Process for the production of natural flavors, using a basidiomycete of the genus trametes and a food ingredient

Description

PROCESS FOR THE PRODUCTION OF NATURAL FLAVORS, USING A BASIDIOMYCETE OF THE GENUS TRAMETES AND A FOOD INGREDIENT Field of invention

[001] The present invention relates to a process for the biological production of flavoring agents, involving the growth of fungi on plant substrates, the use of this process to obtain said flavoring agents and the food ingredient obtained.

[002] The application areas of the present invention are the food, animal feed, pharmaceutical, cosmetics and cleaning products industries. Fundamentals of the invention

[003] Aroma is a crucial element in the acceptability of a product, and even though it is a response to a series of multisensory factors, aroma is still considered a key attribute for its perception. Currently, the production of aromas for food is mostly carried out through chemical synthesis.

[004] Synthetic processes for the production of aromas are considered hostile to the environment due to the use of reagents from non-renewable sources, such as petroleum derivatives (xylene, benzene, toluene, etc.), metals as catalysts, large amounts of energy, as well as the generation of waste.

[005] According to the legislation of several countries, aromas obtained through chemical synthesis are not considered natural. This context represents a challenge for a wide variety of industries, such as food, animal feed, pharmaceuticals and cosmetics, considering the Petition 870250011428, dated 12 / 02 / 2025, page 9 / 33 2 / 14 growing consumer preference for products with natural ingredients.

[006] Standards from multiple countries define natural aroma as that obtained by extraction from natural sources or produced by biotechnological means. Extraction of aromas from natural sources is the traditional process, but it has technical limitations such as low concentrations of the target compound, high cost, long processing time, and susceptibility to seasonality.

[007] In contrast, production via biotechnology does not present the limitation of seasonality and allows for the reduction of process costs through the use of agro-industrial waste and by-products as a substrate in solid-state fermentation processes.

[008] In this context, the present invention provides a biotechnological process that makes it possible to obtain natural aromas, particularly similar to those of passion fruit and meat.

[009] Passion fruit aroma can be obtained directly from the fruit through an extraction process. However, this process has certain limitations, such as low concentrations of the target compound, high cost, long processing time, and susceptibility to seasonality. Passion fruit is harvested 6 to 8 months after planting. In the state of São Paulo, for example, planting takes place between October and November, and harvesting occurs between January and March.

[0010] Another option is the production of passion fruit aroma through an enzymatic process. This type of process makes it possible to obtain natural aromas through reactions catalyzed by enzymes from various sources such as bacteria, fungi, Petition 870250011428, dated 12 / 02 / 2025, page 10 / 33 3 / 14 animals, etc. The main limitation associated with the production of aromas by enzymatic processes is the high cost of enzymes, which greatly increases the cost of the process. Passion fruit aroma and meat aroma can be produced through chemical synthesis. This process consists of a series of chemical reactions until a compound with an aroma similar to the desired one is obtained. However, one of the main limitations of chemical synthesis is that the aroma obtained is not considered natural by the legislation of several countries.

[0011] Meat aroma can be obtained synthetically, see, for example, US3761287A, through a process based on heating a mixture of amino acids, such as proline, methionine, cysteine, etc. Problems and limitations of these technologies consist in obtaining a non-natural aroma, which goes against consumers' desire for products with natural ingredients, and the need for many amino acids for aroma production.

[0012] Submerged fermentation, such as that taught in US2024130408A1, is another option for producing aromas that resemble the aroma of meat. However, there are certain limitations such as the recovery of these aromas and the quantity produced. Because they are in conditions different from those found in nature, microorganisms may produce aromas in smaller quantities.

[0013] The solution presented here has as its main differentiating factor the obtaining of natural aromas, as defined by the legislation of several countries, such as Regulation (EC) No 1334 / 2008; US Code of Federal Regulations Title 21, Section 101.2, through a sustainable process. Petition 870250011428, dated 12 / 02 / 2025, page 11 / 33 4 / 14

[0014] The present invention overcomes the shortcomings of technologies that obtain non-natural aromas from passion fruit and meat, such as those obtained by chemical processes. It also overcomes the shortcomings and limitations of extraction from natural sources, as it is a sustainable process and not susceptible to seasonality.

[0015] The inventors evaluated several strains of wild basidiomycete fungi, occurring in Brazilian biodiversity, exposed to various distinct plant substrates, and selected strains of basidiomycetes capable of fermenting these substrates, generating aromas reminiscent of passion fruit and meat when exposed to specific processing conditions.

[0016] Basidiomycetes are recognized as microorganisms that are a source of enzymes for the production of aromas with food applications. They can be used for the isolation of enzymes of interest, applicable in biochemical aroma synthesis processes, or as living reactors that, after the production of the aromas of interest, can be processed (e.g., dried, cooked, etc.) or used as a food ingredient in whole form (Berger and Ersoy, 2022).

[0017] Exposure of basidiomycetes to plant substrates rich in lignin, a plant polymer containing aromatic ring clusters, favors the production of aromas. When degraded by these fungi, the constituents of lignin can be transformed into compounds with aromas of vanilla, almonds, orange blossom, strawberry and anise (Lomascolo et al, 1999). Petition 870250011428, dated 12 / 02 / 2025, p. 12 / 33 5 / 14

[0018] The application of basidiomycetes to produce meat-like aromas is known. Initially, this was done by exposing the microorganisms to agro-industrial substrates containing sulfur (onion peel, garlic, etc.) in liquid fermentation processes, see, for example, US20230329298.

[0019] However, there is no prior description of the application of specific strains of basidiomycetes for the production of aromas reminiscent of passion fruit or meat, particularly through solid-state fermentation.

[0020] The solid-state fermentation process used in the present invention simulates the natural habitat of the selected basidiomycetes, enabling them to produce compounds of interest, such as aromas, in greater quantities than those found in submerged fermentation processes. Furthermore, it allows the use of agro-industrial waste and by-products as a substrate, providing a more suitable destination for such waste. Finally, the proposed process allows the fermented substrate to be used directly in the formulation of food, animal feed, and cosmetic products, without the need for a prior recovery and purification process of the compounds of interest. Brief description of the invention.

[0021] The present invention relates to the biological production of flavoring agents, as well as a process for producing such flavors and food ingredients involving the growth of the fungus on plant substrates.

[0022] The invention aims at the production of natural aromas of passion fruit and meat products. The term aroma, as used herein, means a specific odor and / or flavor. Petition 870250011428, dated 12 / 02 / 2025, page 13 / 33 6 / 14 The aroma comes from a flavoring substance or a mixture of flavoring substances.

[0023] Macrofungi (Basidiomycetes), known as support fungi, are a group of fungi belonging to the subkingdom Dikarya, in the kingdom Fungi. Their mycelium consists of microscopically thin filamentous hyphae. Macrofungi (Basidiomycetes) can be used for the method according to the invention and are edible basidiomycetes for humans.

[0024] The process, according to the invention, is a biotechnological process in which natural aroma is obtained from plant starting materials (substrates), with the aid of selected fungal strains.

[0025] The term “passion fruit aroma”, as used in the present invention, refers to an aroma that people generally associate with the passion fruit and its processed derivatives, such as juices and sweets.

[0026] The term “meat aroma” or “meat flavor,” as used throughout the text, refers to an aroma that people generally associate with meat products. The meat can be, for example, chicken, turkey, beef, veal, lamb, or pork. In particular, meat aroma encompasses flavors from forms of meat prepared for consumption, such as cooked, roasted, or fried meat.

[0027] The embodiment of the present invention is achieved by means of a process for the solid-state fermentation of edible filamentous fungi initiated from filamentous fungal spores. The process comprises sterilizing the solid fermentation medium, which may be a vegetable substrate, and inoculating a solution containing fungal spores. Petition 870250011428, dated 12 / 02 / 2025, page 14 / 33 7 / 14 selected from the sterile solid medium and incubate the inoculated medium at near ambient temperature for a specified period. Detailed description of the invention

[0028] The present invention relates to the biological production of flavoring agents, as well as a process for producing such flavors and food ingredients involving the growth of the fungus on plant substrates.

[0029] The invention comprises a process consisting of two main phases. The first phase involves the solid-state fermentation of a plant substrate by a basidiomycete, resulting in the production of an aroma similar to that of passion fruit. The second phase consists of the heat treatment of the product obtained in the first phase, leading to the optional formation of an aroma resembling that of meat products.

[0030] Therefore, the invention provides a biological process for the production of natural flavors. This process comprises the following steps: a) provide a plant substrate in a closed container that allows gas exchange; b) subject the container containing the plant substrate (a) to autoclaving; c) prepare a spore suspension of a basidiomycete of the genus Trametes in a 0.85% w / v saline solution; d) inoculate the container containing the plant substrate (a) with the spore suspension (c); e) Incubate the container from step d) at a temperature between 18°C ​​and 30°C, preferably 25°C, for a period of 2 to 20 days, preferably 10 to 15 days; f) to obtain the natural aroma of passion fruit; Petition 870250011428, dated 12 / 02 / 2025, page 15 / 33 8 / 14 g) Subject the product from the incubation in step e) to heat treatment by frying, baking, cooking or heating processes at temperatures between 80 and 250°C for 30s to 30 min; h) To obtain a meat aroma.

[0031] In a particular embodiment of the invention, the plant substrate is potato peel, oat hull or açaí residue.

[0032] The spore suspension of a basidiomycete of the genus Trametes contains basidiomycetes T. villosa. The inoculum containing basidiomycete spores contains 3x10⁵ to 1x10⁷ spores / mL of suspension.

[0033] The incubation stage lasts approximately 2 to 20 days, preferably between 10 and 15 days. In a particular and optional embodiment of the invention, the biological process for the production of natural flavors includes the stage of thermally treating the product of the incubation stage (g) by processes of frying, baking, cooking or heating at temperatures of 80 to 250°C for 30s to 30 min.

[0034] The invention includes the use of a basidiomycete of the genus Trametes in a process for producing natural aromas, in which the basidiomycete of the genus Trametes ferments in the presence of plant substrates. The basidiomycete is T. villosa and the plant substrate is potato peel, oat husk or açaí residue.

[0035] The invention also comprises a food, pharmaceutical or cosmetic ingredient that corresponds to the whole, a part or a processed derivative of a basidiomycete fermented by means of the process described herein. Petition 870250011428, dated 12 / 02 / 2025, page 16 / 33 9 / 14

[0036] Aroma production occurs throughout fungal growth in the substrate, beginning a few hours after inoculation and potentially extending for several weeks. The aromas formed are detected and recognized olfactorily by a technician in the field, without requiring specific training or methods.

[0037] The embodiment of the invention demonstrates that the Trametes villosa culture, when fermenting potato peel, oat husk or açaí residue (dry residue consisting of seeds, fibers and other solids resulting from the depulping of açaí) (e), produces an aroma similar to that of passion fruit (f).

[0038] Furthermore, the identification of aroma compounds indicated the absence of substances classically related to passion fruit aroma according to the literature, namely, ethyl butanoate, ethyl hexanoate and butyl ethanoate, potentially indicating that an aroma compound related to passion fruit aroma, not yet identified, is produced through fermentation.

[0039] In addition to the passion fruit aroma, the inventors sought to produce aromas reminiscent of meat products by fermenting T. villosa using sulfur-rich substrates, without, however, achieving success. Aware of the importance of heat treatment for the formation of meat aroma compounds, the hypothesis of heat-treating the product obtained from fermentation with vegetable substrates that generated passion fruit aromas was evaluated. Thus, it was found that, by heating the product of T. villosa fermentation with oat hulls (g), an aroma similar to that of meat products is obtained (h). Petition 870250011428, dated 12 / 02 / 2025, p. 17 / 33 10 / 14

[0040] Within the scope of the present invention, the product itself from fermentation with T. villosa can be considered a food product for immediate application, ready for human or animal consumption. Alternatively, it can be used as an ingredient (for example, in dry and powdered form) in the formulation of a food product, imparting the aroma of passion fruit or meat product to the product in which it is added, in addition to potentially providing other advantageous properties such as, for example, nutritional improvement and emulsification capacity.

[0041] Additionally, the aroma produced can be extracted using established volatile recovery techniques, such as solvent extraction (ethanol, ethyl acetate, etc.), followed by a solvent removal process and recovery of the desired aroma (e.g., rotary evaporation, low-temperature drying, or fractional distillation). This extract can be used in food formulations as a flavoring agent, provided that a solvent compliant with food processing legislation has been used. Examples of Implementation

[0042] The invention will now be demonstrated by means of examples which provide a complete description of how the natural flavorings of this invention are prepared. A person skilled in the art, in the light of this disclosure, will recognize that many alterations can be made to the specific embodiments disclosed and still obtain a similar or equivalent result without departing from the spirit and scope of the invention. Example 1: Production of passion fruit and meat aromas Petition 870250011428, dated 12 / 02 / 2025, p. 18 / 33 11 / 14

[0043] The passion fruit aroma production process comprises at least the aaf steps mentioned above and detailed below. The fermented fungus resulting from the aforementioned steps may optionally be subjected to an additional heating step (g), which gives rise to the meat aroma. Preparation of substrates for fermentation.

[0044] For fermentations, each substrate (potato peels, oat husks, açaí residue, orange peel, jaboticaba peel, onion peel and pineapple peel) is added to a container (e.g., 50 mL Erlenmeyer flask until it reaches approximately 40% of its volume). These flasks are closed to allow gas exchange (e.g., with a cotton plug) (a) and sterilized (e.g., autoclaving at 121°C for 15 minutes) (b). After cooling to room temperature, the media are inoculated with specific cultures, e.g., by adding a spore suspension (c). Preparation of spore suspension

[0045] To prepare the spore suspension (c), 10 mL of sterile 0.85% w / v saline solution is added to a fungal culture (e.g., in a Petri dish grown for fourteen days) selected from those listed in Table 1, followed by scraping (e.g., with a Drigalski loop) of the fungal culture. The suspension is filtered (e.g., through sterile gauze) to remove hyphae. In order to standardize the suspension (e.g., at a concentration of 1x107 spores / mL), a serial dilution of the spore suspension is performed, followed by cell counting in a Neubauer chamber. Petition 870250011428, dated 12 / 02 / 2025, page 19 / 33 12 / 14

[0046] The spore suspension preparation protocol was used to evaluate the potential for forming aromas of interest from 8 (eight) wild fungal species (Table 1), each applied to 7 (seven) different substrates (Table 2). The identity of each species was confirmed by phylogenetic identification using ITS (Internal Transcribed Spacer), revealing the actual genetic constitution of the fungi tested.

[0047] ITS identification utilizes specific regions of ribosomal DNA located between ribosomal RNA-coding genes (18S, 5.8S, and 28S). For this, fungi cultivated in Petri dishes were scraped and their DNA was extracted using a DNA extraction kit. The extracted genetic material was subjected to a PCR reaction, in which the ITS region was amplified with the ITS1 and ITS4 primers. Subsequently, the amplified DNA was sequenced to determine the nucleotide order. The obtained sequence was compared with databases such as NCBI (GenBank) in order to identify similarities with known sequences. Finally, the ITS sequences were analyzed to reconstruct evolutionary relationships using phylogenetic algorithms, such as Neighbor-Joining or Maximum Likelihood, allowing confirmation of the fungi's identity. Original identification (before molecular identification) Code ITS Identification Lentinus striguellus 1464 Lentinus sajor-caju Panus sp. 1671 Trametes cubensis Trametes versicolor 695 Trametes víllosa Petition 870250011428, dated 12 / 02 / 2025, page 20 / 33 13 / 14 Auricularía sp. 1508 Bj erkandera míkrofumosa Lentínus sp. 1856 Lentinus citrus Panus sp. 2008 Panus sp. Gloeophyllum sp. 1956 Trametes villosa Favolus sp. 1872 Favolus sp. Table 1: Microorganisms evaluated Substrate inoculation

[0048] After standardization of the spore suspension, it is added (e.g., 3 x 10⁵ spores) to the flasks containing the fermentation medium (d), previously sterilized and cooled. Subsequently, the flasks are incubated (e) (e.g., in a BOD type incubator at between 18 and 30°C, preferably 25°C for two weeks) until fungal growth and production of the desired aroma (f). Optional heat treatment

[0049] As a way to modify the aroma of the fermented product obtained in step (f), one can optionally proceed with a heat treatment, for example, in a microwave at a power between 800 and 1000 W for one minute (g). In the case of the microorganism Trametes villosa grown, for example, on oat husks, the heat treatment alters the aroma of the passion fruit ferment to an aroma similar to that of meat (h).

[0050] The results of aroma production by fermentation of residues shown in Table 2 below demonstrate the versatility of basidiomycetes of the species Trametes villosa (strain 1956) for aroma production, particularly of passion fruit, being able to achieve the desired technical effect from a series of distinct plant substrates. Petition 870250011428, dated 12 / 02 / 2025, page 21 / 33 14 / 14 Substrate Abiotic control (without microorganisms) Microorganisms tested (according to code in Table 1) 1464 1671 695 1872 1856 2008 1956 1508 Onion peel Caramelized onion NC NC NC NC NC NC NC NC NC Potato peel Potato Dried grass Pea Lemon balm tea SAP Grass / leaf Grass Passion fruit Floral Jaboticaba peel Jaboticaba NC NC NC NC NC NC NC NC NC Pineapple peel Pineapple Mate tea Cut grass SAP NC SAP SAP Citrus NC Orange peel Orange NC SDC NC Tangerine SDC NC SDC NC Oat husk Damp wood NC SAP NC NC NC NC Passion fruit pie Mold / guaiacol Açaí residue Feed Raw beet Pea Soil SAP SAP SAP Passion fruit Damp wood NC = Did not grow; SAP = No perceptible aroma; SDC = No difference from the control.

Claims

1. Process for the production of natural aromas, characterized by comprising the following steps: a) providing a plant substrate in a closed container that allows gas exchange; b) subjecting the container containing the plant substrate (a) to autoclaving; c) preparing a spore suspension of a basidiomycete of the genus Trametes in a 0.85% w / v saline solution; d) inoculating the container containing the plant substrate (a) with the spore suspension (c); e) incubating the container from step d) at a temperature between 18°C ​​and 30°C, preferably 25°C, for a period of 2 to 20 days, preferably 10 to 15 days; f) obtaining the natural passion fruit aroma; g) subjecting the product of the incubation from step e) to heat treatment by frying, baking, cooking or heating processes at temperatures between 80 and 250°C for 30s to 30 min; and h) obtaining a meat aroma.

2. Process, according to claim 1, characterized in that the plant substrate is potato peel, oat hull or açaí residue.

3. Process according to claim 1, characterized in that the inoculum contains 3x10⁵ to 1x10⁷ spores / mL of suspension.

4. Process, according to claim 1, characterized in that the geh steps are additional and optional. Petition 870250011428, dated 12 / 02 / 2025, page 23 / 33 2 / 2 5. Use of a basidiomycete of the genus Trametes, characterized by its ability to produce natural passion fruit or meat flavoring and its addition to food products.

6. Food ingredient, obtained by the process according to claim 1, steps a) af), characterized by having a passion fruit aroma.

7. Food ingredient, obtained by the process according to claim 1, steps a) to a) , characterized by having a meat aroma.

8. Food ingredient, obtained by the process according to claim 1, characterized by deriving, in whole or in part, from a substrate fermented by a basidiomycete.