Gamma-nonanolactone-producing covered saccharomyces fibuligerus and application of covered saccharomyces fibuligerus in Daqu for brewing feng-flavor liquor

Abstract

The invention discloses a gamma-nonanolactone-producing covered yeast and application thereof in production of yeast for brewing feng-flavor liquor, and belongs to the technical field of processing of brewed liquor. The strain can significantly improve the flavor and quality of Baijiu, especially feng-flavor Baijiu. By introducing the strain to culture the yeast for making hard liquor, the content of gamma-nonalactone in the yeast for making hard liquor is remarkably improved and is far higher than that of a control strain, so that the taste of white liquor is improved, the yeast for making hard liquor fully utilizes the metabolic characteristics of Saccharomyces fibulligera Sa XF Sa-P5, the quality of the yeast for making hard liquor and liquor products is improved, and the fermentation efficiency and the raw material utilization rate are also improved. In addition, the invention also discloses a mechanism and a metabolic pathway of the Saccharomyces fibuligera Sa XF Sa-P5 for forming the gamma-nonanolactone, a new microbial resource and a theoretical basis are provided for the white spirit brewing industry, and further improvement of the quality of the white spirit and intelligent development of the brewing technology are promoted. The invention also discloses a method for preparing the gamma-nonanolactone by using the Saccharomyces fibuligera Sa XF Sa-P5 and a method for preparing the gamma-nonanolactone by using the Saccharomyces fibuligera Sa XF Sa-P5.

Description

Technical Field

[0001] The present invention belongs to the technical field of processed Chinese liquor brewing, and relates to flavor substances produced by microorganisms in Daqu for Chinese liquor brewing and their application in Daqu production. Specifically, it relates to a strain of Saccharomycopsis fibuligera producing γ-nonalactone isolated from Daqu and its application in the production of Daqu for Feng-flavor Chinese liquor brewing. Background Art

[0002] There are five major flavors of Chinese liquor, namely strong flavor type, Maotai flavor type, light flavor type, rice flavor type and Feng flavor type. It is a unique distilled liquor in China, with a long history and unique flavor. Chinese liquor is a distilled liquor mainly made from cereal grains such as barley, wheat, peas and sorghum, using Daqu as the saccharifying and fermenting agent, and is made through cooking, saccharification, fermentation and distillation. Daqu, determined by unique geographical location, environment, raw materials and technology, etc., is the key to the formation of the flavor of Chinese liquor, and the unique "microbial flora" in Daqu is the core factor for the formation of the flavor type of Chinese liquor. 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 flora, establishing an "bacterial system" evaluation system for Daqu, and through the detection and control of the "bacterial system", the stability of the flavor type of Chinese liquor can be fundamentally guaranteed. Therefore, by exploring more and more core microbial flora producing flavor substances in Daqu, the inherent essence of various flavor types of Chinese liquor can be gradually revealed, promoting the transformation of Chinese liquor from traditional to intelligent.

[0003] Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), is a yeast widely present in various Daqu, with strong starch liquefaction ability, saccharification ability, fermentation ability, esterification ability and proteolysis ability. Through its powerful enzyme system, it can hydrolyze macromolecular substances such as starch and protein in raw materials into small molecular substances such as fermentable sugars, amino acids and organic acids, providing material guarantee for the metabolism of itself and other microbial flora to produce flavor substances such as alcohols, aldehydes, acids, esters, ketones and aromatics, and plays a crucial role in Chinese liquor brewing and the formation of flavor substances. γ-Nonanolactone (Chinese name: γ-Nonanolactone; English name: γ-Nonanolactone; alias: γ-Nonalactone, coconut aldehyde, etc., CAS: 104-61-0), is an organic compound with a unique coconut flavor, mainly present in plant tissues such as coconut oil and coconut shell. In Daqu for Chinese liquor brewing, through detection, it is found that there is abundant γ-nonalactone produced, thus endowing Chinese liquor with a unique fragrance and flavor. However, at present, which microorganism produces it through what pathway has not been reported. Summary of the Invention

[0004] Although the fungal biomass in Daqu is large and plays an important role in saccharification, fermentation, esterification, etc., there is currently a lack of research on the composition of core fungi in Fengxiang Daqu, their contributions to Daqu, and their action mechanisms. The purpose of the present invention is to provide a Saccharomycopsis fibuligera strain producing γ-nonalactone and its application in the production of Daqu for brewing Fengxiang Baijiu.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions: The present invention provides a Saccharomycopsis fibuligera strain ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 producing γ-nonalactone, which is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC No. 41633 and the preservation date of November 20, 2024.

[0006] A Daqu for brewing Fengxiang Baijiu includes the above-mentioned Saccharomycopsis fibuligera strain ( Saccharomycopsis fibuligera ) Sa XF Sa-P5.

[0007] The viable count of the Saccharomycopsis fibuligera strain ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 in the Daqu for brewing Fengxiang Baijiu is 8×10 5 ~8×10 6 CFU / g.

[0008] A method for preparing the above-mentioned Daqu for brewing Fengxiang Baijiu includes: Inoculating the above-mentioned Saccharomycopsis fibuligera strain ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 into a liquid medium for cultivation to obtain a seed solution; Crushing cereal raw materials, adding water to make the water content reach 35% - 45%, and steaming and sterilizing to obtain processed Daqu raw materials; Inoculating the seed solution into the processed Daqu raw materials in proportion, stirring evenly, and placing in a fermentation chamber for fermentation to obtain Daqu for brewing Fengxiang Baijiu.

[0009] The liquid medium is: 200 g of potato, 20 g of glucose, and 1000 mL of distilled water.

[0010] The cultivation conditions are: cultivating at 25 - 38°C for 3 - 6 days.

[0011] Further, the cultivation conditions are: cultivating at a constant temperature of 32°C for 5 days.

[0012] The cereal raw materials are barley: wheat: pea with a mass ratio of 6:1:3.

[0013] The inoculation ratio of the seed liquid is 3% to 10% of the mass of the processed Daqu raw materials.

[0014] Furthermore, the inoculation ratio of the seed liquid is 5% of the mass of the processed Daqu raw materials.

[0015] The fermentation conditions are: culturing at 28 - 32°C for 7 - 9 days.

[0016] Furthermore, the fermentation conditions are: culturing at 35°C for 8 days.

[0017] The present invention provides the application of the above-mentioned Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 that produces γ-nonalactone or the above-mentioned Daqu for brewing fen-flavor liquor in the brewing of fen-flavor liquor.

[0018] Compared with the prior art, the present invention has the following beneficial effects: A strain of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 that produces γ-nonalactone provided by the present invention has the ability to produce γ-nonalactone, making this strain have potential application value in liquor brewing. Especially for those fen-flavor liquors that require γ-nonalactone to add specific flavors, the discovery of this strain provides new microbial resources for the brewing of fen-flavor liquor and significantly improves the quality of Daqu and the flavor of liquor.

[0019] A Daqu for brewing fen-flavor liquor provided by the present invention, by introducing Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 with high yield of γ-nonalactone to culture the Daqu, the relative content of γ-nonalactone in the Daqu reaches a significant level, far higher than that of control strains such as Aspergillus chevalieri and Thermoascus aurantiacus, significantly improving the quality of the Daqu, and then improving the flavor and taste of the liquor; it is expected to bring new breakthroughs to the liquor brewing industry and meet the needs of consumers for high-quality liquor.

[0020] The preparation method provided by the present invention, by making full use of the metabolic characteristics of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, can significantly improve the flavor and quality of the Daqu and the final liquor product, and at the same time improve the fermentation efficiency and raw material utilization rate; after introducing Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, it is necessary to appropriately adjust and optimize the existing Daqu preparation process to ensure that it can give full play to the advantages of producing flavor substances such as high-yield γ-nonalactone.

[0021] The application provided by the present invention, by introducing Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera), Saccharomycopsis fibuligera Sa XF Sa-P5, and reveals its formation mechanism and metabolic pathway, providing new microbial resources and theoretical basis for the Baijiu brewing industry, and is expected to promote the further improvement of Baijiu quality and the intelligent development of brewing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the fungal composition of Fengxiang Daqu at the genus level and species level; among them, (a) is at the genus level, and (b) is at the species level; Figure 2 shows the colony morphology of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention on a PDA plate; Figure 3 shows the colony morphology of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention under a stereomicroscope; Figure 4 shows the cell morphology of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention at different magnifications of a scanning electron microscope; Figure 5 shows the phylogenetic tree of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention based on the ITS rRNA sequence; Figure 6 shows the results of the whole-genome ANI / AAI analysis of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention; Figure 7 shows the HS-SPME-GC-MS spectrum of the cells of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention; Figure 8 is Aspergillus chevalieri the HS-SPME-GC-MS spectrum of the cells; Figure 9 is Thermoascus aurantiacus the HS-SPME-GC-MS spectrum of the cells; Figure 10 is the HS-SPME-GC-MS spectrum of the PDA medium; Figure 11 is the HS-SPME-GC-MS spectrum of 7 lactone reference substances and the internal standard substance 2-octanol; Figure 12 shows the HS-SPME-GC-MS spectrum for the quantitative detection of γ-nonalactone in the cells of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 of the present invention; Figure 13For the strain of the present invention, Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), the stereomicroscopic sensory images at different times after being mixed into the raw materials of Daqu for cultivation; Figure 14 For the strain of the present invention, Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), the HS-SPME-GC-MS spectrum after being mixed into the raw materials of Daqu for cultivation; Figure 15 For Aspergillus chevalieri the HS-SPME-GC-MS spectrum after being mixed into the raw materials of Daqu for cultivation; Figure 16 For Thermoascus aurantiacus the HS-SPME-GC-MS spectrum after being mixed into the raw materials of Daqu for cultivation; Figure 17 the HS-SPME-GC-MS spectrum of the raw materials of Daqu; Figure 18 For the strain of the present invention, Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), the HS-SPME-GC-MS spectrum for the quantitative detection of γ-nonalactone 8 days after being mixed into the raw materials of Daqu for cultivation; Figure 19 For the strain of the present invention, Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), the metabolic pathway for producing γ-nonalactone. Detailed implementation manners

[0023] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the 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 the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained from commercial channels.

[0024] Aspergillus chevalieri ( Aspergillus chevalieri ) adopted in the present invention is preserved in the China General Microbiological Culture Collection Center. The preservation date is November 20, 2024, and the preservation number is CGMCC No. 41632; Thermoascus aurantiacus ( Thermoascus aurantiacus ) is purchased from the China National Center for Industrial Culture Collection, and the preservation number is CICC 41655.

[0025] The composition of the potato dextrose medium adopted is: 200 g of potato, 20 g of dextrose, and 1000 mL of distilled water.

[0026] The PDA medium used consisted of: 200 g of potato, 20 g of glucose, 15 g of agar, and 1000 mL of distilled water.

[0027] Example 1 This example provides the isolation, purification and identification of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 with the ability to produce γ-nonalactone. The specific process is as follows: (I) Isolation and purification First, the PacBio SMRT ITS sequencing technology was used to determine that Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) was a high-abundance core flora in Fengxiang Daqu; PacBio full-length fungal sequencing was performed on Fengxiang Daqu samples, and the results were as Figure 1 shown.

[0028] From the attached Figure 1 data, it can be seen that at the genus classification level, the top three genera in terms of abundance ratio were Thermoascus (60.96%), Saccharomycopsis (33.38%), Aspergillus (5.31%) ( Figure 1 a); at the species classification level, the top three species in terms of abundance ratio were Saccharomycopsis fibuliger (31.69 %) 、Aspergillus chevalieri (5.70 %), Thermoascus aurantiacus (62.22 %). The results showed that Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) 、 Aspergillus chevalieri ( Aspergillus chevalieri ), Thermoascus aurantiacus ( Thermoascus aurantiacus ) were the three dominant fungi in Fengxiang Daqu. Thus, it was determined that Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) was a high-abundance core flora in Fengxiang Daqu.

[0029] The Daqu sample was placed under a stereomicroscope, and the snow-white target bacteria on the surface of the Daqu sample's koji skin were picked to prepare a bacterial suspension. The bacterial suspension was inoculated onto a PDA plate medium and cultured for 3 - 7 days to obtain a plate culture of the target bacteria. The target bacteria on the plate culture were picked and inoculated onto the corresponding plate medium, and cultured at the corresponding temperature for 3 - 7 days to obtain a pure plate culture of the target bacteria. The medium was the PDA medium.

[0030] (II) Identification The Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) isolated from Fengxiang Daqu was identified by morphological, ITS sequencing and fungal genome de novo sequencing technologies to further accurately determine Saccharomycopsisfibuligera Classification status

[0031] 1. Morphological identification The morphology of the strain was observed by three methods: plate culture, stereomicroscopy and scanning electron microscopy. The colony morphology on the plate, the colony morphology under the stereomicroscope and the cell morphology under the scanning electron microscope are shown in the attached figure Figure 2-6 as follows

[0032] As can be seen from the attached Figure 2-6 figure, the colony is round, milky white, shiny, relatively flat, with a neat and fringed edge, and a radial elevation on the surface ( Figure 2 ), and it grows relatively fast; the cells are round or oval; usually reproduce asexually by budding multilaterally, with well-developed pseudohyphae, and the cells are attached to the ends or surfaces of the pseudohyphae; in the initial stage of growth, pseudohyphae are dominant, growing radially in a creamy white color, and in the middle and late stages of growth, cells are dominant, and a large number of cells are attached to the ends or surfaces of the pseudohyphae ( Figure 3 , Figure 4 ). The morphological characteristics are in line with Saccharomycopsis fibuligera the morphological characteristics of

[0033] 2. Molecular biology identification (1) Yeast DNA extraction: (2) Yeast genomic PCR amplification: The primers selected for the amplification of the ITS rRNA gene of yeast are ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) (as shown in SEQ ID NO.1) and ITS4R (5’-TCCTCCGCTTATTGATATGC-3’) (as shown in SEQ ID NO.2). The PCR reaction system is 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. Each sample has 3 replicates. The PCR reaction conditions are: 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 GeneAmp® 9700 type).

[0034] (3) Take the purified PCR product for DNA sequencing Using MEGA 6.0 software for cluster analysis, it was found that the strain belongs to the same branch as Saccharomycopsis fibuligera MH855892.1. Therefore, the strain and Saccharomycopsis fibuligeraHighly similar, phylogenetic tree of strain Sa XF Sa-P5 based on ITS rRNA sequences by the neighbor-joining method, as shown in Figure 5 shown

[0035] 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 whole-genome sequence of strain Sa XF Sa-P5 was aligned with the whole-genome sequence of Saccharomycopsis fibuligera (GCA 001936155.1) for ANI, and the results are as shown in Figure 6 shown. The ANI between Sa XF Sa-P5 and Saccharomycopsis fibuligera was 98.55%, and the ANI was greater than 96%. Therefore, it was determined that Sa XF Sa-P5 and Saccharomycopsis fibuligera were of the same species, indicating that strain Sa XF Sa-P5 was Saccharomycopsis fibuligera .

[0036] Based on the above biological characteristics, the above-mentioned strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 was deposited. The 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. The deposit date was November 20, 2024, and the deposit number was CGMCC No. 41633. It is recommended to be taxonomically named as: Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ).

[0037] The ITS rRNA sequence NCBI accession number of the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 is PQ851635; the whole-genome sequence NCBI accession number is JBKKHO000000000.

[0038] Example 2 In this example, based on Example 1, the strain Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 isolated in Example 1 was activated and inoculated into PDA medium for cultivation. At the same time, Aspergillus chevalieri ( Aspergillus chevalieri ) and Thermoascus aurantiacus ( Thermoascus aurantiacus ) were used as controls to qualitatively and quantitatively detect the volatile substances and γ-nonalactone produced by the cells of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5.

[0039] 1. Test materials Strains: Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), Sa XF Sa-P5, Aspergillus chevalieri ( Aspergillus chevalieri ), Thermoascus aurantiacus ( Thermoascus aurantiacus ).

[0040] Medium: PDA (Potato Dextrose Agar) medium.

[0041] Instruments: HS-SPME (Headspace Solid Phase Microextraction) device, GC-MS (Gas Chromatography-Mass Spectrometry) instrument (GC-MS-QP2020 NX, Shimadzu Corporation, Japan).

[0042] Standard and internal standard substances: γ-nonalactone standard, appropriate internal standard substance.

[0043] 2. Test procedures (1) Bacterial culture Inoculate Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), Sa XF Sa-P5, Aspergillus chevalieri and Thermoascus aurantiacus into PDA medium respectively; culture Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), Sa XF Sa-P5 and Aspergillus chevalieri at 30 °C, Thermoascus aurantiacus at 48 °C; culture Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ), SaXF Sa-P5 until specific time points (such as 4 days, 9 days, etc.), and culture other strains as controls until the same or appropriate time points.

[0044] (2) Extraction of volatile substances Use the HS-SPME device to extract volatile substances from the cultured bacterial samples, and record the extraction conditions.

[0045] (3) GC-MS analysis Set the parameters of the GC-MS instrument to separate and detect volatile substances; obtain and save the mass spectra.

[0046] (4) Qualitative and quantitative analysis Compare the mass spectra to qualitatively analyze γ-nonalactone. Use the internal standard method to draw a linear regression equation based on the peak area ratio, and calculate the content of γ-nonalactone (5) r Calculation of OAV value Calculate the r OAV value according to the content and threshold of γ-nonalactone.

[0047] The results are shown in Appendix Figure 7 ~Appendix Figure 10 .

[0048] Using HS-SPME-GC-MS volatile substance analysis technology, qualitatively and quantitatively detect the volatile substances and γ-nonalactone produced by the cells of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5. The results are as follows: The γ-nonalactone produced by the cells of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 is significantly higher than that of Aspergillus chevalieri ( Aspergillus chevalieri ), Thermoascus aurantiacus ( Thermoascus aurantiacus ) and the control. When Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 is cultured in PDA medium for 4 days, the relative content of γ-nonalactone can increase from below the detection limit to 14.584 μg / kg, reaching the highest ( Figure 7 ). The relative content of γ-nonalactone in Aspergillus chevalieri cells and Thermoascus aurantiacus cells is below the detection limit ( Figure 8-10 ).

[0049] Accurately quantify the γ-nonalactone in the cell samples during the culture process of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, and calculate its r OAV value. The results are as follows: The retention time of γ-nonalactone is 38.337 min. According to the peak area ratio with the internal standard substance, draw a linear regression equation, which is y = 0.4813x + 0.006 (r 2 = 0.992) ( Figure 11 ). After calculation, γ-nonalactone in the PDA medium control is not detected. The content of γ-nonalactone in the cell samples during the culture process of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 is below the detection limit at the beginning of the culture, reaches the highest on the 9th day, and the content is 18.213 μg / kg, and its r OAV value is 7.285 ( Figure 12 ).

[0050] Example 3 Mix the seed liquid of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 into the Daqu raw materials at 5% of the mass of the treated Daqu raw materials, ferment at 30 °C for 8 days to obtain Daqu for brewing Fengxiang-flavor Baijiu. Using HS-SPME-GC-MS volatile substance analysis technology, qualitatively and quantitatively detect the volatile substances and γ-nonalactone. Use a stereomicroscope to observe the growth of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 on the Daqu raw materials, and record the observation results. See Appendix Figure 13 ~Appendix Figure 18 .

[0051] (1) Qualitative and quantitative detection of volatile substances and γ-nonalactone Accurately weigh 2.0000 g of solid sample (accurate to 0.0001 g), add 4 mL of distilled water, 1.5 g of NaCl and 20 μL of 2-octanol standard solution (50 μg / mL), 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 5 min. The heating program is: 45℃ for 3 min, then increase the temperature to 230℃ at a rate of 4℃ / min and keep it for 6 min; carrier gas He, flow rate 3 mL / min, non-split injection. EI ionization source, electron energy -70 eV, ion source temperature 210℃, injection port temperature 250℃, mass scanning range 35.0~350.0 m / z. The data from the gas chromatography-mass spectrometry instrument 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.

[0052] The results were: Saccharomycopsis fibuligera ) After Sa XF Sa-P5 was mixed with Daqu raw materials and cultured, the γ-nonalactone produced by Daqu was significantly higher than that of Aspergillus shevarii ( Aspergillus chevalieri )、Thermoascus aurantiacus( Thermoascus aurantiacus ) and control.

[0053] The capsule coated yeast ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 was mixed with Daqu raw materials and cultured at 30℃ for sensory observation under a stereo microscope. The results showed that after 2 days of culture, the surface of the Daqu raw materials grew vigorously with white mycelium in the shape of snowflakes. The mycelium was covered with white mycelium. After 2 days, the surface of the Daqu raw materials was covered with the white mycelium and became stable. Figure 13 ).

[0054] The capsule coated yeast ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 was added to the Daqu raw material at a rate of 5%, and the volatile substances and γ-nonalactone 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 γ-nonalactone reached 12405.518 ug / kg (6 days after being added to the Daqu raw material for cultivation). Figure 14 ), γ-nonalactone in Aspergillus chevalieri , Thermoascus aurantiacus The relative contents in the control group and Daqu raw materials were all below the detection limit ( Figures 15 - 17 ).

[0055] The capsule coated yeast ( Saccharomycopsis fibuligera)Accurately quantify the γ-nonalactone in the Daqu samples at different times after Sa XF Sa-P5 is mixed into the Daqu raw materials, and calculate its r OAV value. After calculation, γ-nonalactone in the Daqu raw materials was not detected, and Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5 was below the detection limit at the beginning of the culture, and reached the highest content of 2177.527 ug / kg on the 8th day of culture, and its r OAV value was 871.019 ( Figure 18 ).

[0056] (2)The formation mechanism and metabolic pathway of γ-nonalactone produced by Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5 Based on the functional annotations and metabolic relationships in the whole genome of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5 and the γ-nonalactone metabolome database produced, the formation mechanism and metabolic pathway of γ-nonalactone were determined, further confirming the ability of this strain to produce γ-nonalactone.

[0057] Synthesis pathway: According to the qualitative and quantitative analysis of the metabolome of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5, four synthesis pathways of γ-nonalactone were found in KEGG, including hydroxylation of fatty acids, β-oxidation and lactonization, reduction of unsaturated lactones, Baeyer-Villiger oxidation of cyclic ketones to form lactones, and a,ω-oxidation of alkanes or fatty acids to form a,ω-dicarboxylic acids, and then lactonization of a,ω-dicarboxylic acids to form macrocyclic lactones. According to the functional gene annotation results in the whole genome of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5, 17 key enzyme-encoding genes related to the production of γ-nonalactone, such as long-chain acyl-CoA synthetase (ACSL), fatty acid synthase subunit beta, fungi type (FAS1), acyl-coenzyme A thioesterase (ACOT), etc. Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera )Sa XF Sa-P5 encodes 10 ACSL (gene0449, gene0622, gene0450, gene0707, gene1349, gene1502, gene2652, gene2773, gene3343, gene3921), 3 FAS1 (gene1122, gene5708, gene0411), and 2 ACOT (gene2655, gene5543) genes.Saccharomycopsis fibuligera Under the catalysis of FAS1, Sa XF Sa-P5 generates palmitic acid, which is catalyzed by ACSL to form palmitoyl-CoA. Through a series of elongation and desaturation reactions, it is converted into linoleoyl-CoA. Linoleoyl-CoA is converted into linoleic acid under the catalysis of various enzymes such as ACOT. Linoleic acid undergoes hydroxylation to form (9Z,11E,13S)-13-hydroxyoctadec-9,11-dienoic acid, which is an important precursor for the synthesis of (S)-γ-nonalactone. In the previous experiments on the determination of the cellular metabolome of Saccharomycopsis fibuligera Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, these precursors were detected in high abundance in the samples by non-targeted metabolomics technology. Finally, after several cycles of β-oxidation and lactonization, (S)-γ-nonalactone is synthesized, which is also Figure 19 ) the main pathway for Sa XF Sa-P5 to synthesize γ-nonalactone.

[0058] Meanwhile, Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 converts and synthesizes γ-decalactone. First, using hydroxy fatty acids, non-hydroxy fatty acids, and fatty acid esters as substrates, and then through β-oxidation reaction to generate the precursor 4-hydroxydecanoic acid. Finally, 4-hydroxydecanoic acid forms γ-dodecalactone through the dehydration lactonization reaction of the hydroxyl group and carboxyl group. Figure 19 )

[0059] In summary, in the metabolic pathway of γ-nonalactone in Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, based on the catalytic effects of 15 ACSL, FAS1, and ACOT encoding genes in the metabolic process of γ-nonalactone, it is speculated that these 15 genes are Saccharomycopsis fibuligera the key genes for Sa XF Sa-P5 to produce γ-nonalactone metabolism.

[0060] In the present invention, a strain of Saccharomycopsis fibuligera ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 with the ability to produce γ-nonalactone was isolated, purified, and identified, and it was confirmed that it has the ability to produce high yields of γ-nonalactone both in PDA medium and in Daqu after being cultured with Daqu raw materials. At the same time, through the analysis of the whole genome and metabolome databases, the formation mechanism and metabolic pathway of the strain Sa XF Sa-P5 to produce γ-nonalactone were revealed, and the key enzyme encoding genes were determined. The present invention not only provides microbial resources with high yields of γ-nonalactone for the liquor brewing industry, but also provides a theoretical basis and technical support for improving the quality of liquor through microbial regulation, promoting the intelligent development of liquor brewing technology.

[0061] 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 γ-nonalactone-producing yeast ( Saccharomycopsis fibuligera ) Sa XF Sa-P5, characterized in that, The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.41633.

2. A Daqu for brewing Fengxiang-style liquor, characterized in that: A γ-nonalactone-producing yeast according to claim 1 ( Saccharomycopsis fibuligera )Sa XF Sa-P5.

3. The Daqu for brewing Fengxiang-style liquor according to claim 2, characterized in that: The capsule-coated yeast ( Saccharomycopsis fibuligera )The viable count of Sa XF Sa-P5 was 8×10 5 ~8×10 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 γ-nonalactone-producing yeast according to claim 1 ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 was inoculated into liquid culture medium to obtain seed solution; The grain raw materials are crushed, water is added to make the moisture content reach 35% to 45%, and steamed and sterilized to obtain the processed Daqu raw materials; The seed liquid is inoculated into the processed Daqu raw material according to a certain proportion, the mixture is fully stirred, and the mixture is placed in a fermentation room for fermentation to obtain Daqu for brewing Fengxiang-style 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, 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 25-38°C for 3-6 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 3% to 10% 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 25-38° C. for 3-9 days.

10. The γ-nonalactone-producing yeast according to claim 1 ( Saccharomycopsis fibuligera ) Sa XF Sa-P5 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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