Lepista fungus strain LV17 capable of efficiently synthesizing various natural characteristic flavor substances and application of lepista fungus strain LV17

Abstract

The invention discloses a mushroom strain LV17 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.42460 and an application of the mushroom strain LV17. The strain can efficiently synthesize up to 64 key aroma substances in liquid fermentation, the yield of a core flavor substance dihydro-2-methyl-3 (2H)-furanone stably reaches milligram per liter, and the relative odor activity value is up to about 3.1 * 10 < 5 >; high-valence terpenoids such as beta, beta-ionone and the like are also remarkably enriched, so that strong and pure flower fragrance-fruit fragrance-sweet fragrance composite characteristics are jointly formed. Electronic nose and GC-MS (gas chromatography-mass spectrometry) analysis show that the overall aroma intensity of the strain is obviously superior to that of a control strain, and the response values of bad sulfides and amines are not abnormal. The strain can be applied to the fields of natural spices, food aroma enhancement and the like, and an efficient microbial cell factory is provided for solving the problem of production of natural characteristic aroma substances.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary technical field of microbial fermentation engineering, food biotechnology and natural product chemistry, and specifically relates to a mushroom strain LV17 that can efficiently synthesize a variety of natural characteristic aroma substances and its applications. Background Technology

[0002] With consumers' growing demand for "natural sources" and "health and safety," the global market for natural fragrances and flavors is experiencing rapid growth. Currently, the acquisition of natural fragrances mainly relies on: plant extraction methods such as rose essential oil and citrus oil, but these are limited by factors such as planting area, climate conditions, and low extraction rates, resulting in high prices and unstable supply; animal-derived extraction methods such as musk and civet, which involve ethical issues and are scarce; and microbial fermentation, which is considered the most sustainable development potential method, especially suitable for producing rare and expensive natural aroma compounds.

[0003] Characteristic aroma compounds are core elements in shaping high-end flavorings and enhancing the flavor and quality of food and beverages. However, the current production technology of these key substances faces significant bottlenecks, severely restricting the development and application of natural, high-quality flavor products. Currently, characteristic aroma compounds are mainly obtained through the following routes, but all have fundamental flaws: Industrial production of compounds such as β-ionone (a key floral aroma compound) and 4-methyl-5-(β-hydroxyethyl)thiazole (a key meat aroma compound) heavily relies on mature chemical synthesis routes. While this method has the advantages of high yield and relatively low cost, its products are legally defined as "artificially synthesized flavorings," failing to meet the market's urgent demand for "all-natural" products. Furthermore, chemical synthesis struggles to selectively obtain isomers with specific optical activities, resulting in a "dull and lifeless" aroma quality, far removed from the delicate and vibrant qualities of natural extracts. For compounds such as (E)-2-hexenal (a characteristic green aroma compound), natural sources mainly rely on solvent extraction from specific plants (such as juniper and phellodendron). While the products obtained by this method possess natural properties, the extremely low extraction rate, complex processes, and exorbitant costs are due to the target substance being present in trace amounts (typically at the ppm or even ppb level) in the raw materials. Furthermore, the supply of raw materials is highly dependent on seasonality and origin, making it completely unsuitable for stable industrial production. Cutting-edge biomanufacturing methods remain in an inefficient research and development stage. To obtain naturally labeled products, biosynthesis and enzymatic conversion have become research hotspots. For example, metabolic engineering of yeast to produce β-ionone, or the conversion of fatty acids into (E)-2-hexenal using a lipoxygenase / lyase system. However, these cutting-edge technologies generally suffer from core problems such as lengthy metabolic pathways, insufficient precursor supply, low or unstable activity of key enzymes, and low product concentrations. This results in production efficiency far from achieving commercially viable levels, and currently remains only at the laboratory research or pilot-scale stages. In summary, existing technologies are caught in a dilemma: using chemical synthesis methods results in the loss of "natural" attributes and competitiveness in the high-end market; using natural extraction methods faces industrialization obstacles such as unstable supply and excessively high costs; and emerging biological / enzymatic methods are hampered by low production efficiency, making it difficult to achieve large-scale production.

[0004] Although there are some commonly used aroma-producing microorganisms in industry, they all have certain limitations: Saccharomyces cerevisiae, a type of yeast, mainly produces higher alcohols and esters, and its aroma is relatively simple, mainly wine-like; filamentous fungi such as Aspergillus oryzae and Penicillium can synthesize aroma components such as lactones and terpenes, but may produce fungal toxins, raising safety concerns; bacteria, represented by lactic acid bacteria and Bacillus, can produce substances such as diacetyl and short-chain acids, but their aroma intensity is usually weak and often accompanied by a sour taste; while macrofungi (mainly referring to some edible fungi) produce aroma substances mainly composed of octane compounds and aldehydes, which also suffer from insufficient aroma intensity and have a high content of sulfur compounds, lacking pleasant floral and fruity notes. In summary, the current application of aroma-producing microorganisms generally faces several core problems: insufficient aroma intensity and complexity, with most strains only able to produce 1-3 main aroma substances, making it difficult to create a rich and layered olfactory experience; numerous unpleasant odor byproducts, such as sulfides, amines, and lower fatty acids, affecting the purity of the aroma; and low metabolic efficiency, resulting in insufficient accumulation of key aroma precursors and obstructed biosynthetic pathways.

[0005] Lepista fungi are a group of edible fungi with typical aroma characteristics. Previous studies have identified abundant volatile aroma compounds in the mycelium and fruiting bodies of Lepista, a member of the same genus, and confirmed that its aroma biosynthesis-related enzyme system is relatively complete. However, to date, no research has successfully screened and developed Lepista-specific strains and related technologies that can systematically solve the aforementioned industry bottlenecks and possess both ultra-high aroma production capacity and commercial production potential. Discovering such strains not only has significant scientific value, but also means seizing the high ground in emerging technologies for the biomanufacturing of natural fragrances and developing core raw materials with independent intellectual property rights and market competitiveness.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a *Lentinula* fungal strain LV17 capable of efficiently synthesizing a variety of natural aroma compounds. This strain can act as a highly efficient "cell factory" to synthesize various natural aroma compounds, thereby solving the supply bottleneck of high-end natural fragrance raw materials.

[0008] The second objective of this invention is to provide a fermentation product obtained from the above-mentioned *Lentinula* fungal strain LV17.

[0009] A third objective of this invention is to provide a microbial agent containing the above-mentioned fermentation products.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned *Lentinula edodes* fungal strain LV17, fermentation products, or inoculants.

[0011] The fifth objective of this invention is to provide a method for preparing the above-mentioned fermentation products.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a *Lepista* fungal strain LV17 capable of efficiently synthesizing a variety of natural characteristic aroma substances. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42460. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 13, 2026. It is classified and named *Lepista saeva*, and the strain is referred to as LV17.

[0013] In the aforementioned *Lentinula* fungal strain LV17 from the first aspect, further, The culture temperature for the *Lentinula* fungal strain LV17 is 24-28℃, preferably 25℃; And / or, the activation medium for the *Lentinula* fungal strain LV17 is PDA; And / or, the propagation medium for the *Lentinula* fungal strain LV17 is a solid plate medium, the solvent of which is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, peat moss 5-8 g / L, agar 15-25 g / L; preferably: glucose 20 g / L, brewer's yeast powder 3 g / L, peat moss 8 g / L, agar 20 g / L; And / or, the *Lentinula* fungal strain LV17 is cultured in the dark.

[0014] Based on morphological observation and molecular identification, this fungal strain LV17 belongs to Lepistasaeva.

[0015] A second aspect of the present invention provides a fermentation product obtained by liquid fermentation culture of the *Lentinula* fungal strain LV17 described in the first aspect of the present invention.

[0016] In the fermentation products of the second aspect mentioned above, further, The solvent of the liquid fermentation culture medium is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L. And / or, the *Lentinula* fungal strain LV17 added to the liquid fermentation culture medium for fermentation culture is a mycelium-covered block; And / or, the conditions for the liquid fermentation culture include: culture in the dark for 10-16 days, preferably 14 days, at a shaking speed of 120-180 r / min, preferably 150 r / min, at 24-28℃, preferably 25℃; And / or, the fermentation product is a fermentation broth obtained after liquid fermentation culture, or a fermentation supernatant obtained after filtering the fermentation broth, or a crude extract of natural fragrance obtained after extracting and enriching volatile substances from the fermentation supernatant, or a fragrance base obtained by further refining the crude extract of natural fragrance.

[0017] The flavor base is a mixture of various monomeric flavorings. The filtration separates the mycelium from the supernatant in the fermentation broth. The methods for extracting and enriching volatile substances can be conventional methods in the art, including but not limited to simultaneous distillation extraction (SDE), headspace solid-phase microextraction (HS-SPME), or organic solvent liquid-liquid extraction. The purification methods can be conventional separation and purification methods, including but not limited to molecular distillation or preparative column chromatography.

[0018] A third aspect of the present invention provides a microbial agent comprising the fermentation product of the second aspect described above. The microbial agent is a formulation with aroma substances as its active ingredient.

[0019] In the aforementioned third aspect of the microbial agent, the microbial agent further includes a carrier; if the microbial agent is used in the food field, the carrier may be an edible solid carrier or a liquid carrier, such as at least one of the following for encapsulating stable β-cyclodextrin, for oil-soluble systems, for drying and shaping maltodextrin / modified starch, and for freeze-drying protection, such as skim milk powder / sucrose, glycerol, etc., which may be selected according to their dosage form and use.

[0020] The main functions of adding a carrier to a microbial agent include: Standardization and dilution: Standardize the concentration of high-intensity fragrance substances to facilitate accurate measurement and use.

[0021] Stabilization: Protects active ingredients (fragrance substances) from the effects of light, heat, and oxygen, prevents volatilization and degradation, and extends shelf life.

[0022] Functionalization: Changing the physical form of active ingredients (e.g., from liquid to solid powder) to facilitate processing, storage, transportation, and application in different dosage forms (e.g., granules, capsules).

[0023] Furthermore, the dosage form of the bacterial agent is lyophilized powder, suspension, emulsion, granules, or capsules.

[0024] In the microbial agent, the active ingredient may be in the form of live or dead cells of the cultured strain, or a mixture of live or dead cells and fermentation supernatant, or fermentation supernatant, or a crude extract of natural fragrance obtained by crude extraction of fermentation broth, or a fragrance base that has been further refined.

[0025] The "active ingredients" here primarily refer to "fragrance substances" (such as β-ionone) present in the aforementioned forms, produced through the fermentation process. The bacterial cells (whether live or dead) primarily act as "producers" or "carriers" of these fragrance substances. Live cells of the cultured strain: may be used in special cases where a slow, continued aroma production is required in the product. A mixture of dead cells and fermentation supernatant: dead cells may contain intracellular fragrance substances, while the supernatant contains extracellular fragrance substances, providing a complete fermentation aroma spectrum. The fermentation supernatant, after removing water-soluble and partially dispersed fragrance substances, provides a clear liquid fragrance base. For crude extracts of natural fragrances obtained from fermentation broth, the aroma components are concentrated and preliminarily purified, removing most water and non-volatile substances, providing a strong, small-volume, and more stable fragrance base. For further refined fragrance bases, the purity of specific key aroma components (such as β-ionone) is higher, allowing for high-end applications or precise formulations requiring specific aroma profiles.

[0026] Furthermore, the microbial agent also includes at least one of a surfactant, a stabilizer, and a pH adjuster; Depending on the needs, surfactants (such as Tween 20, Tween 80, etc.), stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0027] The fourth aspect of this invention provides the application of the above-mentioned *Lentinula* fungal strain LV17, the above-mentioned fermentation product, or the above-mentioned inoculum in any of the following aspects 1)-4): 1) Application in the preparation of food or food additives; 2) Applications in the preparation of fragrances and flavorings; 3) Applications in the preparation of cosmetics or daily chemical products; 4) Application in the production of natural characteristic aroma substances.

[0028] The fermentation products or microbial agents provided by this invention can be used as "source raw materials" or "flavor functional raw materials" for aroma substances. Under the premise of complying with relevant safety and quality standards, the strain is an edible fungus and the fermentation substrate is a food-permitted raw material. The microbial agent can be directly applied to food systems as an overall flavor raw material (such as fermented flavor base, flavoring base liquid or natural flavor enhancer), or it can be directly used as a fermentation source raw material in daily chemical or cosmetic formulations under the conditions of passing safety assessment, microbial inactivation and ingredient traceability. Its application emphasizes the overall aroma characteristics and flavor complexity.

[0029] In addition, the strain of the present invention not only produces a special aroma after fermentation, but also has antioxidant function. Therefore, the application of the strain of the present invention provides new possibilities for the development of compound products with both aroma and antioxidant functions, and expands its commercial application dimensions and value space.

[0030] The natural characteristic aroma substances contain at least one of the following substances: dihydro-2-methyl-3(2H)-furanone, β-ionone, (E)-2-hexenal, 1-octen-3-ol, phenethyl alcohol, hexanal, linalool, 4-methyl-5-(β-hydroxyethyl)thiazole, 2-acetylpyrrole, γ-nonanolide, and maltol.

[0031] Based on the above applications, the present invention also provides a product containing the above-mentioned *Lentinula edodes* fungal strain LV17, the above-mentioned fermentation product, or the above-mentioned inoculant. The product may be food, flavoring, cosmetic, or daily chemical product, and these products contain aroma substances produced by the strain.

[0032] The fifth aspect of the present invention provides a method for preparing the above-mentioned fermentation product, comprising: inoculating a block of mushroom strain LV17 of the genus Lepidium into a liquid fermentation medium for fermentation culture to obtain a fermentation broth.

[0033] Furthermore, the solvent of the liquid fermentation medium is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L; Furthermore, the fermentation culture conditions include: light-protected culture at 24-28℃ with an oscillation speed of 120-180 r / min; Furthermore, the fermentation culture time is 10-16 days; Furthermore, the fermentation culture was carried out at 25°C in the dark for 14 days with an oscillation speed of 150 r / min. Furthermore, the method for preparing the mycelial blocks of the *Lentinula* strain LV17 includes: inoculating the activated *Lentinula* strain LV17 into a solid plate culture medium for cultivation to form dense mycelial blocks; Furthermore, in the method for preparing the mycelial blocks, the culture is carried out in the dark at 24-28°C for 18-22 days; Furthermore, the solvent of the solid plate culture medium is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, peat moss 5-8 g / L, agar 15-25 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L, peat moss 8 g / L, agar 20 g / L; Furthermore, the activated *Lentinula* strain LV17 was obtained by inoculating *Lentinula* strain LV17 into PDA medium and culturing it in the dark at 24-28°C for 10-16 days. Furthermore, the method for preparing the fermentation product also includes filtering the fermentation broth to obtain a fermentation supernatant and mycelium.

[0034] Optionally, the method for preparing the fermentation product further includes extracting and enriching the fermentation supernatant to obtain a crude extract of natural fragrance; optionally, the crude extract of natural fragrance is refined to obtain a fragrance base.

[0035] The *Lentinula* strain LV17 provided by this invention can produce one or more of the following aroma compounds: dihydro-2-methyl-3(2H)-furanone, β-ionone, (E)-2-hexenal, 1-octen-3-ol, phenethyl alcohol, hexanal, linalool, 4-methyl-5-(β-hydroxyethyl)thiazole, 2-acetylpyrrole, γ-nonanolide, and maltol. Preferably, the aroma compounds comprise dihydro-2-methyl-3(2H)-furanone, β-ionone, and (E)-2-hexenal.

[0036] The beneficial effects of this invention are as follows: This invention marks the first time that a specific strain of Lepista (LV17) with extremely high aroma synthesis capacity has been screened from Lepista fungi. This strain can efficiently synthesize up to 64 key aroma compounds (rOAV ≥ 10) during liquid fermentation, with a stable yield of the core flavor compound dihydro-2-methyl-3(2H)-furanone reaching milligrams per liter (~1.5 mg / L), and a relative odor activity (rOAV) value as high as approximately 3.1 × 10⁻⁶. 5 High-valent terpenoids such as β-ionone were also significantly enriched (rOAV reached 2.2 × 10⁻⁶). 4These components combine to form a rich and pure floral-fruity-sweet aroma complex. Electronic nose and GC-MS analysis showed that its overall aroma intensity was significantly better than the control strain, and no abnormalities were found in the response values ​​of undesirable sulfides and amines. This invention achieves efficient, green, and large-scale production of various high-value natural aroma substances through microbial fermentation, breaking through the bottlenecks of high cost and unstable supply in traditional natural extraction methods, while avoiding the shortcomings of chemical synthesis methods that cannot obtain the 'natural' label. This strain can be applied to the fields of natural fragrances and food flavoring, providing an efficient microbial cell factory for the production of natural characteristic aroma substances. The strain of this invention can achieve efficient, reliable, green, and large-scale production of various high-value natural aroma substances through microbial fermentation, and has direct prospects for industrial application.

[0037] The most commercially groundbreaking aspect of this invention lies in the ability of strain LV17 to produce key, scarce natural flavor components. Taking dihydro-2-methyl-3(2H)-furanone, a core flavor compound that shapes caramel, fruit, and sweet aromas, as an example, this substance is present in extremely small amounts in nature. Traditional plant extraction methods can only yield trace amounts, and obtaining commercially pure products is extremely costly. Currently, the market mainly relies on chemical synthesis. Under basic liquid fermentation conditions (20 g / L glucose, 3 g / L yeast extract), strain LV17 of this invention can efficiently biosynthesize this substance, achieving a stable yield of ~1.5 mg / L in the fermentation broth (see Example 4 and Table 2). This production level of "milligrams per liter" represents a qualitative leap from "trace detection" to "microbial mass production," signifying: a breakthrough in the barriers to large-scale production of natural sources; providing a stable, sustainable, and scalable production pathway for this scarce natural ingredient, freeing it from dependence on unstable plant raw materials; significant cost optimization potential: compared to extremely expensive natural extracts, fermentation production has enormous potential for cost reduction and controllability; and providing core raw materials for high-premium products: the product possesses complete "natural" attributes and can be directly used to formulate high-end foods, beverages, and cosmetics with "all-natural" labels, replacing chemically synthesized counterparts, meeting the clean label trend, and enhancing the added value of end products.

[0038] The *Lentinula* strain LV17 provided by this invention can efficiently yield a variety of key characteristic aroma substances, such as dihydro-2-methyl-3(2H)-furanone and β-ionone. These substances together constitute a natural and balanced complex aroma combination, which can be used as a high-quality raw material for preparing high-end natural fragrances and flavorings.

[0039] In addition to producing specific aroma substances, the strains of this invention also have strong antioxidant capabilities. Attached Figure Description

[0040] Figure 1The growth morphology of strain LV17, with preservation number CGMCC No.42460.

[0041] Figure 2 Phylogenetic trees of strain LV17 (CGMCC No. 42460) and control strain were constructed based on ITS.

[0042] Figure 3 The results of electronic nose testing of fermentation broths of strain LV17 (CGMCC No. 42460) and control strain.

[0043] Figure 4 GCMS total ion chromatograms of fermentation broths of strain LV17 (CGMCC No. 42460) and control strain.

[0044] Figure 5 rOAV of key aroma compounds in the fermentation broth of strain LV17 (CGMCC No. 42460) and control strain.

[0045] Figure 6 Aroma wheel of fermentation broth for strain LV17 (CGMCC No. 42460). (Based on the corresponding quantity of aroma compounds of LV17 and rOVA value) Figure 7 LCMS total ion chromatograms of fermentation broths of strain LV17 (CGMCC No. 42460) and control strain.

[0046] Figure 8 A bar chart showing the main metabolites in the fermentation broth of strain LV17 (CGMCC No. 42460) which is superior to the control strain. The vertical axis of the chart represents the peak area of ​​the substance determined by non-target metabolism after log 10 data transformation. In the statistical results of a certain metabolite, the data between LV17 and different strains represent the difference fold of the peak area of ​​the substance between them.

[0047] Figure 9 The enzyme activities of the aroma-producing pathway of strain LV17 (CGMCC No. 42460) and the control strain are shown. For comparison of data between different strains, if the letters are different, it indicates that there is a significant difference between the two (p<0.01).

[0048] Figure 10 The total antioxidant capacity of mycelia of strain LV17 (CGMCC No. 42460) and control strain was measured. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0050] Unless otherwise specified, the experimental methods used in these examples are conventional methods in the field and can be found in the following books: *Microbiology Experiments*, 4th Edition (edited by Shen Ping and Chen Xiangdong), *Plant Molecular Biology Experimental Handbook*, *Molecular Cloning: A Laboratory Manual* (4th Edition), and *Solid Phase Microextraction: Principles and Applications*. All reagents used are commercially available, and the instructions for use should be followed according to the manufacturer's recommendations.

[0051] Example 1: Isolation and identification of *Lentinula spp.* strain LV17 I. Source and Acquisition Methods of Strains The *Lepista* strain LV17 (CGMCC No. 42460) described in this invention is classified as *Lepista saeva*, with the classification and nomenclature referenced at the following URL: https: / / www.speciesfungorum.org / names / names.asp?strGenus=Lepista&GSD=Yes. This strain was obtained by tissue isolation from fresh fruiting bodies of wild *Lepista saeva* collected in Weichang, Hebei Province. Specifically, under aseptic conditions, internal tissue blocks were excised from the fruiting bodies and inoculated onto potato dextrose agar (PDA) plates, and cultured at 25°C in the dark. After mycelial germination, the mycelial tips were repeatedly purified to obtain a pure culture, named LV17.

[0052] II. Culture and Morphological Characteristics of Strain LV17 Culture of strain LV17: It was inoculated onto PDA medium and cultured in the dark at 25°C for 14 days. Colony morphology was then observed.

[0053] Colony morphology: Colonies are 40-50 mm in diameter. This strain exhibits circular, radial growth with neat edges, and hyphae radiating outwards from the center. The overall colony texture is dense, with a fluffy to cottony surface, slightly raised in the center, creating a distinct layered appearance. The colony surface is milky white to light off-white with transparent edges. The hyphae are flexible, dense, and grow uniformly, emitting a slight sweet or floral aroma during cultivation. See [link to relevant documentation]. Figure 1 .

[0054] III. Molecular phylogenetic analysis of the strains The following are the *Tricholoma* strains used as controls: *Tricholoma floribunda* LS2 (CCMSSC05218) and LS3 (CCMSSC05150), *Tricholoma lilacinus* LN3 (CCMSSC05221) and LN16 (CCMSSC05222), and *Tricholoma purpureum* LV13 (CCMSSC05219). All of these control strains are deposited in the National Standard Collection of Edible Fungi (CCMSSC).

[0055] Genomic DNA was extracted from the mycelium of all *Mushroom* strains using a modified CTAB method. PCR amplification was performed using universal primers for fungal intraribosomal transcription spacers (ITS) such as ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', sequence 10 in the sequence listing) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', sequence 11 in the sequence listing). The amplified products were purified and sequenced. The sequencing results for strain LV17 are shown in Sequence 1 of the sequence listing; the sequencing results for strain LS2 are shown in Sequence 2 of the sequence listing; the sequencing results for strain LV13 are shown in Sequence 3 of the sequence listing; the sequencing results for strain LN16 are shown in Sequence 4 of the sequence listing; the sequencing results for strain LS3 are shown in Sequence 5 of the sequence listing; and the sequencing results for strain LN3 are shown in Sequence 6 of the sequence listing. The obtained sequences were aligned to the GenBank database using BLAST and analyzed using the neighbor-joining phylogenetic tree method. This confirmed that strain LV17 clustered with Lepista saeva, with a support rate higher than 98%, thus completing the molecular identification.

[0056] ITS sequence characteristics: The ITS sequence of LV17 is 99.26% similar to that of the PQ653025.5 Lepista saeva strain in GenBank (as shown in Sequence 7 in the sequence listing). A maximum likelihood tree (bootstrap=1000) was constructed based on the ITS sequence. LV17 clusters independently with PQ653025.5 Lepista saeva, and its genetic distance from closely related species is >0.02. See the phylogenetic tree of LV17 and other control strains. Figure 2 . Figure 2 The ITS sequence of strain OR668710.1:9-689 Lepista sordida is shown in sequence 8 of the sequence listing, and the ITS sequence of strain LC370443.1:9-679 Lepista nuda is shown in sequence 9 of the sequence listing.

[0057] Based on BLAST alignment and phylogenetic tree analysis, the fungal strain LV17 provided in this invention can be identified as Lepista saeva.

[0058] Example 2: Liquid fermentation culture of strain LV17 – the technological basis for commercial production The preserved strain LV17 was inoculated into PDA medium and activated by incubation in the dark at 25°C for 15 days. The activated strain LV17 was then inoculated into solid agar plates and incubated in the dark at 25°C for 20 days. The solid agar plate formulation consisted of 20 g / L glucose, 3 g / L brewer's yeast extract, 8 g / L peat moss, and 20 g / L agar, with water as the solvent. The bacterial culture blocks were then placed in a liquid fermentation medium for fermentation. The liquid fermentation medium consisted of 20 g / L glucose, 3 g / L brewer's yeast extract, and water as the solvent; the pH was not adjusted. The specific fermentation method is as follows: Ten mycelial blocks (approximately 0.1 cm in diameter) were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of the above-described liquid fermentation medium. The flasks were placed in a shaker and cultured at 25°C and 150 r / min in the dark for 14 days. After fermentation, the mycelium and fermentation supernatant were separated by filtration. The mycelium was washed three times with pre-cooled sterile water, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent metabolomics and enzyme activity analysis. The fermentation supernatant was immediately used for volatile component analysis.

[0059] The fermentation method of the strain of this invention uses a simple culture medium with low cost and mild conditions, which has the basic conditions for large-scale scale-up and cost control.

[0060] To conduct comparative analysis of subsequent fermentation products, this embodiment also used the liquid fermentation culture method of the above-mentioned strain LV17 to culture the following Lepista genus strains as controls: Lepista sordida LS2 (CCMSSC05218) and LS3 (CCMSSC05150), Lepista nuda LN3 (CCMSSC05221) and LN16 (CCMSSC05222), and Lepista saeva LV13 (CCMSSC05219). These control strains are all deposited in the National Standard Fertilizer Library for Edible Fungi (CCMSSC).

[0061] After fermentation with the control strain, the fermentation broth was separated to obtain the corresponding mycelium and fermentation supernatant, which were used as control samples for subsequent analysis of strain LV17.

[0062] Example 3: Electronic nose analysis of aroma profile of fermentation products from strain LV17 I. Instruments and Samples The overall aroma profile of fermentation broths from strain LV17 and other control strains was analyzed using a PEN3 portable electronic nose system. This system is equipped with an array of 10 metal oxide semiconductor sensors, each selectively responding to specific classes of volatile compounds.

[0063] II. Sample Pretreatment and Testing Methods Take 5 mL of the fermentation broth from each strain prepared in Example 2 and place it in a 20 mL headspace vial, then seal immediately. Place the vial in a 60°C water bath for 30 minutes to equilibrate, allowing volatile components to fully escape into the headspace. During testing, use filtered clean air as the carrier gas at a flow rate of 300 mL / min. The headspace gas is drawn into the sensor array chamber, and sampling takes place for 60 seconds, followed by a 90-second clean air purge to restore the sensor baseline. Three independent replicates are performed for each sample.

[0064] III. Data Analysis and Results The electronic nose system records the conductivity change of each sensor during the sampling process, and its response value is expressed as G / G0 (the ratio of the conductivity G at sampling to the initial baseline conductivity G0). The analysis results are as follows... Figure 3 As shown, the R2 (W5S), R8 (W2S), and R1 (W1C) sensors mainly respond to aroma components such as esters, floral and fruity aromas, sweet aromas, citrus aromas, and aromatic aldehydes, located in the upper right, lower left, and upper right areas of Figure 3, respectively. The response values ​​of the various strains on the R1 sensor are not significantly different, indicating that this channel has weak selectivity for different samples. In contrast, LS2 and LS3 showed the highest responses on the R2 channel, while the response value of LV17, although lower than these two, was significantly higher than LN3, LN16, and LV13, indicating that its fermentation products are rich in aromatic and ester compounds. LV17 showed strong responses on the R2 (W5S) sensor (targeting nitrogen oxides and aromatic compounds), the R6 (W1S) channel (targeting short-chain alkanes), and the R8 (W2S) channel (targeting alcohols and aldehydes / ketones), especially on the R8 channel, where the response value was slightly higher than most control strains, only lower than LN3. In contrast, LV17 did not show abnormally high responses in the R7 (W1W) and R3 (W3C) channels, which primarily respond to unpleasant odor substances such as sulfides and amines. The signal on R7 was close to maximum but still within the normal range, while the responses on R3 were largely consistent across strains. Overall, the electronic nose response pattern of LV17 indicates that its fermentation products have a high aroma intensity, significant floral and fruity characteristics, and are rich in compounds associated with pleasant aromas in their volatile components, with few off-odor impurities, exhibiting a relatively harmonious and pleasant aroma profile and sensory quality. In other words, the electronic nose results chemically validate that LV17 possesses an aroma profile dominated by floral and fruity notes and with few off-odors, providing a basis for subsequent analysis.

[0065] Example 4: GC-MS analysis of volatile components in fermentation products of strain LV17 I. Extraction of Volatile Substances Volatile organic compounds were extracted from the fermentation supernatant using headspace solid-phase microextraction (HS-SPME). Saturated NaCl solution and 20 μL (10 μg / mL) 3-hexanone-2,2,4,4-d4 were added as internal standards. The samples were extracted using an automated headspace solid-phase microextraction (HS-SPME) system for GC-MS analysis.

[0066] GC-MS analysis conditions HS-SPME sampling conditions: Under constant temperature of 60℃, shake for 5 min, insert a 120 μm DVB / CWR / PDMS extraction tip into the sample headspace vial, perform headspace extraction for 15 min, followed by resolution at 250 ℃ for 5 min, and then perform GC-MS separation and identification. Before sampling, the extraction tip was aged at 250 ℃ for 5 min in a Fiber Conditioning Station. Note: New extraction tips were aged in a Fiber Conditioning Station for 2 h before extraction. Furthermore, an SPME Arrow was used, whose sensitivity is up to 10 times that of traditional SPME fiber tips.

[0067] Chromatographic conditions: DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas high-purity helium (purity not less than 99.999%), constant flow rate 1.2 mL / min, injection port temperature 250°C, splitless injection, solvent delay 3.5 min. Temperature program: 40 °C held for 3.5 min, increased to 100 °C at 10 °C / min, then increased to 180 °C at 7 °C / min, and finally increased to 280 °C at 25 °C / min, held for 5 min.

[0068] Mass spectrometry conditions: Electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, mass spectrometry interface temperature 280°C, electron energy 70 eV, scan mode selected ion detection mode (SIM), qualitative and quantitative ion precise scan (GB 23200.8-2016).

[0069] III. Compound Identification and Screening of Key Aroma Substances Volatile compounds were identified by comparing their retention time and characteristic ions with a standard library built by Maiwei Metabolism, and by calculating retention indices and matching them with the NIST17 mass spectrometry library. Semi-quantitative analysis was performed using the internal standard method. To screen for key aroma active substances, the relative odor activity (rOAV) of each compound was calculated using the formula: rOAV = compound concentration / its sensory threshold. Compounds with rOAV ≥ 10 were defined as key aroma substances.

[0070] IV. Analysis Results The raw data from mass spectrometry analysis were processed using MassHunter software for qualitative and quantitative analysis. Figure 4 The image shows the total ion current (TIC) chromatogram of the sample. GC-MS analysis of the fermentation products of strain LV17 identified 64 key aroma compounds (rOAV ≥ 10), demonstrating significantly superior aroma complexity and layering compared to the control strain. Figure 5 In particular, the rOAV value of dihydro-2-methyl-3(2H)-furanone can reach 3.1 × 10⁻⁶. 5 The compound exhibited the highest rOAV value among all tested strains in the strains of this invention, imparting a strong sweet, caramel, and fruity aroma to the fermentation broth. The rOAV value of β-ionone can reach 2.2 × 10⁻⁶. 4 (Table 1) demonstrates its extremely high aroma intensity, highly consistent with its electronic nose results. Furthermore, an aroma wheel belonging to strain LV17 was constructed using the classification quantity of key aroma compounds and their relative aroma activity values. (See Table 1 for details.) Figure 6 The LV17 strain of this invention possesses irreplaceable metabolic characteristics. Its unique advantage lies in the highly efficient expression of key aroma-synthesizing enzyme systems in its genome, making it difficult to replicate through simple screening or genetic engineering. Table 2 shows that the content of dihydro-2-methyl-3(2H)-furanone in the LV17 fermentation broth is 1.54409 μg / mL (i.e., ~1.54 mg / L). This data quantitatively confirms that the invention achieves a "milligram per liter" yield under unoptimized conditions, providing a key benchmark for evaluating the economics of its commercial production. These data collectively constitute the core indicators for evaluating the commercial efficiency (output / input) of this strain as a cell factory for aroma raw materials.

[0071] Table 1. rOAV of key aroma compounds detected by GCMS in fermentation broths of all strains.

[0072] Table 2. Relative contents (μg / ml) of key aroma compounds in the fermentation broth of all strains as determined by GCMS.

[0073] Example 5: Non-targeted metabolomics analysis of strain LV17 mycelium I. Sample Pretreatment Mycelial samples were freeze-dried in a Scientz-100F freeze dryer for 63 hours. They were then ground into powder using a grinder (MM400, Retsch) at 30 Hz for 1.5 minutes. 50 mg of the sample powder was weighed using an electronic balance (MS105DM) and added to 1200 μL of pre-cooled 70% methanol-water internal standard extraction solution at -20℃. The internal standard extraction solution was prepared by dissolving 1 mg of 2-chloro-phenylalanine as a standard in 1 mL of 70% methanol-water to prepare a 1000 μg / mL stock solution. This stock solution was further diluted with 70% methanol to prepare a 250 μg / mL internal standard solution. The sample was vortexed every 30 minutes for 30 seconds, for a total of 6 vortexes. After centrifugation (12000 rpm, 3 minutes), the supernatant was collected and filtered through a 0.22 μm pore membrane. The sample (size) was filtered and stored in a vial for UPLC-MS / MS analysis.

[0074] II. LC-MS Analysis Conditions Chromatographic separation was performed using a Waters ACQUITY UPLC system. Specific chromatographic conditions were as follows: Column: Waters ACQUITY UPLC HSS T3 Column (1.8 µm, 2.1 mm × 100 mm); Column temperature: 40 °C; Flow rate: 0.40 mL / min; Injection volume: 4 µL. Mobile phase A was ultrapure water containing 0.1% (vol) formic acid, and mobile phase B was acetonitrile containing 0.1% (vol) formic acid. A gradient elution program was used: 0–5.0 min, phase B increased from 5% to 65%; 5.0–6.0 min, phase B rapidly increased to 99% and held until 7.5 min; at 7.6 min, it returned to the initial proportion (5% phase B) and equilibrated to 10.0 min, with a total run time of 10.0 minutes. Mass spectrometry was performed on a SCIEX TripleTOF 6600+ instrument, using an electrospray ionization source that switches between positive and negative ions and an information-dependent acquisition mode.

[0075] III. Data Analysis and Results The raw data underwent peak extraction, alignment, and normalization using XCMS. The total ion chromatogram of the sample is shown below. Figure 7As shown. Analysis revealed that the primary metabolism (glycolysis, TCA cycle, core amino acids) of the strains remained stable across different strains, but their secondary metabolic pathways underwent significant specific reprogramming. The levels of specialized metabolites such as diterpenes, triterpenes, and isoquinoline alkaloids in the LV17 mycelium were enriched by hundreds to thousands of times compared to the control strain (e.g., (-)-oleuropein 11-methyl ester, chelidonine, etc.). Figure 8 As shown, these enriched secondary metabolic precursors provide a solid material basis for the efficient synthesis of characteristic aroma compounds in LV17. These data, from a systems biology perspective, reveal the intrinsic mechanism of LV17's high aroma production, demonstrating the stability and heritability of its traits. This is of great significance for ensuring the performance consistency of industrial production strains, reducing production risks, and providing a stable guarantee for long-term commercial application.

[0076] Example 6: Determination of enzyme activity in key aroma synthesis pathway of strain LV17 I. Enzyme Solution Preparation Fresh mycelium was collected, ground in an ice bath with the appropriate pH buffer, and the supernatant was collected after centrifugation as crude enzyme solution. When using an ELISA kit, the pH buffer was physiological saline; when using a biochemical kit, the pH buffer was PBS buffer (pH=7.4).

[0077] II. Enzyme Activity Assay The contents of carotenoid cleavage dioxygenase and terpene synthase were determined using a commercially available ELISA kit. Lipoxygenase activity was determined using a biochemical kit. All assays were performed strictly according to the manufacturer's instructions, with three technical replicates for each sample.

[0078] III. Measurement Results The enzyme activity assay results corroborated the metabolomics and volatile matter spectroscopy data. The results are as follows: Figure 9 As shown, there were significant differences in the activity levels of key aroma-related enzymes among different strains, with strain LV17 exhibiting a clear advantage in multiple aroma-related metabolic pathways. The activity of carotenoid cleavage dioxygenases (CCDs) was significantly higher in LV17 than in other tested strains (p < 0.01). This enzyme is responsible for the degradation of carotenoids to produce C-type compounds such as β-ionone. 13- A key rate-limiting enzyme for isoprene volatiles. This result is highly consistent with the significant enrichment of terpene aroma compounds such as β-ionone in LV17 observed in metabolomics and volatile matter profiling, indicating that the high enzyme activity of CCDs is an important biochemical basis for their high terpene aroma production. The terpene synthase (TPS) activity of LV17 was also the highest among all strains (p < 0.01), indicating a stronger synthetic capacity in the terpene skeleton construction stage. This enzyme activity advantage further supports the results of increased levels of various terpene volatiles detected in metabolomics and volatile matter profiling, indicating that LV17 has a systemic metabolic advantage in the "generation-accumulation" process of terpene aroma compounds. Lipoxygenase (LOX) activity assays showed a significant enhancement in LV17 (p < 0.01). LOX is a key enzyme in the oxidative cleavage of fatty acids to generate C6–C9 aldehydes, alcohols, and other aroma compounds derived from fatty acids. Its enhanced enzyme activity indicates that LV17 possesses stronger fatty acid metabolic potential. This is consistent with the increased content of fatty acid-derived aroma components in the volatile matter spectrum, further demonstrating that fatty acid metabolic pathways also play an important role in the aroma formation of LV17.

[0079] In summary, LV17 exhibits significantly enhanced key enzyme activities in multiple aroma compound formation pathways, including carotenoid degradation, terpene synthesis, and fatty acid oxidation. These enzymatic activity advantages, along with metabolomics and volatile matter spectroscopy data, form a highly consistent chain of evidence, collectively constituting the core biochemical mechanism by which LV17 produces a wide variety of natural aroma compounds.

[0080] Example 7: Application of strain LV17 in the preparation of natural fragrance substances The LV17 fermentation broth obtained in Example 2 was subjected to volatile substance extraction and enrichment according to the sample pretreatment steps in Example 3, resulting in a crude natural fragrance extract rich in key characteristic aroma compounds such as dihydro-2-methyl-3(2H)-furanone and β-ionone. This crude extract can be used directly or after further purification (e.g., molecular distillation or column chromatography) as a natural, high-intensity fruity-floral fragrance base in the formulation of baked goods, beverages, candies, dairy products, and high-end daily chemical fragrances, effectively enhancing the natural flavor attributes and aroma complexity of the products. The crude natural fragrance extract obtained from the LV17 fermentation broth through appropriate extraction and enrichment can be used directly as a high-intensity fruity-floral fragrance base. This application example demonstrates a complete and concise path from strain fermentation to obtaining an intermediate product that can be directly used in downstream formulations, shortening the technology-to-product transformation chain.

[0081] Example 8: Study on the functional characteristics of strain LV17 I. Sample Preparation Take 0.1 g of fresh mycelium filtered after fermentation in Example 2, add 1 mL of pre-cooled phosphate buffer (pH 7.4) as the extraction solution, homogenize in an ice bath, centrifuge at 10000 g for 5 min at 4℃, and place the supernatant on ice for testing.

[0082] II. Determination of Total Antioxidant Capacity The total antioxidant capacity (T-AOC) of six bacterial strains was determined using the FRAP method with a Beijing Box Biochemical Kit. All assays were performed strictly according to the instructions, and three technical replicates were set for each sample.

[0083] III. Measurement Results Significant differences in antioxidant activity were observed among different strains (p < 0.05). For example... Figure 10 As shown, strain LN16 had the highest T-AOC value (10.47 U / mL), significantly superior to all other strains. Notably, strain LV17 exhibited the second highest antioxidant activity after LN16 (4.34 U / mL), and its three replicate values ​​were highly consistent (4.366, 4.348, 4.313), demonstrating outstanding data stability. Although the activity of LV17 was significantly lower than that of LN16, it was significantly higher than that of the other four strains (p < 0.05), forming a clear second tier of activity. Among the remaining strains, the T-AOC values ​​of LS2, LV13, and LN3 were at an intermediate level (1.92, 1.69, 1.16 U / mL), with no significant differences among them; LS3 had the lowest activity (0.44 U / mL), significantly lower than all other strains. In summary, LN16 is the strain with the strongest antioxidant capacity, while LV17 also exhibits stable and significant high antioxidant activity. It can be studied as a potential superior strain, which provides the possibility for developing compound products that combine flavor enhancement and functionality (such as antioxidant preservation and skin care), expanding its commercial application dimensions and value space.

[0084] In summary, the novel *Lentinula spp.* strain LV17, possessing an exceptionally high aroma synthesis capability, provided by this invention not only lays the foundation for addressing the bottleneck in the supply of natural spices but also opens up new pathways for the high-value utilization of edible fungi, thus possessing significant scientific and commercial value.

Claims

1. A fungal strain of the genus *Lentinula* LV17, capable of efficiently synthesizing a variety of natural characteristic aroma substances, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42460.

2. The *Lentinula* fungal strain LV17 according to claim 1, characterized in that, The culture temperature for the *Lentinula* fungal strain LV17 is 24-28℃, preferably 25℃; And / or, the activation medium for the *Lentinula* fungal strain LV17 is PDA; And / or, the propagation medium for the *Lentinula* fungal strain LV17 is a solid plate medium, the solvent of which is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, peat moss 5-8 g / L, agar 15-25 g / L; preferably: glucose 20 g / L, brewer's yeast powder 3 g / L, peat moss 8 g / L, agar 20 g / L; And / or, the *Lentinula* fungal strain LV17 is cultured in the dark.

3. A fermentation product, characterized in that, The fermentation product is obtained by liquid fermentation culture of the *Lentinula* fungal strain LV17 as described in claim 1 or 2.

4. The fermentation product according to claim 3, characterized in that, The solvent of the liquid fermentation culture medium is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L. And / or, the *Lentinula* fungal strain LV17 added to the liquid fermentation culture medium for fermentation culture is a mycelium-covered block; And / or, the conditions for the liquid fermentation culture include: incubation in the dark for 10-16 days at 24-28°C, preferably 25°C, with an oscillation speed of 120-180 r / min, preferably 150 r / min; And / or, the fermentation product is a fermentation broth obtained after liquid fermentation culture, or a fermentation supernatant obtained after filtering the fermentation broth, or a crude extract of natural fragrance obtained after extracting and enriching volatile substances from the fermentation supernatant, or a fragrance base obtained by further refining the crude extract of natural fragrance.

5. A microbial agent, characterized in that, The microbial agent comprises the fermentation product as described in claim 3 or 4.

6. The microbial agent according to claim 5, characterized in that, The microbial agent also includes a carrier; And / or, the microbial agent further includes at least one of a surfactant, a stabilizer, and a pH adjuster; And / or, the dosage form of the microbial agent is a lyophilized powder, suspension, emulsion, granule or capsule.

7. The use of the *Lentinula* fungal strain LV17 according to claim 1 or 2, the fermentation product according to claim 3 or 4, or the inoculum according to claim 5 or 6 in any of the following aspects 1)-4): 1) Application in the preparation of food or food additives; 2) Applications in the preparation of fragrances and flavorings; 3) Applications in the preparation of cosmetics or daily chemical products; 4) Application in the production of natural characteristic aroma substances.

8. A method for preparing the fermentation product according to claim 3 or 4, characterized in that, The preparation method includes: inoculating a block of mushroom strain LV17 of the genus Lepidium into a liquid fermentation medium for fermentation culture to obtain a fermentation broth.

9. The preparation method according to claim 8, characterized in that, The solvent of the liquid fermentation medium is water, and the components and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L. And / or, the fermentation culture conditions include: light-protected culture at 24-28℃ with an oscillation speed of 120-180 r / min; And / or, the fermentation culture time is 10-16 days; And / or, the method for preparing the mycelial blocks of the *Lentinula* strain LV17 includes: inoculating the activated *Lentinula* strain LV17 into a solid plate culture medium for cultivation to form dense mycelial blocks; And / or, the method for preparing the fermentation product further includes filtering the fermentation broth to obtain a fermentation supernatant and mycelium.

10. The preparation method according to claim 9, characterized in that, The fermentation culture was carried out at 25°C and with a shaking speed of 150 r / min in the dark for 14 days. And / or, in the method for preparing the mycelial blocks, the culture is carried out in the dark at 24-28°C for 18-22 days; And / or, the solvent of the solid plate culture medium is water, and the components contained therein and their concentrations are as follows: glucose 15-30 g / L, brewer's yeast powder 2-6 g / L, peat moss 5-8 g / L, agar 15-25 g / L, preferably: glucose 20 g / L, brewer's yeast powder 3 g / L, peat moss 8 g / L, agar 20 g / L; And / or, the activated *Lentinula* strain LV17 is obtained by inoculating *Lentinula* strain LV17 into PDA medium and culturing it in the dark at 24-28°C for 10-16 days; And / or, the method for preparing the fermentation product further includes extracting and enriching the fermentation supernatant for volatile substances to obtain a crude extract of natural fragrance, and optionally, refining the crude extract of natural fragrance to obtain a fragrance base.

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