics. Submerged culture is preferred, wherein the culture includes stirring or agitation.

[0067] The culture is preferably carried out in the dark.

[0068] A second aspect of the present invention relates to a method for preparing a natural seasoning with a meaty flavor, comprising the following steps:

[0069] A culture medium comprising one or more components is provided, the culture medium supporting the growth of fungi of the Basidiomycota phylum and being convertible into a natural seasoning with a meaty flavor;

[0070] Basidiomycetes fungi are cultured in or on the culture medium under conditions that support fungal growth and flavoring formation; and

[0071] Optionally, the seasoning can be recycled.

[0072] The culture medium comprises food ingredients and / or food side streams, wherein the food ingredients and / or food side streams are selected from the group consisting of vegetables and their parts, fruits and their parts, grains and their parts, microbial cultures and their parts, and animal parts.

[0073] The fungus is preferably mycelium.

[0074] The second aspect of the invention is primarily based on the discovery that food sidestreams contain highly interesting seasoning precursors that can be biocatalytically converted into meat-flavored seasonings. Furthermore, the second aspect is characterized by providing the possibility of using otherwise disposed food sidestreams as a source of high-quality seasoning substances, and thus advantageously integrating food sidestreams into the value chain of existing food-related preparation methods.

[0075] Although the term "seasoning" is used herein in the singular form, the invention should not be construed as limited to the preparation of a single seasoning, but also includes the preparation of mixtures of two or more seasonings. This also applies to the terms "food sidestream" and "food ingredient," which can consist of food sidestream or food ingredient from a single source, or a mixture of food sidestream or food ingredient from two or more (different) sources.

[0076] Preferred food ingredients and / or food side streams include one or more of the following components:

[0077] Plants or parts thereof from the Allium genus, preferably onions, garlic, or leeks.

[0078] Plants or parts thereof derived from celery, carrots, or apples.

[0079] Animal-derived, preferably chicken meat, fat, and bones.

[0080] yeast.

[0081] Optionally, the food ingredients and / or food sidestreams exist in a processed form, such as residue, oil, molasses, flour, powder, isolate, concentrate, extract, or malt. Examples in this regard include (cereal) malt, malt extract, corn starch, yeast extract, concentrated vegetable juice, concentrated fruit juice, meat meal, and oil / broth powder. Specific food ingredients and / or food sidestreams include or consist of: onion residue, chicken meal (CMP), chicken fat, chicken broth powder, dehydrated chicken, chicken fat, concentrated onion juice, leek powder, celery powder, onion oil, garlic oil, or mixtures thereof. In one embodiment, the sidestream comprises or consists of chicken meat (waste). In another embodiment, the sidestream comprises or consists of: portions of plants from the Allium genus, preferably onions, garlic, and / or leeks. Particularly preferred food ingredients and / or food sidestreams include or consist of: onion residue and / or chicken fat.

[0082] In a highly preferred embodiment of the invention, the culture medium comprises or consists of: onion residue and / or chicken fat; an optional sulfur source, preferably thiamine; and an optional antioxidant and / or organic acid, preferably ascorbic acid.

[0083] A third aspect of the invention relates to the use of a culture medium for increasing fungal biomass and / or inhibiting oxalic acid formation and / or for the biotechnology-based preparation of one or more compounds, said fungi being selected from sulfur bacteria, dark-colored fungi, etc. Fungi, dark brown streaks Fungi and Alpine The culture medium comprises a group of polypores, wherein the compounds are selected from the group consisting of unsaturated aldehydes, unsaturated lactones and organosulfur compounds, and wherein the culture medium contains a carbon source, a nitrogen source and an optional sulfur source (as described herein).

[0084] The fourth aspect of the invention relates to a dark brown texture Fungi, dark Fungi or Alpine Uses of pore fungi in the preparation of seasonings based on biotechnology.

[0085] The fifth aspect of the invention relates to a composition, particularly a composition prepared (or prepared) according to the method described herein, comprising at least one compound selected from the group consisting of unsaturated aldehydes, at least one compound selected from the group consisting of unsaturated lactones, and at least one compound selected from the group consisting of organosulfur compounds.

[0086] A sixth aspect of the invention relates to the use of the compositions as described herein as nutritional preparations or in nutritional preparations, particularly as seasonings. Therefore, the invention also relates to nutritional or edible preparations comprising or consisting of the compositions according to the invention as described herein.

[0087] Regarding the preferred features and specific embodiments of the second to sixth aspects of the present invention, reference is made to the first aspect of the present invention, which defines the corresponding features and embodiments of the second to sixth aspects of the present invention.

[0088] The invention will now be described in more detail below with reference to selected embodiments.

[0089] Example

[0090] 1. Methods and Materials

[0091] Table 1 provides an overview of the tested fungi, their abbreviations, and sources (Germany Smart Microbial Culture Collection (DSMZ): Leibniz Institute DSMZ-German Microbial and Cell Culture Collection, Braunschweig, Germany; Netherlands Culture Collection (CBS): CBS-KNAW (Dutch Institute of Ecology) Culture Collection, Westerdijk Institute for Fungal Biodiversity, Utrecht, Netherlands). Table 2 shows the tested sulfur bacteria strains and their sources. Table 3 summarizes the tested culture media. Table 4 shows the tested food matrices and their sources.

[0092] Table 1. Cultured Basidiomycetes

[0093]

[0094]

[0095] Table 2 Screening of different sulfur bacteria strains (the strains used)

[0096] Internal naming conventions Source LSU 24 DSMZ Collectible No. 1014 LSU 79 DSMZ Collection No. 2785 LSU 319 Separated from the University of Giessen

[0097] Table 3. Composition of the culture medium used for culturing

[0098]

[0099] Table 4 Food bases used

[0100]

[0101]

[0102] Aqueous media were autoclaved prior to inoculation. Unless otherwise specified, cultures were incubated in shake flasks in the dark at 24°C, initial pH 2, and agitation at 150 rpm. These conditions were kept substantially consistent across all experiments unless parameters were intentionally varied to test their effects (e.g., pH). Temperature-sensitive supplements tested (e.g., thiamine hydrochloride, L-ascorbic acid, L-cysteine, L-glutathione, L-methionine) were dissolved in water, aseptically filtered, and added to the master medium after autoclaving.

[0103] 2. Screening of Basidiomycetes

[0104] Basidiomycetes fungi (Table 1) in hyphal form were cultured in shake flasks and MEP medium (Table 3) in the dark, in submerged cultures. Cultures were grown until maximum biomass was achieved, i.e., for 5 days (MAL), 7 days (MPR, MSC, LED), or 14 days (LSU, LPO, LPER), respectively. Ten trained panel members examined the supernatant formed after fermentation to obtain sensory characteristics. Sensory analysis was performed by direct smelling of the supernatant. Results are as follows: Figure 1 As shown.

[0105] Subsequently, the recovered supernatant was subjected to headspace-solid phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS) to determine aroma compounds. The results are as follows: Figure 2 As shown.

[0106] Table 5 shows the results of descriptive sensory analysis performed by trained panel members on different sulfur bacteria strains (Table 2) cultured in deep culture in ME or MEP medium (Table 3).

[0107] Table 5. Descriptive sensory analysis of different sulfur bacteria strains

[0108] strain / culture medium Sensory analysis (olfactory impressions) LSU 319MEP Meaty, sweet (pastry), oily, delicious LSU 319ME Spicy, meaty, chicken broth, delicious LSU 24MEP Spicy, meaty (somewhat unpleasant), chicken broth, Maggi seasoning, sweet LSU 24ME Pastries, meaty, sweet LSU 79MEP Fresh milk, cream, sweet LSU 79ME Fresh milk, cream, hot, curry

[0109] Then, the supernatants obtained from different sulfur bacteria strains cultured in ME or MEP media were examined by headspace-SPME-GC-MS. The results are as follows: Figure 3 As shown.

[0110] 3. Identification of flavor compounds formed by the hyphae of *Sulphurella* subspecies

[0111] Aroma (extraction) dilution analysis was performed on sulfur bacteria cultured in MEPM medium. Odor-active compounds were identified based on the determination of flavor dilution (FD) factors. Two analytical methods were compared for this purpose. First, aroma dilution analysis was performed on a nonpolar DB-5ms GC column using a stir bar adsorption extraction (SBSE)-thermal desorption (TDU)-GC-MS-olfaction assay, as described in the literature published by Trapp et al. (Trapp, T., DA, Fraatz, MA, et al., Development and validation of a novel method for aroma dilution analysis by stir bar adsorption extraction, *European Journal of Food Research and Technology*, 244, 949-957 (2018) (Trapp, T., DA, Fraatz, MA et al. Development and validation of a novel method for aroma dilution analysis by means of stir barsorptive extraction. Eur Food Res Technol 244, 949–957 (2018)). Then, aroma dilution analysis was performed based on liquid-liquid extraction of an azeotropic mixture of pentane and diethyl ether, combined with solvent-assisted flavor evaporation (SAFE). Based on the determination of the FD factor, the identified odor-active compounds are summarized in Table 6.

[0112] Table 6. Aroma extraction dilution analysis of sulfur bacteria cultured in MEPM medium. Odor-active compounds identified based on flavor dilution (FD) factor determination by GC-MS-olfaction assay (polar column: VF-WAX). RI = Retention Index; FD = Flavor Dilution Factor.

[0113]

[0114]

[0115] 4. Optimization of biomass production

[0116] 4.1 sulfur source

[0117] LSU mycelia were cultured in MEPM medium supplemented with different sulfur sources (17 mM each): L-methionine, thiamine hydrochloride, L-glutathione, and L-cysteine. After 13 days of culture, the biomass produced was measured. Furthermore, olfactory analysis was performed by directly smelling the supernatant.

[0118] The culture supplemented with thiamine produced the largest biomass, followed by the culture supplemented with glutathione. Furthermore, olfactory analysis revealed significant differences between samples, with the thiamine-supplemented sample exhibiting the most appealing "meaty" aroma. The results are shown in Table 6.1.

[0119] Table 6.1 Olfactory analysis of LSU cultured in MEPM medium supplemented with different sulfur sources (17 mM each) after different culture times.

[0120]

[0121] 4.2 organic acids

[0122] In another set of experiments, LSU mycelia were cultured in deep layers in MEPM medium with initial pH values ​​adjusted to 2.5 and 3.0 using different acidifiers. After 7 days of culture, the mass of the generated biomass was measured. The results are as follows: Figure 4 As shown in the figure. The results indicate that all acids led to an increase in biomass, thus demonstrating that organic acids were superior to inorganic acids.

[0123] 4.3 pH value

[0124] Next, LSU mycelia were cultured in deep layers in MEP medium adjusted to different initial pH values. Experiments were conducted with and without thiamine hydrochloride. After 13 days of culture, the mass of the generated biomass was determined twice using gravimetric analysis. Results are as follows... Figure 5 As shown in the figure. The results indicate that the lower the initial pH, the greater the biomass generated. This effect was observed in both the presence and absence of thiamine hydrochloride.

[0125] In addition, the amount of oxalic acid was determined by ion-exchange chromatography, and the amount of oxalic acid was described relative to the initial pH value, such as... Figure 6 As shown in the figure. From these results, we can conclude that the lower the pH value, the greater the biomass, which may be due to the inhibition of oxalic acid production at lower pH values.

[0126] 4.4 Thiamine and ascorbic acid

[0127] In another round of experiments, the effects of thiamine and / or ascorbic acid on LSU mycelial growth were further tested. The results of the concentrations and combinations tested after 12 days of culture, as well as the dry biomass (DBM), are shown below. Figure 7 As shown.

[0128] 4.5 Kinetic analysis of dry biomass, pH and thiamine levels

[0129] Kinetic analysis was performed on the mycelia of *Sulphurella* subsp. *sulfuronide* cultured in MEPM medium. For this purpose, dry biomass and pH were measured at several time points over a total culture period of 14 days. Results are as follows: Figure 8 As shown.

[0130] Figure 9 The thiamine levels are shown as of incubation time in culture media with or without added thiamine.

[0131] 4.6 Bioprocess optimization

[0132] Methods were developed and optimized to improve fungal biomass formation. To this end, in the Design of Experiments (DoE), the selected bioprocess parameters were treated as categorical or numerical variables, varying with different levels. DoE minimizes the required sample size and allows for the investigation of statistical interactions between factors. The optimized variables and levels of the bioprocess parameters, along with their respective ranges, are shown in Table 7.

[0133] Table 7. Optimization of biological processes in LSU deep culture: Design of Experiments (DoE): Customized design of response surface methodology. DoEs were created using the statistical analysis tool JMP.

[0134]

[0135] The effects of biological process conditions on fungal growth as determined by DoE, such as Figure 10 As shown.

[0136] The same DoE-based approach was implemented to optimize the culture medium composition for LSU submerged culture. Specifically, the concentrations of yeast extract, malt extract, peptone, glucose, and thiamine hydrochloride were varied, with the results shown below. Figure 11 As shown.

[0137] 5. Optimization of 2,4-decadienal generation

[0138] The formation of 2,4-decadienal over time during the deep culture of LSU was investigated. 2,4-decadienal was evaluated using SPME-GC-MS. Cultures were performed with and without thiamine. Figure 12 It was found that more 2,4-decadienal was formed in the presence of thiamine than in the absence of thiamine. The optimal concentration of (E,E)-2,4-decadienal was discovered on day 7. No (E,E)-2,4-decadienal was formed in the absence of fungi.

[0139] Based on DoE, a model for optimizing biological processes in deep LSA culture was developed. Figure 13 The effects of bioprocess conditions on selected analytes, namely 3-phenylpropanol, 2-heptanone, 5-butyl-2(5H)-furanone and E,E-2,4-decadienal, are shown. Figure 14 The predicted effects of culture medium composition are shown using analyte (E,E)-2,4-decadienal as an example.

[0140] Figure 15 The results of LSU submerged culture in MED medium supplemented with different types of yeast extracts (15 g / L each) are presented again. Interestingly, SPME-GC-MS analysis using (E,Z)-(2-6)-nonadienal as an internal standard revealed that additional nitrogen sources such as yeast extracts reduced the formation of 2,4-decadienal. This was also observed in the model obtained via DoE, thus confirming the model's validity.

[0141] 6. Optimization of rare unsaturated lactone formation

[0142] The formation of lactones by LSU during 15 days of deep culture in MEP medium was determined by SBSE-GC-MS. The results and fungal growth parameters are summarized in Table 8.

[0143] Table 8 Kinetic analysis of lactone formation during LSU deep culture in MEP medium: Fungal growth parameters and lactone concentration (ppb) were determined by SBSE-GC-MS with 2-nonanol as an internal standard.

[0144]

[0145]

[0146] The effect of culture medium composition on the formation of 5-butyl-2(5H)-furanone via LSU submerged culture was predicted using DoE. Results are as follows: Figure 16 As shown.

[0147] 7. Optimization of the generation of organic sulfur aroma compounds

[0148] The effect of culture medium composition on the formation of 2-methyl-3-(meththio)-furan via LSU submerged culture was predicted using DoE. Results are as follows: Figure 17 As shown, a significant positive correlation was found between the amount of 2-methyl-3-(meththio)-furan formed and the concentration of yeast extract or thiamine hydrochloride.

[0149] 7.1 The effect of sulfur source on the formation of organic sulfur aroma compounds

[0150] The effects of cysteine ​​(CYS), glutathione (GSH), and thiamine hydrochloride, as examples of different sulfur sources, on the formation of organosulfur aroma compounds in LSU submerged cultures in MED medium were investigated. The cultures were incubated for 7 days. Figure 18 and Figure 19 The results for 2-methyl-3-(methylthio)-furan and 2-methyl-5-(methylthio)-furan are shown respectively.

[0151] 7.2 The effects of ascorbic acid and thiamine on the formation of organosulfur aroma compounds

[0152] The effects of L-ascorbic acid (asc.) and thiamine hydrochloride on the formation of organosulfur aroma compounds, particularly 2-methyl-3-(methylthio)-furan and 2-methyl-3-furanthiol, in LSU submerged cultures and LPER submerged cultures in ME medium were determined. Results are as follows: Figure 20 and 21 As shown.

[0153] 7.3 Kinetic analysis of the formation of organic sulfur aroma compounds

[0154] The formation of 2-methyl-3-(meththio)-furan during 10-day deep culture of LSU in MEPM medium was determined by SPME-GC-MS. Cultures were performed with and without thiamine hydrochloride. Results are as follows: Figure 22 As shown.

[0155] Then, the formation of 2-methyl-3-(meththio)-furan during the first 30 hours of LSU submerged culture in ME medium was determined by SPME-GC-MS. The medium was supplemented with thiamine hydrochloride and ascorbic acid. The results are as follows: Figure 23 As shown.

[0156] Interestingly, the concentration of 2-methyl-3-(methylthio)-furan reached its maximum after 22 hours, then began to decline sharply until day 3. On day 6, the concentration began to increase slightly again until the end of the incubation.

[0157] 7.4 Aroma characteristics of cultured basidiomycetes

[0158] Aroma characteristics of LSU, LPER, and LPO were determined after 14 days of culture in MEPM medium with or without thiamine hydrochloride, using a descriptive sensory analysis conducted by a trained panel (n=11) on an intensity scale from 0 ("attribute not detected") to 5 ("very strong"). The results are as follows: Figure 24 As shown in Figures 25 and 26.

[0159] By adding label-free and label-labeled [materials] to the deep culture of LPER 13 The thiamine isomer of C was used to elucidate the metabolic formation of 2-methyl-3-(methylthio)furan. The addition of thiamine hydrochloride-(4-methyl- 13 C-thiazolyl-5-yl- 13 The mass spectrum of 2-methyl-3-(methylthio)furan formed after C3) shows a +3 mass fragment shift (data not shown).

[0160] 2,5-Dimethyl-3-methylthiothiophene was identified in LSU cultured in a liquid medium supplemented with 500 ppm of 2,5-dimethylthiophene (20 g / L malt extract, 5 g / L thiamine hydrochloride, 2 g / L L-ascorbic acid). SPME-GC-MS chromatograms of chemical control groups with / without supplementation of LSU and without inoculation with LSU were compared. The measured mass spectra of 2,5-dimethyl-3-methylthiothiophene (retention index on polar column: 1221) were compared with spectra from an internal database (data not shown).

[0161] 8. Basidiomycelium cultured in side stream from the food industry

[0162] The effects of deep culture of LSU in different media containing side streams from the food industry were examined.

[0163] 8.1 Biotransformation in culture media containing vegetables or fruits

[0164] First, LSU was cultured in different media containing vegetables or fruits. Then, trained panel members performed sensory evaluations of the fermented samples based on "meat" aroma (grades: 0 ("no meat aroma") - 3 ("strong meat aroma"). The media are shown in Table 9, where the first column is the corresponding food side flow and the last three columns are the auxiliary media compositions M1 to M3.

[0165] Table 9. Submerged culture of sulfur bacteria in different media containing side streams from the food industry. Trained panel members performed sensory evaluation of the fermented samples on the "meat" aroma (grades: 0 ("no meat aroma") - 3 ("strong meat aroma").

[0166]

[0167]

[0168] The compositions of the auxiliary culture media M1 to M3 are as follows:

[0169]

[0170] 8.2 Biotransformation of culture medium containing chicken meat

[0171] LSU was then cultured in submerged medium containing chicken meal (CMP) (18 g / L), chicken fat / broth powder (10 g / L), or dehydrated chicken (10 g / L) as the matrix. Other technical aids in the medium were L-ascorbic acid (2 g / L) and thiamine hydrochloride (5 g / L). After culture, SPME-GC-MS chromatograms were obtained. As a control, corresponding chromatograms were obtained from uninoculated medium (data not shown). The peak areas of the identified organosulfur compounds obtained by SPME-GC-MS are summarized in Table 10.

[0172] Then, a similar experiment was performed using pure chicken oil. Specifically, LSU was cultured in a biphasic medium containing chicken oil (50 g / L CMP; 10 g / L malt extract; 2 g / L L-ascorbic acid; 5 g / L thiamine hydrochloride). SPME-GC-MS chromatograms of the culture medium and the uninoculated medium (as a control group) were obtained (data not shown).

[0173] Table 10. Biotransformation of culture media containing different parts of chicken via LSU deep culture: peak areas of identified organosulfur compounds obtained by SPME-GC-MS. Comparison between the uninoculated chemical control group and the culture media after LSU biotransformation. Culture medium composition: CMP, 20 g / L; L-ascorbic acid, 2 g / L; thiamine hydrochloride, 5 g / L.

[0174]

[0175] 8.3 Biotransformation of culture media containing a portion of Allium species

[0176] Next, this paper uses onion residue as an example to study the composition of a culture medium for preparing basidiomycete-based seasonings, using a portion of the Allium genus as an example. The amino acid composition of the tested onion residue is as follows: Figure 27 As shown.

[0177] LSU was then cultured in submerged medium containing onion residue (30 g / L fresh onion residue; 2 g / L L-ascorbic acid; 5 g / L thiamine hydrochloride). SPME-GC-MS chromatograms of the culture medium and the uninoculated medium (as a control group) were recorded after incubation. The chromatograms are shown below. Figure 28 As shown, biotransformation of deep cultures using LSU resulted in a significant change in the composition of volatile aroma compounds. Compared to the medium (control group), cultures using LSU showed a significant reduction in aroma compounds that impart an "onion" flavor, such as dimethyl trisulfide and several isomers of dimethylthiophene. This demonstrates that fungal biotransformation can reduce undesirable or unpleasant flavors. Furthermore, the biotransformation enriched cultures containing organosulfur aroma compounds with a "meaty" flavor, such as 2-methyl-3-(methylthio)-furan and, after preliminary identification, dimethylthiophene isomers. As described above, this further confirms the hypothesis that dimethylthiophene is biotransformed into dimethylthiophene using the tested fungal hyphae.

[0178] Then, a similar experiment was conducted using a culture medium composed of celery powder (20 g / L), leek powder (20 g / L), onion extract (15 g / L-55 g / L), and fresh onion residue (30 g / L-35 g / L) as the Allium genus-based culture medium composition. The culture medium also contained 1 g / L-2 g / L L-ascorbic acid and 3 g / L-5 g / L thiamine hydrochloride. The peak areas of flavor compounds in each culture solution after identification, determined by SPME-GC-MS, were plotted as box plots, as shown below. Figure 29 As shown. For simplification, the peak areas of the isomers of dimethylthiophene and dimethylmethylthiothiophene are summed, respectively.

[0179] Another type of LSU was cultured using MYD medium supplemented with onion oil (7.5 μl / L). The SPME-GC-MS chromatograms of the culture medium obtained after culture and the uninoculated blank medium (as a control group) are shown below. Figure 30 As shown. At the same time, as Figure 28 As shown, the developed fermentation platform altered the composition of volatile aroma compounds in the onion oil used. Undesirable flavor molecules were reduced, while highly efficient compounds with desired flavor properties were generated through metabolic transformation by fungal hyphae.< / x> < / x> < / x>

Claims

1. A method for preparing one or more seasonings, wherein the seasonings are selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds, the method comprising the following steps: A culture medium comprising one or more components is provided, the culture medium supporting the growth of fungi selected from the Basidiomycota phylum of sulfur bacteria, *Phellinus tarda*, *Phellinus alpinus*, and *Phellinus tarda*, and convertible into one or more seasonings selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds, wherein the culture medium contains food ingredients and thiamine. The fungi are cultured in or on the culture medium under conditions that support the growth of fungi selected from sulfur bacteria, diatomaceous porphyria, alpine porphyria, and brown-striped porphyria, and the formation of one or more seasonings, wherein the one or more seasonings include at least 2,4-decadienal.

2. The method of claim 1 further includes recycling the one or more seasonings.

3. The method according to claim 1 or 2, wherein the fungus is a hyphae.

4. The method of claim 1, wherein the one or more seasonings comprise at least two compounds selected from two different groups of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds.

5. The method according to claim 4, wherein one of the at least two compounds is selected from the unsaturated lactone group.

6. The method of claim 1, wherein the one or more seasonings comprise at least one compound selected from each of the groups of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds.

7. The method according to claim 1, wherein the fungus is *Phellinus glomeratus*.

8. The method according to claim 1, wherein, The thiamine concentration is in the range of 0.1 g / L to 10 g / L relative to the total volume of the culture medium.

9. The method according to claim 8, wherein, The thiamine concentration is in the range of 0.2 g / L to 10 g / L relative to the total volume of the culture medium.

10. The method according to claim 9, wherein, The thiamine concentration is in the range of 0.5 g / L to 5 g / L relative to the total volume of the culture medium.

11. The method according to claim 1, wherein, The culture medium also contains at least one of an antioxidant and an organic acid.

12. The method according to claim 11, wherein, At least one of the antioxidants and organic acids includes L-ascorbic acid.

13. The method according to claim 12, wherein, The L-ascorbic acid concentration is in the range of 0.1 g / L to 10 g / L relative to the total volume of the culture medium.

14. The method according to claim 1, wherein, The pH value during the cultivation process is in the range of 1.5 to 6.

15. The method according to claim 1, wherein, The culture process lasts for at least 12 hours.

16. The method according to claim 1, wherein, The temperature during the cultivation process is in the range of 15℃ to 35℃.

17. The method according to claim 1, wherein, The culture is carried out by submerged fermentation, hydrothermal fermentation, or solid-state fermentation.

18. The method according to claim 1, wherein, The culture was carried out in the dark.

19. The method according to claim 1, wherein, The culture is carried out under stirring or agitation.

20. The method according to claim 1, wherein, The culture process shall not exceed 20 days.

21. The method according to claim 1, wherein, The temperature during the cultivation process is in the range of 20℃ to 28℃.

22. A method for preparing a natural seasoning with a meaty flavor, comprising the following steps: A culture medium containing one or more components is provided, the culture medium supporting the growth of fungi selected from the Basidiomycota phylum such as sulfur bacteria, diatomite, alpine diatomite, and brown-striped diatomite, and can be converted into a natural seasoning with a meaty flavor. The fungi are cultured in or on the culture medium under conditions that support the growth of fungi selected from sulfur bacteria, diatomaceous porphyria, alpine porphyria, and brown-striped porphyria and the formation of the seasoning. The culture medium contains food ingredients and thiamine, wherein the food ingredients are selected from the group consisting of vegetables and their parts, fruits and their parts, grains and their parts, microbial cultures and their parts, and animal parts. The natural flavorings mentioned therein include at least 2,4-decadienal.

23. The method according to claim 22, wherein, The fungus is hyphae.

24. The method according to claim 22 or 23, wherein, The vegetables and their parts include plants or parts of plants from the Allium genus.

25. The method of claim 24, wherein the Allium genus includes onion, garlic, or leek.

26. The method according to claim 24, wherein, The animal's parts include its meat, fat, and bones.

27. The method of claim 24, wherein the animal comprises a chicken.

28. The method according to claim 24, wherein, The microbial culture and a portion thereof contain yeast.

29. The method of claim 22, wherein the culture medium comprises at least one selected from onion residue and chicken fat.

30. The method of claim 29, wherein the culture medium comprises at least one selected from antioxidants and organic acids.

31. The method of claim 30, wherein the antioxidant or the organic acid comprises L-ascorbic acid.

32. Use of a culture medium for the bio-based preparation of one or more compounds, said compounds being selected from the group consisting of unsaturated aldehydes, unsaturated lactones, and organosulfur compounds, wherein said one or more compounds comprise at least 2,4-decadienal, wherein, The culture medium supports the growth of fungi selected from the Basidiomycota phylum, including sulfur bacteria, *Phellinus datura*, *Phellinus alpinus*, and *Phellinus datura*. The culture medium contains a carbon source, a nitrogen source, and an organic sulfur source, wherein the organic sulfur source includes thiamine.

33. The use of *Phellinus davidii*, *Phellinus datura*, or *Phellinus alpinus* in the preparation of seasonings based on biotechnology by the method according to any one of claims 1 to 31.

34. A composition prepared by the method of any one of claims 1 to 31, the composition comprising at least one compound selected from the group consisting of unsaturated aldehydes, at least one compound selected from the group consisting of unsaturated lactones, and at least one compound selected from the group consisting of organosulfur compounds, wherein the composition comprises at least 2,4-decadienal.

35. Use of the composition as defined in claim 34 as a nutritional preparation or in a nutritional preparation.

36. Use of the composition as defined in claim 34 as a seasoning.

Images