Aspergillus scherweri for producing linalool and application of Aspergillus scherweri in brewing of liquor yeast for making hard liquor

Abstract

The invention discloses an Aspergillus scherweri strain for producing linalool and an application of the Aspergillus scherweri strain in production of yeast for making hard liquor, and belongs to the technical field of processing of brewed white liquor. The aspergillus scherweri provided by the invention is separated from white spirit yeast, the strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.41632. The bacterial colony of the strain is bright yellow to deep yellow or yellowish brown, the texture is compact, and the linalool content in yeast for making hard liquor can be remarkably increased when the strain is introduced into yeast culture, so that the taste of liquor is optimized. Meanwhile, the invention discloses a metabolic mechanism and a way for synthesizing the linalool by using the Aspergillus scherweri As MHR2, and a new theoretical support is provided for the application of the Baijiu brewing microorganisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of processed Baijiu brewing, and relates to flavor substances produced by microorganisms in Daqu for Baijiu brewing and their application in Daqu production. Specifically, it relates to an Aspergillus chevalieri strain producing linalool isolated from Daqu and its application in Daqu production. Background Art

[0002] Baijiu is a distilled liquor made by using Daqu as saccharifying and fermenting agent through cooking, saccharification, fermentation and distillation. The unique "microbial community" in Daqu is the core factor for the formation of Baijiu fragrance types. However, at present, the evaluation indexes of Daqu quality are only sensory characteristics and physical and chemical properties. As the core of Daqu, the microbial community, establishing an "ecological system" evaluation system for Daqu, through the detection and control of the "ecological system", can fundamentally ensure the stability of Baijiu fragrance types. Therefore, by exploring more and more core microbial communities producing flavor substances in Daqu, the inherent essence of various fragrance types of Baijiu can be gradually revealed, promoting the transformation of Chinese Baijiu from traditional to intelligent.

[0003] Aspergillus chevalieri is a fungus widely existing in nature, especially commonly existing in traditional fermented tea and wine koji. It has strong adaptability such as drought tolerance, high temperature tolerance and high osmotic pressure tolerance; it can form a unique "bacterial flower fragrance", and can secrete various enzymes (such as amylase, lipase, protease, etc.), which is beneficial to the digestion of starch and protein; it has a complex enzyme system and can produce flavor functional substances, thus affecting the material transformation in tea and wine koji, and ultimately affecting the quality of tea and wine. In addition, this bacterium can also produce antibacterial substances, which can effectively prevent the growth and pollution of spoilage bacteria during the fermentation process of tea and wine koji, and is an important guarantee for the quality of tea and wine koji. Linalool (English name: Linalool; alias: linalol, coriandrol, linalool, etc., CAS No.: 78-70-6) belongs to chain-like terpenoid alcohols and is a plant secondary metabolite. It is generally considered to be the substance with the strongest aroma intensity among monoterpenoids. It can endow agarwood fragrance and is an important aroma component in tea and wine. Its content varies in alcoholic beverages of different fragrance types, making Baijiu of different fragrance types have their own unique flavor characteristics. However, at present, whether Aspergillus chevalieri can produce linalool has not been reported. Summary of the Invention

[0004] In Daqu, the fungal biomass is large and plays an important role in saccharification, fermentation, esterification, etc. However, there is currently a lack of research on the composition of core fungi in Fengxiang Daqu, their contributions to Daqu, and the mechanism of action. Aspergillus chevalieri is a common fungus in Daqu, widely distributed and with a high abundance, but its aroma-producing function in Daqu has not been clarified. The purpose of the present invention is to provide an Aspergillus chevalieri As MHR2 that produces linalool and its application in the production of Daqu for brewing Fengxiang-flavor Baijiu.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] The present invention provides an Aspergillus chevalieri As MHR2 that produces linalool, and the strain is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 41632.

[0007] The present invention provides a Daqu for brewing Fengxiang-flavor Baijiu, which includes the above-mentioned Aspergillus chevalieri As MHR2 that produces linalool.

[0008] The viable count of Aspergillus chevalieri As MHR2 that produces linalool in the Daqu for brewing Fengxiang-flavor Baijiu is 1×10 5 ~1×10 6 CFU / g.

[0009] The present invention provides a preparation method for the above-mentioned Daqu for brewing Fengxiang-flavor Baijiu, including:

[0010] Inoculating the above-mentioned Aspergillus chevalieri As MHR2 that produces linalool into a liquid medium for cultivation to obtain a seed liquid;

[0011] Crushing cereal raw materials, adding water to make the water content reach 35 - 45%, cooking and sterilizing to obtain processed Daqu raw materials;

[0012] Inoculating the seed liquid into the processed Daqu raw materials in proportion, stirring evenly, and placing in a fermentation chamber for fermentation to obtain Daqu for brewing Fengxiang-flavor Baijiu.

[0013] The liquid medium is: 200 g of potato, 20 g of glucose, 1 g of yeast extract, and 1000 mL of distilled water.

[0014] The cultivation conditions are: constant temperature cultivation at 25 - 35°C for 5 - 9 days.

[0015] Furthermore, the culture conditions are: constant temperature culture at 30°C for 5 days.

[0016] The cereal raw materials are barley: wheat: peas with a mass ratio of 6:1:3.

[0017] The inoculation ratio of the seed liquid is 5% - 15% of the mass of the processed Daqu raw materials.

[0018] Furthermore, the inoculation ratio of the seed liquid is 10% of the mass of the processed Daqu raw materials.

[0019] The fermentation conditions are: culture at 25 - 35°C for 5 - 9 days.

[0020] Furthermore, the fermentation conditions are: culture at 30°C for 8 days.

[0021] The present invention provides the use of the above-mentioned Aspergillus chevalieri As MHR2 producing linalool or the above-mentioned Daqu for brewing Fengxiang-flavor Baijiu in the brewing of Fengxiang-flavor Baijiu.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides an Aspergillus chevalieri As MHR2 producing linalool, which is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC No. 41632. The Aspergillus chevalieri As MHR2 provided by the present invention is screened and isolated from Fengxiang-flavor Daqu. Through ITS sequence phylogenetic analysis and morphological analysis, the strain Aspergillus chevalieri As MHR2 belongs to Aspergillus chevalieri. When Aspergillus chevalieri As MHR2 is cultured in PDA medium for 8 days, the linalool content can reach 1.583 μg / g, which is significantly higher than that of Saccharomycopsis fibuligera, Thermoascus aurantiacus and the control.

[0024] A Daqu for brewing Fengxiang-flavor Baijiu provided by the present invention is cultured by introducing Aspergillus chevalieri As MHR2 that produces linalool, so that the relative content of linalool in the Daqu reaches a significant level, far exceeding that of control strains such as Saccharomycopsis fibuligera and Thermoascus aurantiacus. This method significantly improves the quality of the Daqu, and further optimizes the flavor and taste of Baijiu, providing a new technological breakthrough for the Baijiu brewing industry.

[0025] A preparation method of a Daqu for brewing Fengxiang-flavor Baijiu provided by the present invention significantly increases the content of linalool in the Daqu by making full use of the metabolic characteristics of Aspergillus chevalieri As MHR2. In order to give full play to the advantages of Aspergillus chevalieri As MHR2 in producing linalool and other flavor substances, the existing Daqu preparation process can be targeted adjusted and optimized to achieve more efficient synthesis of flavor substances and process adaptation.

[0026] The application provided by the present invention introduces Aspergillus chevalieri As MHR2 that produces linalool, and clarifies the metabolic mechanism and pathway of linalool synthesis, providing new microbial resources and theoretical support for the Baijiu brewing industry, and contributing to the overall improvement of Baijiu quality and the intelligent upgrading of brewing technology. Description of the Drawings

[0027] Figure 1 Fungal composition of Fengxiang-flavor Daqu at the genus level and species level; among them, (a) is the genus level and (b) is the species level;

[0028] Figure 2 Colony morphology of the strain Aspergillus chevalieri As MHR2 of the present invention on a PDA plate;

[0029] Figure 3 Colony morphology of the strain Aspergillus chevalieri As MHR2 of the present invention under a stereomicroscope;

[0030] Figure 4 Morphology of the cells of the strain Aspergillus chevalieri As MHR2 of the present invention at different magnifications under a scanning electron microscope;

[0031] Figure 5 Phylogenetic tree of the strain Aspergillus chevalieri As MHR2 of the present invention based on the ITS rRNA sequence;

[0032] Figure 6 This is the result of the whole-genome ANI / AAI analysis of the strain Aspergillus chevalieri As MHR2 of the present invention;

[0033] Figure 7 This is the HS-SPME-GC-MS spectrum of the cells of the strain Aspergillus chevalieri As MHR2 of the present invention;

[0034] Figure 8 This is the HS-SPME-GC-MS spectrum of the cells of the strain Saccharomycopsis fibuligera;

[0035] Figure 9 This is the HS-SPME-GC-MS spectrum of the cells of the strain Thermoascus aurantiacus;

[0036] Figure 10 This is the HS-SPME-GC-MS spectrum of the PDA medium;

[0037] Figure 11 This is the HS-SPME-GC-MS spectrum of linalool reference substance and the internal standard substance 2-octanol;

[0038] Figure 12 This is the HS-SPME-GC-MS spectrum for the quantitative detection of linalool in the cells of the strain Aspergillus chevalieri As MHR2 of the present invention;

[0039] Figure 13 This is the stereomicroscopic sensory map of the strain Aspergillus chevalieri As MHR2 of the present invention at different times after being mixed into the raw materials of Daqu for cultivation;

[0040] Figure 14 This is the HS-SPME-GC-MS spectrum of the strain Aspergillus chevalieri As MHR2 of the present invention after being mixed into the raw materials of Daqu for cultivation;

[0041] Figure 15 This is the HS-SPME-GC-MS spectrum of Saccharomycopsis fibuligera after being mixed into the raw materials of Daqu for cultivation;

[0042] Figure 16HS-SPME-GC-MS chromatogram after Thermoascus aurantiacus was mixed into the raw materials of Daqu for cultivation;

[0043] Figure 17 HS-SPME-GC-MS chromatogram of the raw materials of Daqu;

[0044] Figure 18 HS-SPME-GC-MS chromatogram for quantitative detection of linalool in the raw materials of Daqu;

[0045] Figure 19 HS-SPME-GC-MS chromatogram for quantitative detection of linalool after the strain Aspergillus chevalieri As MHR2 of the present invention was mixed into the raw materials of Daqu for cultivation;

[0046] Figure 20 Linalool metabolic pathway of the strain Aspergillus chevalieri As MHR2 of the present invention. Detailed implementation manners

[0047] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods without special instructions; the materials, reagents, etc. used, without special instructions, can all be reagents and materials obtained from commercial channels.

[0048] The strain of Saccharomycopsis fibuligera used in the present invention was deposited on November 20, 2024 at the International Depository Authority of Microorganisms under the Budapest Treaty: China General Microbiological Culture Collection Center (CGMCC), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Deposit Number: CGMCC No. 41633, and the proposed taxonomic name is: Saccharomycopsis fibuligera, alive. Thermoascus aurantiacus was purchased from China National Center for Industrial Culture Collection of Microorganisms, and the deposit number is CICC 41655.

[0049] The composition of the potato dextrose medium used is: 200 g of potato, 20 g of dextrose, 1 g of yeast extract, and 1000 mL of distilled water.

[0050] The PDA medium used consisted of: 200 g of potato, 20 g of glucose, 15 g of agar, and 1000 mL of distilled water.

[0051] Example 1

[0052] This example provides the isolation, purification, and identification of a strain of Aspergillus chevalieri As MHR2 that produces linalool. The specific process is as follows:

[0053] (I) Isolation and purification

[0054] First, the PacBio SMRT ITS sequencing technology was used to determine that Aspergillus chevalieri is a high-abundance core microbiota in Fengxiang-flavor Daqu. PacBio full-length fungal sequencing was performed on Fengxiang-flavor Daqu samples, and the results are as Figure 1 shown.

[0055] From the attached Figure 1 data, at the genus classification level, the top three genera in terms of abundance ratio are Thermoascus (60.96%), Saccharomycopsis (33.38%), and Aspergillus (5.31%) ( Figure 1 a); at the species classification level, the top three species in terms of abundance ratio are Aspergillus chevalieri (5.70%), Saccharomycopsis fibuliger (31.69%), and Thermoascus aurantiacus (62.22%). It shows that Aspergillus chevalieri, Saccharomycopsis fibuligera, and Thermoascus aurantiacus are the three dominant fungi in Daqu. Thus, Aspergillus chevalieri was determined to be a high-abundance core microbiota in Fengxiang-flavor Daqu.

[0056] Collect Fengxiang-flavor Daqu samples. Place the Daqu samples under a stereomicroscope, pick out the koji core samples with yellow spots in the samples to prepare a bacterial suspension, inoculate the bacterial suspension on a PDA plate medium, and culture it at 30 °C for 3 - 7 d to obtain a plate culture of the target bacterium. Pick out the target bacterium on the plate culture, inoculate it on a PDA plate medium, and culture it at 30 °C for 3 - 7 d to obtain a pure plate culture of the target bacterium. Pick out the target bacterium in the pure plate culture, inoculate it on a PDA slant medium, and culture it at 30 °C for 3 - 7 d to obtain the isolated and purified target bacterium.

[0057] The culture medium is PDA culture medium.

[0058] (II) Identification

[0059] Aspergillus chevalieri As MHR2 isolated from Fengxiang Daqu was identified by morphological, ITS sequencing and fungal genome de novo sequencing techniques to further accurately determine the taxonomic status of Aspergillus chevalieri As MHR2.

[0060] 1. Morphological identification

[0061] The morphology of the strain was observed by three methods: plate culture, stereomicroscope and scanning electron microscope. The colony morphology on the plate, the colony morphology under the stereomicroscope and the mycelium morphology under the scanning electron microscope are shown in the attached figure Figures 2-6 as shown.

[0062] As can be seen from the attached Figures 2-6 , the colony is bright yellow to dark yellow or yellowish brown, with a compact texture, difficult to pick, a rough surface, and slow spreading growth ( Figure 2 ); the hyphae are slender and sparsely reticulated between the hyphae ( Figure 4 a)); usually sexual reproduction, forming spiral ascogonia on the aerial hyphae, with obvious ascocarp primordia. The ascocarp is a cleistothecium, bright yellow to dark yellow or yellowish brown, spherical or nearly spherical, with a thin wall, smooth, naked or surrounded by a sparse hyphal net. There are many asci in the ascocarp. The asci are spherical or nearly spherical, containing 8 ascospores, and the ascus wall is easily decomposed; the ascospores are biconvex lens-shaped, with obvious grooves, ridges or cristate protrusions, and the convex surface is smooth or relatively smooth ( Figure 3 , Figure 4 ). The morphological characteristics are close to those of Aspergillus chevalieri.

[0063] 2. Molecular biology identification

[0064] (1) Bacterial DNA extraction

[0065] (2) PCR amplification: The primers selected for ITS rRNA gene amplification were ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS4R (5’-TCCTCCGCTTATTGATATGC-3’). The PCR reaction system was as follows: 4 μL of 5×FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of FastPfu polymerase, 0.2 μL of BSA, 10 ng of template DNA, and made up to 20 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min, 27 cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s), then stable extension at 72°C for 10 min, and finally stored at 4°C (PCR instrument: ABI Model 9700).

[0066] (3) Take the purified PCR product for DNA sequencing

[0067] Using MEGA 6.0 software for cluster analysis, it was found that this strain was in the same branch as Aspergillus chevalieri MZ573106.1, Aspergillus amstelodami MN187972.1, and Aspergillus montevidensis NR137449.1. It indicates that the ITS rRNA gene phylogenetic tree can classify the strain As MHR2 as Aspergillus amstelodami, Aspergillus montevidensis, or Aspergillus chevalieri at the species level, but cannot clearly classify it as a single species. The results are as Figure 5 shown.

[0068] Results of whole-genome sequencing analysis: The default algorithm Ortho Average Nucleotide Identity (ANI) was selected to evaluate the phylogenetic relationship between fungal species at the whole-genome level. The average nucleotide identity (ANI) of the whole-genome sequence of strain MHR2 was compared with the whole-genome sequences of Aspergillus amstelodami (GCA 027569315.1), Aspergillus montevidensis (GCA020826735.1), and Aspergillus chevalieri (GCA016861735.1). The results are as Figure 6As shown in the figure. The ANI between strain As MHR2 and Aspergillus chevalieri was 99.7%, and the ANI was greater than 96%. The ANI values of the remaining strains and strain As MHR2 were all lower than 90%. Therefore, it was determined that strain As MHR2 and Aspergillus chevalieri were the same species, indicating that strain As MHR2 was Aspergillus chevalieri.

[0069] Based on the above biological characteristics, the above strain Aspergillus chevalieri AsMHR2 was deposited. This strain was deposited at the International Depository Authority of Microorganisms under the Budapest Treaty: China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Deposit number: CGMCC No. 41632. The proposed taxonomic name is: Aspergillus chevalieri.

[0070] The NCBI accession number of the ITS rRNA sequence of strain Aspergillus chevalieri As MHR2 (abbreviated as: Aspergillus chevalieri MHR2) is PQ851626; the NCBI accession number of the whole gene sequence is JBKJAG000000000.

[0071] Example 2

[0072] Based on Example 1, the strain Aspergillus chevalieri As MHR2 isolated in Example 1 was activated and inoculated into a PDA medium for cultivation. At the same time, Saccharomycopsis fibuligera and Thermoascus aurantiacus were used as controls to qualitatively and quantitatively detect the volatile substances and linalool produced by the cells of Aspergillus chevalieri As MHR2.

[0073] 1. Test materials

[0074] Strains: Aspergillus chevalieri As MHR2, Saccharomycopsis fibuligera, Thermoascus aurantiacus.

[0075] Culture medium: PDA (Potato Dextrose Agar) medium.

[0076] Instruments: HS-SPME (Headspace Solid Phase Microextraction) device, GC-MS (Gas Chromatography-Mass Spectrometry) instrument (GC-MS-QP2020NX, Shimadzu Corporation, Japan).

[0077] Standards and internal standards: Linalool standard, 2-Octanol standard.

[0078] 2. Test procedures

[0079] (1) Bacterial culture

[0080] Aspergillus chevalieri As MHR2, Saccharomycopsis fibuligera, and Thermoascus aurantiacus were respectively inoculated into PDA medium; Aspergillus chevalieri As MHR2 and Saccharomycopsis fibuligera were cultured at 30 °C, and Thermoascus aurantiacus was cultured at 48 °C; Aspergillus chevalieri As MHR2 was cultured until specific time points (such as 4 days, 9 days, etc.), and other strains were cultured as controls until the same or appropriate time points.

[0081] (2) Extraction of volatile substances

[0082] The HS-SPME device was used to extract volatile substances from the cultured bacterial samples, and the extraction conditions were recorded.

[0083] (3) GC-MS analysis

[0084] The parameters of the GC-MS instrument were set to separate and detect volatile substances; the mass spectra were obtained and saved.

[0085] (4) Qualitative and quantitative analysis

[0086] The mass spectra were compared to qualitatively analyze linalool. The internal standard method was used to draw a linear regression equation based on the peak area ratio and calculate the content of linalool.

[0087] (5) Calculation of rOAV value

[0088] The rOAV value was calculated based on the content and threshold of linalool.

[0089] The results are shown in Appendix Figure 7 ~AppendixFigure 10 as shown

[0090] Using HS-SPME-GC-MS volatile substance analysis technology, qualitatively and quantitatively detect the volatile substances and linalool produced by the cells of Aspergillus chevalieri As MHR2. The results are as follows: The linalool produced by the cells of Aspergillus chevalieri As MHR2 is significantly higher than that of the two dominant bacteria, Saccharomycopsis fibuligera and Thermoascus aurantiacus, and the control. When Aspergillus chevalieri As MHR2 is cultured in PDA medium for 8 days, the relative content of linalool can reach 345.202 μg / kg, reaching the highest ( Figure 7 ). The relative contents of linalool in the cells of Saccharomycopsisfibuligera and Thermoascus aurantiacus are both lower than the detection limit ( Figures 8-10 ).

[0091] Accurately quantify the linalool in the cell samples during the cultivation of Aspergillus chevalieri As MHR2 and calculate its rOAV value. The results are as follows: The retention time of linalool is 24.735 min. According to the peak area ratio of linalool to the internal standard substance, a linear regression equation is plotted as y = 1008.7x - 0.11 (r 2 = 0.9919) ( Figure 11 ). After calculation, the linalool content in the PDA medium control is lower than the detection limit. The linalool content in the cell samples during the cultivation of Aspergillus chevalieri As MHR2 is lower than the detection limit at the beginning of cultivation and reaches the highest on the 8th day, with a content of 1.583 μg / g and an rOAV value of 263.833 ( Figure 12 ).

[0092] Example 3

[0093] Mix the seed liquid of Aspergillus chevalieri As MHR2 into the raw materials of Daqu at an addition amount of 5% of the mass of the raw materials of Daqu. For the cultured Daqu, use HS-SPME-GC-MS volatile substance analysis technology to qualitatively and quantitatively detect the volatile substances and linalool. Use a stereomicroscope to observe the growth of Aspergillus chevalieri As MHR2 on the raw materials of Daqu and record the observation results. The test results are shown in the appendixFigure 13 -Attached Figure 19 .

[0094] (1) Qualitative and quantitative detection of volatile substances and linalool

[0095] Accurately weigh 2.0g of solid sample and add it to the headspace bottle. Before loading, add 20μL of internal standard 2-octanol (concentration 50.0μg / mL), 4mL of distilled water, and 1.5g of NaCl to the headspace bottle, and use a solid phase microextraction autosampler equipped with an extraction head (DVB / C-WR / PDMS) to perform headspace solid phase microextraction on volatile substances, and analyze at 250℃ for 5min. Heating program: 45℃ for 3min, increase the temperature to 230℃ at a rate of 4℃ / min, and maintain for 6min; carrier gas He, flow rate 3mL / min, non-split injection. EI ionization source, electron energy -70eV, ion source temperature 210℃, injection port temperature 250℃, mass scanning range 35.0~350.0m / z. The data from the gas chromatography-mass spectrometer were processed using GC-MS solution 4.52. The results with a matching degree greater than 80% were retained for qualitative analysis through mass spectrometry analysis and comparison with the NIST 20.0 standard spectral library.

[0096] The results showed that the linalool produced by mixing Aspergillus chevalieri As MHR2 with Daqu raw materials was significantly higher than that of Saccharomycopsis fibuligera, Thermoascus aurantiacus and the control.

[0097] Aspergillus chevalieri As MHR2 was mixed into the Daqu raw material and cultured at 30℃ for sensory observation under a stereomicroscope. The results showed that after 2 days of culture, the number of bright yellow ascocarps on the surface of the Daqu raw material increased and grew vigorously with the extension of culture time, until it almost covered the surface of the Daqu raw material and tended to be stable ( Figure 13 ).

[0098] Aspergillus chevalieri As MHR2 was added to the Daqu raw material at a rate of 10%, and the volatile substances and linalool in the Daqu were qualitatively and quantitatively detected using HS-SPME-GC-MS volatile substance analysis technology. The results showed that the relative content of linalool could reach 1.511ug / g (8 days after mixing with the Daqu raw material for cultivation). Figure 14), significantly higher than the two dominant bacteria, Saccharomycopsis fibuligera and Thermoascus aurantiacus, and the control ( Figures 15-17 ).

[0099] Linalool in the Daqu samples at different times after Aspergillus chevalieri As MHR2 was mixed into the Daqu raw materials was accurately quantified, and its rOAV value was calculated. After calculation, the linalool content in the Daqu raw material control group was 0.268 μg / g ( Figure 18 ), and the linalool content in the samples during the cultivation of Aspergillus chevalieri As MHR2 in the Daqu raw materials reached the highest of 1.511 μg / g on the 8th day of cultivation, and its rOAV value was 251.833 ( Figure 19 ).

[0100] (2) Formation mechanism and metabolic pathway of linalool produced by Aspergillus chevalieri As MHR2

[0101] Based on the functional annotations and metabolic relationships in the whole genome of Aspergillus chevalieri As MHR2 and the linalool metabolome database, the formation mechanism and metabolic pathway of linalool were determined, further confirming the ability of this strain to produce linalool.

[0102] Formation mechanism of linalool: The strain Aspergillus chevalieri As MHR2 has the ability to produce linalool. The synthesis of linalool mainly depends on the MVA (mevalonic acid) pathway, which is catalyzed by a series of key enzymes in the strain. These key enzymes include HMGCS (3-hydroxy-3-methylglutaryl-CoA synthase), MVK (mevalonate kinase), PMVK (phosphomevalonate kinase), MVD (diphosphomevalonate decarboxylase), and FDPS (farnesyl diphosphate synthase). These enzymes play a crucial role in the synthesis of linalool. According to the qualitative and quantitative analysis of the metabolome of Aspergillus chevalieri As MHR2, the key enzyme-encoding genes related to linalool production, such as hydroxymethylglutaryl-CoA synthase (HMGCS), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate decarboxylase (MVD), and farnesyl diphosphate synthase (FDPS), were found in KEGG( Figure 20 ).

[0103] Metabolic pathway:

[0104] Initial stage: The metabolic pathway starts with acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). These substances synthesize mevalonic acid (MVA) under the catalysis of HMGCS and MVK.

[0105] Intermediate stage: MVA then undergoes phosphorylation and decarboxylation reactions to form isopentenyl pyrophosphate (IPP). This step is a key transformation in the MVA pathway and provides the basis for subsequent terpene synthesis.

[0106] Key step: IPP is further converted to geranyl pyrophosphate (GPP) under the catalysis of FDPS. GPP has a C10 backbone and is one of the direct precursors of terpene compounds. In the strain Aspergillus chevalieri As MHR2, GPP enters the terpene backbone biosynthesis pathway and finally generates (+)-linalool and (-)-linalool under the action of (3S)-linalool synthase or (3R)-linalool synthase (LIS), respectively.

[0107] Key encoding genes: Through functional annotation of the whole-genome and metabolome databases, the key enzyme-encoding genes related to linalool production were identified. The strain Aspergillus chevalieri As MHR2 encoded one HMGCS gene (gene5148), two MVK genes (gene1526 and gene9308), one PMVK gene (gene3319), one MVD gene (gene4251), and five FDPS genes (gene2013, gene2119, gene4469, gene4631, gene6373).

[0108] Metabolic pathway confirmation: The strain Aspergillus chevalieri As MHR2 was not annotated with enzymes related to the DOXP / MEP pathway, indicating that it participates in linalool synthesis through the MVA pathway rather than the DOXP / MEP pathway, further confirming the importance of the MVA pathway in linalool production by the strain Aspergillus chevalieri As MHR2.

[0109] In summary, in the linalool-producing metabolic pathway of Aspergillus chevalieri As MHR2, based on the catalytic roles of the HMGCS, MVK, PMVK, MVD, and FDPS encoding genes in the linalool metabolism process, these 10 genes may be the key genes for linalool metabolism in the strain Aspergillus chevalieri As MHR2. These key enzymes play important roles in the catalytic reaction and are key nodes in the linalool metabolic pathway, providing an important basis for in-depth understanding of the mechanism of linalool production by the strain Aspergillus chevalieri As MHR2.

[0110] The present invention isolated, purified, and identified a strain Aspergillus chevalieri As MHR2 with the ability to produce linalool, and confirmed its high linalool-producing ability in both PDA medium and Daqu made from Daqu raw materials after cultivation. At the same time, through analysis of the whole-genome and metabolome databases, the formation mechanism and metabolic pathway of linalool production by Aspergillus chevalieri As MHR2 were revealed, and the key enzyme-encoding genes were determined. The present invention not only provides microbial resources with high linalool production for the liquor brewing industry, but also provides a theoretical basis and technical support for improving liquor quality through microbial regulation, promoting the intelligent development of liquor brewing technology.

[0111] The above content is only for explaining the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A strain of Aspergillus shevarii producing linalool ( Aspergillus chevalieri ) As MHR2, characterized in that The strain is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC No.41632.

2. A Daqu for brewing Fengxiang-style liquor, characterized in that: A linalool-producing Aspergillus shevarii strain according to claim 1 ( Aspergillus chevalieri )As MHR2.

3. The Daqu for brewing Fengxiang-style liquor according to claim 2, characterized in that: The linalool-producing Aspergillus shevarii in the Daqu used for brewing Fengxiang-style liquor ( Aspergillus chevalieri )The viable count of As MHR2 is 1×10 5 ~1×10 6 CFU / g.

4. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 2 or 3, characterized in that: include: The linalool-producing Aspergillus shevarii of claim 1 ( Aspergillus chevalieri ) As MHR2 is inoculated into liquid culture medium and cultured under suitable conditions to prepare seed solution; After the grain raw materials are crushed, the moisture content is adjusted to 35-45%, and the raw materials are obtained by steaming and sterilizing; The seed liquid is inoculated into the processed Daqu raw material in a certain proportion, mixed thoroughly and evenly, and then transferred to the fermentation room for fermentation to obtain Daqu for brewing Fengxiang-type liquor.

5. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 4, characterized in that: The liquid culture medium comprises: 200 g potato, 20 g glucose, 1 g yeast extract, and 1000 mL distilled water.

6. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 4, characterized in that: The culture conditions are: constant temperature culture at 25-35° C. for 5-9 days.

7. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 4, characterized in that: The cereal raw material is barley:wheat:pea in a mass ratio of 6:1:

3.

8. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 4, characterized in that: The seed liquid inoculation ratio is 5% to 15% of the mass of the processed Daqu raw material.

9. The method for preparing Daqu for brewing Fengxiang-style liquor according to claim 4, characterized in that: The fermentation conditions are: culturing at 25-35° C. for 5-9 days.

10. The linalool-producing Aspergillus shevarii strain of claim 1 ( Aspergillus chevalieri ) As MHR2 or the use of a Daqu for brewing Fengxiang-style liquor as described in any one of claims 2 to 3 in brewing Fengxiang-style liquor.

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