Absolute quantification method of core yeast in liquor brewing and simultaneous analysis method of metabolic function

Abstract

The application belongs to the technical field of microbial fermentation, and relates to an absolute quantification method and a metabolic function synchronous analysis method of core yeast bacteria in liquor brewing. In view of the problem that the existing quantification method cannot consider absolute quantification of bacteria and metabolic function characterization, the application takes high alcohol synthesis key genes BAT2, ILV2 and ILV3 as molecular targets, designs inter-species specific primers, constructs a qPCR absolute quantification system, realizes rapid and accurate quantification of multiple core yeasts in a complex fermentation system, and simultaneously analyzes the metabolic characteristics of the high alcohol synthesis pathway through gene transcription level analysis. The application can simultaneously obtain yeast absolute content and metabolic function information, and provides technical support for liquor fermentation microbial monitoring, high alcohol regulation and flavor quality optimization.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to an absolute quantitative method and a method for simultaneous analysis of metabolic functions of core yeast strains used in Baijiu brewing. Background Technology

[0002] Solid-state fermentation of baijiu is a complex system involving the synergistic action of multiple microorganisms. The community structure succession and metabolic activity dynamics of the yeast community directly determine the yield and flavor quality of the base liquor. Higher alcohols (including isobutanol, isoamyl alcohol, and reactive amyl alcohol) are important flavor compounds in baijiu, and their content and proportion directly affect the flavor harmony and drinking comfort of the liquor. Excessive content not only leads to a bitter and unbalanced taste but also easily causes adverse reactions such as headaches after drinking. The synthesis of higher alcohols mainly relies on two conserved metabolic pathways: one is the Ehrlich pathway of amino acid catabolism, with the key gene BAT2; the other is the Harris pathway of de novo sugar synthesis, with the key genes ILV2 and ILV3. Saccharomyces cerevisiae is recognized as a major contributor to higher alcohols. However, recent high-throughput macroomics studies have confirmed that non-Saccharomyces cerevisiae, such as Saccharomyces cerevisiae, Isaac's orientalis, and Wickham's anomalous yeast, also make significant contributions to the synthesis of higher alcohols in baijiu fermentation. Furthermore, different yeasts exhibit significant differences in their response patterns and metabolic regulation mechanisms to key environmental factors such as nitrogen source abundance, fermentation acidity, and lactic acid bacteria interactions.

[0003] Currently, quantitative analysis methods for yeast in the Baijiu brewing system are mainly divided into two categories: traditional culture methods and non-culture molecular biology methods. While traditional plate count methods can obtain viable cell counts, they suffer from drawbacks such as long operation cycles (usually 2-3 days), high workload, and low throughput. Furthermore, they are limited by the selectivity of the culture medium and the specific culture conditions, making it impossible to accurately count the large numbers of difficult-to-culture and uncultured yeasts present in the system. To compensate for the shortcomings of traditional methods, various non-culture molecular biology detection technologies have been widely applied, but they still have significant technical limitations: for example, PCR-DGGE technology is affected by the multi-copy effect of ribosomal genes, enabling only semi-quantitative analysis and failing to obtain absolute abundance data; fluorescence in situ hybridization (FISH) technology has complex probe design, cumbersome specificity verification, and limited quantitative sensitivity; while metagenomic methods based on high-throughput sequencing can comprehensively analyze the species diversity and relative abundance of microbial communities, they have long detection cycles, high costs, and complex data analysis, making it difficult to meet the rapid and low-cost daily monitoring needs in industrial production processes.

[0004] Real-time quantitative PCR (qPCR) based on nucleic acid amplification is a recognized rapid quantitative detection method with advantages such as high specificity, high sensitivity, short detection cycle, and moderate throughput. It can effectively overcome the limitations of traditional quantitative methods in endpoint detection and has become a core tool for rapid microbial quantification. However, existing qPCR quantitative methods for yeast in brewing systems mostly target housekeeping genes commonly used for species identification, such as 18S rRNA and ITS. These methods can only reflect the species abundance of the target strain and cannot simultaneously characterize its transcriptional activity and functional contribution potential in specific metabolic pathways (such as the higher alcohol synthesis pathway). Ultimately, this leads to a disconnect between the bacterial population data and functional metabolic data in the analysis of flavor compound formation mechanisms, forming a significant information gap. There is an urgent need in this field to develop a technical method that uses functional genes as detection targets to simultaneously achieve rapid absolute quantification of multiple core yeasts in the brewing system and synchronous analysis of the transcriptional level of the higher alcohol synthesis pathway, providing key technical support for the process-oriented optimization and precise control of flavor quality in baijiu fermentation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an absolute quantitative method for core yeast strains in Baijiu brewing and a method for simultaneous analysis of their metabolic functions.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an absolute quantitative method for the core yeast strain in Baijiu brewing, the method comprising the following steps: (1) Establishing a qPCR quantitative standard curve for the core yeast: Obtain the pure culture of the core yeast and establish a standard correspondence between the absorbance of the culture at 600 nm wavelength and the concentration of live cells; take the culture with known live cell concentration and perform 10-fold serial dilutions, extract the genomic DNA of each gradient culture as a standard template, and perform qPCR amplification using specific primer pairs targeting the higher alcohol synthesis gene of the core yeast to obtain the fluorescence quantitative Ct value, and establish a linear standard curve between the logarithm of different yeast live cell concentrations and the Ct value; (2) Extract genomic DNA from the microorganisms in the sample to be tested; (3) Using specific primer pairs, qPCR amplification was performed with the genomic DNA extracted in step (2) as a template to obtain the fluorescence quantitative Ct value of the sample to be tested. The value was then substituted into the corresponding standard curve established in step (1) to calculate the absolute content of the target core yeast in the sample to be tested.

[0007] Preferably, the core yeast is selected from *Schizosaccharomyces cerevisiae* (Saccharomyces cerevisiae). Schizosaccharomyces pombe ), brewer's yeast ( Saccharomyces cerevisiae ), Oriental Isaac yeast ( Issatchenkia orientalis ) and abnormal Wickham yeast ( Wickerhamomyces anomalusOne or more of the following.

[0008] Preferably, the liquor is brewed using a small-batch, light-aroma type of liquor.

[0009] Preferably, the key gene for higher alcohol synthesis is selected from one or more of the BAT2, ILV2, and ILV3 genes.

[0010] Preferably, the detection of *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, and *Wickhamia lanceolata* uses a specific primer pair for the ILV3 gene; the detection of *Issa mesasura* uses a specific primer pair for the ILV2 gene.

[0011] Preferably, the nucleotide sequences of the specific primer pair are shown in SEQ ID NO:1 to SEQ ID NO:24 in the sequence listing.

[0012] Preferably, the annealing temperature for the qPCR amplification is 55-65℃, more preferably 60±5℃.

[0013] Preferably, the sample to be tested includes a sample from the process of making baijiu koji or the fermentation process of brewing; more preferably, it is a sample of fermented mash.

[0014] Secondly, the present invention provides a method for analyzing the absolute content of core yeast and its higher alcohol synthesis metabolism function, the method comprising the following steps: (1) The absolute content of each core yeast in the sample to be tested is obtained by using the absolute quantification method described in the first aspect; (2) Extract total RNA from microorganisms in the sample to be tested and reverse transcribe it to synthesize cDNA; (3) Using the specific primer pair described in the first aspect, qPCR amplification was performed with the cDNA synthesized in step (2) as a template to obtain transcriptional level data of key genes for higher alcohol synthesis in each core yeast; the absolute content data obtained in step (1) and the gene transcriptional level data obtained in step (3) were correlated to achieve absolute quantification of core yeast and analysis of higher alcohol synthesis metabolism function.

[0015] Compared with the prior art, the present invention has the following advantages: (1) This invention breaks through the technical limitations of traditional qPCR quantitative methods that use housekeeping genes such as 18S rRNA and ITS as detection targets. It uses key genes (BAT2, ILV2, ILV3) of the two core pathways of higher alcohol synthesis as molecular targets and establishes an absolute quantitative method for four core yeasts in the Baijiu brewing system. For the first time, it realizes the simultaneous monitoring and correlation analysis of the absolute abundance of strains and the metabolic function of higher alcohol synthesis.

[0016] (2) This invention provides a rapid, accurate, and high-throughput absolute quantitative method for yeast, which shortens the detection cycle by more than 90% compared to the traditional plate colony counting method (from 2-3 days to 4-6 hours), and can specifically distinguish different core yeasts, making it suitable for real-time dynamic monitoring of industrial brewing processes. The accuracy of the method was verified by a dual-system approach. In the liquid mixed culture system, the Pearson correlation coefficients r between the measured and actual values ​​of the four yeasts were 0.998, 0.857, 0.874, and 0.915, respectively (P < 0.01); in the solid-state fermentation complex system, the r values ​​were 0.932, 0.773, 0.931, and 0.845, respectively (P < 0.01).

[0017] (3) This invention can systematically analyze the regulatory mechanism of different fermentation conditions on the synthesis of higher alcohols in core yeast. In the liquid fermentation system, this method revealed the strain-specific regulatory law of nitrogen source abundance on higher alcohol synthesis: the addition of ammonium dihydrogen phosphate reduced the higher alcohol production of Schizosaccharomyces cerevisiae, Saccharomyces cerevisiae and Wickham's aberrant yeast by 61.9%, 22.1% and 13.1% respectively, while the production of Isaac's orientalis increased by 14.2%; gene expression analysis showed that the BAT2, ILV2 and ILV3 genes of the first three were significantly downregulated, while the above genes of Isaac's orientalis were upregulated, confirming that nitrogen source regulates the production of higher alcohols by regulating two synthetic pathways at the transcriptional level. In a solid-state fermentation system, the effect of *Lactobacillus brevis* interaction on the higher alcohol synthesis flux of different yeasts was elucidated: the addition of *Lactobacillus brevis* decreased the higher alcohol yield of *Schizophyllum commune*, *Saccharomyces cerevisiae*, and *Wickham's aberrant* by 29.9%, 14.6%, and 18.8%, respectively, while slightly increasing the yield of *Issa mesasulata* by 2%. Gene expression analysis showed that both synthetic pathways of *Schizophyllum commune* and *Saccharomyces cerevisiae* were significantly inhibited. In *Wickham's aberrant*, the BAT2 gene was downregulated, but the ILV2 and ILV3 genes were upregulated, indicating that its higher alcohol synthesis mainly depends on the Ehrlich pathway, and the upregulation of the sugar metabolism pathway could not offset the inhibitory effect of the amino acid decomposition pathway. These results indicate that this invention provides key technical support for the targeted optimization of baijiu fermentation process and the precise control of flavor substances. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Agarose gel electrophoresis images showing the primer specificity verification of four key genes for higher alcohol synthesis in core yeast. Among them: (a¹) is an electrophoresis diagram for primer specificity verification of the BAT2 gene of *Schizosaccharomyces cerevisiae*. (a²) is an electrophoresis diagram for primer specificity verification of the ILV2 gene of *Schizosaccharomyces cerevisiae*. (a³) is an electrophoresis diagram for primer specificity verification of the ILV3 gene of *Schizosaccharomyces cerevisiae*. (b¹) is an electrophoresis diagram verifying the specificity of the BAT2 gene primer in Saccharomyces cerevisiae; (b²) is an electrophoresis diagram verifying the primer specificity of the Saccharomyces cerevisiae ILV2 gene; (b³) is an electrophoresis diagram for primer specificity verification of the Saccharomyces cerevisiae ILV3 gene; (c¹) is an electrophoresis diagram verifying the specificity of the BAT2 gene primers in *Issa mesasura*. (c²) is an electrophoresis diagram for primer specificity verification of the ILV2 gene in *Issa mesasura*. (c³) is an electrophoresis diagram for primer specificity verification of the ILV3 gene in *Issa mesasura*. (d¹) is an electrophoresis diagram for primer specificity verification of the abnormal Wickham yeast BAT2 gene; (d²) is an electrophoresis diagram for specificity verification of the abnormal Wickham yeast ILV2 gene primers; (d³) is an electrophoresis diagram for primer specificity verification of the abnormal Wickham yeast ILV3 gene.

[0019] Lane 1 in all electrophoresis images is *Schizosaccharomyces cerevisiae* (Saccharomyces cerevisiae). Schizosaccharomyces pombe BJV11044; Lane 2 contains *Schizosaccharomyces cerevisiae* (BJV11044); Schizosaccharomyces pombe BJV11034; Lane 3 contains brewer's yeast ( Saccharomyces cerevisiae Lane 678; Lane 4 is Saccharomyces cerevisiae AY-12; Lane 5 is Isaac's orientalis (… Issatchenkia orientalis JM-3; Lane 6 is an abnormal Wickham yeast ( Wickerhamomycesanomalus Lane 7 is the negative control, Kazakhstani yeast JM-7; Lane 8 is the negative control, Manchurian Pichia pastoris JM-9; M is DL2000 DNA Marker.

[0020] Figure 2 Standard curves showing the absorbance at 600 nm wavelength and the concentration of live cells for four core yeast cultures.

[0021] Figure 3 This is a comparison chart of the measured values ​​and actual inoculation values ​​obtained by the quantitative method established in this invention in a liquid mixed bacterial system.

[0022] Figure 4 A comparison chart of the measured values ​​and actual inoculation values ​​obtained by the quantitative method established in this invention in a solid-state fermented mash mixed microbial system.

[0023] Figure 5This figure compares the effects of nitrogen source addition on the production of higher alcohols and the expression levels of key genes involved in their synthesis in four core yeasts under liquid fermentation conditions. Specifically: (ad) shows a comparison of higher alcohol production in *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Issa mesosoma orientalis*, and *Wickhamia sapiens*, respectively; (e) shows a comparison of the relative expression levels of the BAT2, ILV2, and ILV3 genes in the four core yeasts.

[0024] Figure 6 For solid-state fermentation conditions, short-lived lactobacillus ( Lactobacillus brevis The effects of the addition of *Saccharomyces cerevisiae* on the production of higher alcohols and the expression levels of key genes involved in their synthesis in four core yeasts are compared. (a) shows the comparison of higher alcohol production in each yeast on day 2 of fermentation; (b) shows the comparison of higher alcohol production in each yeast on day 7 of fermentation; and (c) shows the comparison of the relative expression levels of the BAT2, ILV2, and ILV3 genes in the four core yeasts. Abbreviations in the figures: Sp: *Schizosaccharomyces cerevisiae*; Sc: *Saccharomyces cerevisiae*; Io: *Issarum orientalis*; Wa: *Wickhamia sapiens*; -D2: co-culture group with *Lactobacillus brevis* added. Detailed Implementation

[0025] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0026] Unless otherwise specified, the raw materials involved in the following specific embodiments of the present invention are all conventional materials in the art and can be purchased from commercially available products.

[0027] The relevant information of the primers and primer pairs used in the embodiments of this invention is shown in Table 1: Table 1. Specific primers for different yeast species used in this experiment

[0028] Example 1: Design and Validation of Yeast-Specific Probes Based on Higher Alcohol Synthesis Pathways This invention selects key genes from two core pathways of higher alcohol synthesis as molecular targets: the BAT2 gene of the Ehrlich pathway of amino acid catabolism and the ILV2 and ILV3 genes of the Harris pathway of de novo sugar synthesis. Based on the above gene sequences of different yeast strains, interspecies-specific primers are designed.

[0029] The specific steps are as follows: Retrieve the yeast for winemaking (Schizozyme spp.) from the NCBI database. Schizosaccharomyces pombe NCBI: txid4896), brewer's yeast ( Saccharomyces cerevisiae NCBI: txid4932), Oriental Isaac Yeast ( Issatchenkiaorientalis(NCBI: txid4909) and abnormal Wickham yeast ( Wickerhamomycesanomalus Reference sequences of the BAT2, ILV2, and ILV3 genes (NCBI: txid4927) were obtained. Multiple alignment analysis of homologous gene sequences from different yeast species was performed using SnapGene software. Specific primers were designed in the regions of least sequence conservation, controlling the amplified fragment length to be 150–200 bp. All primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and primer information is detailed in Table 1. This example uses conventional PCR amplification to verify the interspecies specificity of the designed primers. The PCR reaction system is shown in Table 2.

[0030] Table 2 PCR Validation Reaction System

[0031] The PCR procedure is shown in Table 3.

[0032] Table 3 PCR Validation Reaction System

[0033] Genomic DNA from laboratory-preserved *Schizosaccharomyces cerevisiae* BJV11044, *Schizosaccharomyces cerevisiae* BJV11034, *Saccharomyces cerevisiae* 678, *Saccharomyces cerevisiae* AY-12, *Issa mesasura* JM-3, and *Wickhamia lanceolata* JM-5 was used as positive templates, and genomic DNA from *Saccharomyces kazakhstanensis* JM-7 and *Pichia manshuriensis* JM-9 was used as negative controls for PCR amplification and verification.

[0034] The experimental results are as follows: When using SP-BAT2-F / SP-BAT2-R as primer pairs for amplification, only the templates of *Schizosaccharomyces cerevisiae* BJV11044 and BJV11034 amplified the expected specific band (150 bp). No corresponding bands were observed in other yeast templates or the negative control, indicating that this primer pair is specific only to *Schizosaccharomyces cerevisiae*. The specificity of the other primer pairs in Table 1 was verified using the same method. The results showed that all primers amplified only a single band of the expected length in the corresponding target yeast genome, with no non-specific amplification or primer dimer formation. Figure 1 This confirms that the primers designed in this invention have good interspecies specificity.

[0035] Example 2: Establishment of a core yeast absolute quantification method based on qPCR I. Determination of the Standard Curve for Yeast Fluid Absorbance vs. Cell Count Different yeast species exhibit significant differences in cell morphology and size, resulting in varying correlations between the absorbance of their bacterial suspensions at 600 nm and the concentration of viable cells (cfu / mL). This embodiment establishes a linear correlation between the absorbance of bacterial suspensions at 600 nm and the concentration of viable cells for four core yeast species using the dilution-spread plate counting method.

[0036] The specific steps are as follows: (1) Pick the purified single colonies of each yeast strain, inoculate them into 5 mL of YPD liquid medium, label the strain information, and culture them at 30℃ and 180 r / min for 12 h until the logarithmic growth phase.

[0037] (2) In a clean bench, take 1.5 mL of each yeast culture and transfer it to a 2 mL sterile EP tube. Centrifuge at 12000 r / min for 2 min, discard the supernatant, resuspend the bacterial precipitate with sterile physiological saline, and wash once.

[0038] (3) Use sterile physiological saline to adjust each bacterial suspension to a gradient of absorbance of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 at a wavelength of 600 nm. Set up 3 parallel samples for each gradient.

[0039] (4) Perform serial 10-fold dilutions of each grade of bacterial suspension. Take 100 μL of the appropriate dilution and spread it evenly on YPD solid medium plates with sterile glass beads. Invert the plates and incubate them in a 30°C incubator for 2–5 days.

[0040] (5) Select plates with colony counts between 30 and 300 to count the cells, calculate the viable cell concentrations corresponding to each gradient, and use Office Excel 2019 software to plot the standard curve of the absorbance of the bacterial solution at 600 nm wavelength versus the logarithm of the viable cell concentration.

[0041] The experimental results are as follows: The linear relationship between the absorbance of the *Schizosaccharomyces cerevisiae* bacterial suspension at 600 nm and the concentration of viable cells was y = 16.379x - 0.954 (R² = 0.9678); the absorbance of *Saccharomyces cerevisiae*, *Issa mesosoma orientalis*, and *Wickham's aberrant* bacterial suspensions at 600 nm and the concentration of viable cells also showed a significant positive correlation, with R² values ​​of 0.9676, 0.9604, and 0.9758, respectively. Figure 2 The above standard curves allow for rapid estimation of the viable cell concentration of each yeast strain using the absorbance at a wavelength of 600 nm in the bacterial solution.

[0042] II. Determination of the Standard Curve for Yeast Cell Count and CT Value Using the specific primers for key genes involved in the synthesis of higher alcohols designed in this invention, a linear relationship between different yeast viable cell concentrations and quantitative Ct values ​​was established through real-time quantitative PCR (qPCR) technology.

[0043] The specific steps are as follows: (1) Take 50 mL of each yeast culture in the logarithmic growth phase, centrifuge at 6500 r / min for 5 min, discard the supernatant to obtain bacterial sludge, and resuspend and wash twice with sterile physiological saline.

[0044] (2) Measure the absorbance of the bacterial suspension at 600 nm wavelength. Calculate the bacterial concentration based on the standard curve of bacterial suspension absorbance at 600 nm wavelength and live cell concentration established above. Take the corresponding volume of bacterial suspension and collect yeast cells from 10^1 to 10^8. Set up 3 parallel samples for each cell order of magnitude.

[0045] (3) Use a fungal genomic DNA extraction kit to extract total genomic DNA from each gradient of bacteria.

[0046] (4) qPCR amplification was performed using the extracted genomic DNA as a template. The reaction mixture was prepared on ice, and the reaction system is shown in Table 4.

[0047]

[0048] (5) Refer to Table 5 for the procedure of real-time PCR reaction.

[0049] Table 5 qPCR reaction procedure

[0050] (6) After the reaction is complete, export the Ct value data and use Office Excel 2019 software to plot the standard curve of the logarithm of the live cell concentration (x, lg cfu) and the Ct value (y).

[0051] The experimental results are as follows: the standard curves constructed with primers for different genes of each yeast showed good linear correlation (Table 6). In the linear relationship, x is the logarithm of viable cell concentration (lg cfu), and y is the Ct value of qPCR amplification. Among them, the standard curves constructed with primers for the ILV3 gene of *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, and *Wickham's aberrant* showed the highest correlation, with R² values ​​of 0.9311, 0.9114, and 0.9415, respectively. Therefore, primers for the ILV3 gene were selected as the quantitative detection primers for the above three yeasts. The standard curve constructed with primers for the ILV2 gene of *Issa mesasura* showed the highest correlation (R² = 0.9885), therefore, primers for the ILV2 gene were selected as the quantitative detection primers for it.

[0052] Table 6. Standard curves of primers and probes for different yeast strains

[0053] Example 3: Validation of the accuracy of mixed systems in the qPCR absolute quantification method The purpose of this embodiment is to verify the accuracy of the absolute quantitative method established in this invention in a mixed yeast system. Six yeast strains were used: *Schizosaccharomyces cerevisiae* BJV10034, *Saccharomyces cerevisiae* 678, *Issa mesasura* JM-3, *Wickhamia lanceolata* JM-5, *Saccharomyces kazakhstanensis* JM-7, and *Pichia manshuriensis* JM-9. These were blended according to the reported absolute abundance and proportion of yeast strains in different aroma types (light aroma, strong aroma, soy sauce aroma, and northern soy sauce aroma) of the fermented mash (Table 7). Mixed yeast samples were constructed in both liquid culture systems (YPD medium) and solid culture systems (sterilized fermented mash). Genomic DNA was extracted from each mixed system, and qPCR was performed using the optimal specific primer pairs screened in Example 2 (Schizosaccharomyces cerevisiae: SP-ILV3; Saccharomyces cerevisiae: SC-ILV3; Isaac's orientalis: IO-ILV2; Wickham's aberrant: WA-ILV3). The number of cells measured was compared with the actual number of cells inoculated to evaluate the quantitative accuracy of the method in the two systems.

[0054] Table 7. Absolute abundance of core yeasts in the mash of different aroma types of Baijiu

[0055] (1) Validation in liquid culture system To verify the reliability of the established method in a liquid culture system, four core yeasts were inoculated into YPD liquid medium and cultured to the logarithmic growth phase. Different mixed yeast systems were then prepared according to the abundance ratios shown in Table 7. The mixed bacterial sludge was obtained by centrifugation at 6500 r / min for 5 min. Genomic DNA was extracted using the same method as in Example 2, and PCR amplification was performed. The measured Ct values ​​were substituted into the corresponding standard curve to calculate the cell count, which was compared with the actual inoculated cell count. The results are shown below. Figure 3 As shown in Table 8, Pearson correlation analysis was used to assess the accuracy of the quantitative analysis.

[0056] Table 8. Correlation analysis between actual inoculation values ​​and measured values ​​of the liquid mixed culture system.

[0057] The results showed that the measured values ​​of the four core yeasts were all highly significantly positively correlated with the actual inoculation values ​​(P < 0.01). Among them, the quantitative accuracy of *Schizosaccharomyces cerevisiae* was the highest (r = 0.998), and the correlation coefficients of the other yeasts were all > 0.85, indicating that the method of the present invention has high quantitative accuracy in liquid mixed culture systems.

[0058] (2) Validation in solid-state culture system To verify the reliability of the established method in the complex matrix of solid-state fermented mash, fermented light-aroma mash was subjected to autoclaving at 121℃ three times (20 min each time) to completely degrade residual microbial DNA in the mash. Following the mixed culture protocol in Table 7, separately cultured yeast was inoculated into the sterilized mash and thoroughly mixed.

[0059] The following method was used to separate the microbial cell precipitate from the fermented mash: Weigh 5.0 g of inoculated mash sample into a 50 mL sterile centrifuge tube, add 3 sterile glass beads with a diameter of 0.2 cm and 20 mL of sterile PBS buffer, vortex for 10 min; centrifuge at 300×g for 5 min to remove large particles of mash impurities, and collect the supernatant; repeat the washing twice, and combine all supernatants; centrifuge at 9000×g for 5 min, discard the supernatant, and collect the microbial cell pellet.

[0060] Yeast genomic DNA was extracted using the same method as in Example 2, and qPCR was performed. The measured Ct values ​​were substituted into the corresponding standard curve to calculate the number of cells to be measured, and compared with the actual number of cells inoculated. The results are as follows: Figure 4 As shown in Table 9.

[0061] Table 9. Correlation analysis between actual inoculation values ​​and measured values ​​in the solid-state fermentation mash system.

[0062] The results showed that the measured values ​​of the four core yeasts in the solid-state fermentation mash system were all significantly positively correlated with the actual inoculation values ​​(P < 0.01). Among them, the quantitative accuracy of *Schizosaccharomyces cerevisiae* and *Issa mesasura* was relatively high (r > 0.93), indicating that the method of the present invention also has good quantitative accuracy in complex solid-state fermentation systems.

[0063] Example 4: Analysis of the regulatory mechanism of different fermentation conditions on the synthesis pathway of higher alcohols in core yeast. This example analyzes the regulatory rules of nitrogen source abundance in liquid fermentation system and interaction of short-lived Lactobacillus in solid fermentation system on the synthesis pathway of four higher alcohols in core yeast, and verifies the application value of the method of the present invention in the study of flavor compound formation mechanism.

[0064] (1) Regulation of the synthesis pathway of higher alcohols in core yeast by nitrogen source in liquid fermentation This section investigates the effects of exogenous inorganic nitrogen sources on the higher alcohol synthesis flux and key gene transcription levels of different core yeasts. *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Issa mesospora* orientalis, and *Wickhamia sapiens* were used as fermentation strains, with sorghum hydrolysate as the basal fermentation medium.

[0065] The specific experimental method is as follows: Preparation of sorghum hydrolysate culture medium: Sorghum powder and water were mixed at a mass ratio of 1:3 and gelatinized in a 60℃ water bath for 30 min; then transferred to a 90℃ water bath, and liquefied with 1 μL / g sorghum powder liquefying enzyme was added for 30 min; the mixture was then heated to boiling in an induction cooker for 20 min (timing started from boiling); cooled to 60℃, and saccharifying enzyme of 2 μL / g sorghum powder was added, and saccharification was carried out at 60℃ for 4 h; then cooled to 40℃, and acidic protease of 1 μL / g sorghum powder was added, and the mixture was carried out at 40℃ for 2 h; after filtration through eight layers of gauze, the mixture was centrifuged at 8000 r / min and 4℃ for 10 min, and the precipitate was discarded; the sugar concentration of the supernatant was adjusted to 16°Brix, mixed well, and dispensed into 500 mL Erlenmeyer flasks, 150 mL per flask, and autoclaved at 115℃ for 20 min for later use.

[0066] Experimental setup and fermentation conditions: The control group used the above-mentioned sorghum hydrolysate culture medium, supplemented with 1.5 g / L acetic acid and 5 g / L lactic acid to simulate the acidic environment of baijiu fermentation; the experimental group, in addition to the control group, was supplemented with 1.48 g / L ammonium dihydrogen phosphate (approximately 400 mg / L of nitrogen). The fermentation was carried out at a rate of 1×10⁻⁶. 6 Four types of core yeasts were inoculated with a final concentration of cfu / mL and fermented at 30℃. The carbon dioxide loss was measured by weighing every 12 h, and fermentation was considered complete when the difference between two consecutive weighings was ≤0.2 g.

[0067] Determination of higher alcohol content: After fermentation, 50 mL of fermentation broth was distilled under normal pressure, and 100 mL of distillate was collected. The contents of isobutanol, isoamyl alcohol, and active pentanol were determined by gas chromatography. Chromatographic conditions: The chromatographic column was an Agilent CP-WAX capillary column (50 m × 250 μm × 0.25 μm); the carrier gas was high-purity nitrogen (purity > 99.999%), and the column flow rate was 1 mL / min; the injection port temperature was 250℃, and the flame ionization detector (FID) temperature was 260℃; the temperature program was as follows: initial temperature 35℃, held for 1 min, increased to 70℃ at 3℃ / min, held for 15 min, and then increased to 190℃ at 3.5℃ / min, held for 22 min; the injection volume was 1 μL, and the injection was performed by split injection at a split ratio of 30:1. Before the assay, add 10 μL of mixed internal standard (chromatographically pure tert-amyl alcohol, n-amyl acetate, and 2-ethylhexanol, with final concentrations of 16732.1 mg / L, 16732.1 mg / L, and 16043.9 mg / L, respectively) to 1 mL of sample.

[0068] Detection of transcriptional levels of key genes for higher alcohol synthesis: The cell pellet at the end of fermentation was collected, and total RNA was extracted using a fungal total RNA extraction kit. cDNA was synthesized by reverse transcription. The specific primer pairs screened in Example 1 were used for qPCR detection to analyze the relative transcriptional levels of BAT2, ILV2 and ILV3 genes. Three replicates were set up for each sample.

[0069] Experimental results are as follows Figure 5 As shown, the addition of 1.48 g / L ammonium dihydrogen phosphate exhibited significant strain-specific regulation of higher alcohol production. Compared with the control group, the total higher alcohol production of *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, and *Wickham's aberrant* decreased by 61.9%, 22.1%, and 13.1%, respectively, while the total higher alcohol production of *Issa mesasura* increased by 14.2%. Gene expression analysis showed that the BAT2, ILV2, and ILV3 genes were significantly downregulated in the first three strains, while the above three genes were significantly upregulated in *Issa mesasura*. These results confirm that nitrogen sources simultaneously regulate the activity of the Ehrlich and Harris pathways at the transcriptional level, and the direction and intensity of regulation show significant strain-specificity.

[0070] (2) Regulation of the higher alcohol synthesis pathway in core yeast by interaction between short-lived Lactobacillus and solid-state fermentation This section investigates the effects of *Lactobacillus short-lived* (Lactobacillus) under simulated solid-state fermentation conditions of light-aroma baijiu in a laboratory setting. Lactobacillus brevis The effect of interactions on the synthesis of higher alcohols by different core yeasts was investigated. Four core yeasts were used as fermentation strains, Lactobacillus brevis was used as the interacting strain, and fresh lees from sorghum and light-aroma baijiu distilleries were used as solid-state fermentation raw materials.

[0071] The specific experimental method is as follows: Solid-state fermentation process: The process flow is as follows: sorghum → soaking grains → initial steaming → steaming → re-steaming → cooling and adding yeast → saccharification → mash preparation → fermentation → distillation → index determination. Specific operations: After washing the sorghum, soak it in a 72℃ constant temperature water bath for 20 hours (the liquid level should be at least 10 cm above the grain surface); drain the water, and steam it at 105℃ for 20 minutes; add 65℃ hot water to steam the grains for 5 minutes, drain again, and steam at 105℃ for 20 minutes; cool the cooked grains to 20-40℃, and evenly mix in 1% of the original dry weight of yeast (prepared with quantitative inoculation of the above four core yeasts); transfer to a culture container, cover with sterilized damp gauze, and saccharify at 30℃ for 24 hours; sterilize the fresh mash at 105℃ high pressure steam for 20 minutes, mix the saccharified mash and sterilized mash at a mass ratio of 1:2, fill into 500 mL Erlenmeyer flasks, and let ferment at 25℃ for 7 days.

[0072] Experimental setup: The control group underwent fermentation according to the above process; the experimental group received an additional Lactobacillus brevis suspension before saccharification and culture to achieve a final concentration of [missing information]. (Calculated based on the dry weight of the original grain).

[0073] Detection of transcriptional levels of key genes for higher alcohol synthesis: Samples of fermented mash on day 2 were collected, total RNA of microorganisms was extracted and reverse transcribed to synthesize cDNA, and the relative transcriptional levels of BAT2, ILV2 and ILV3 genes were detected by qPCR. Three parallel samples were set up for each sample.

[0074] Determination of higher alcohol content: After fermentation, 10 g of mash was weighed and placed in a distillation apparatus, 300 mL of distilled water was added and distilled under normal pressure, and 100 mL of distillate was collected; the higher alcohol content was determined using the same gas chromatography conditions as the liquid fermentation described above.

[0075] Experimental results are as follows Figure 6 As shown, the interaction between *Lactobacillus short-lived* and *Lactobacillus* produced differential regulatory effects on the synthesis of higher alcohols in different core yeasts. Compared with the control group, the total production of higher alcohols in *Schizophyllum commune*, *Saccharomyces cerevisiae*, and *Wickham's aberrant* decreased by 29.9%, 14.6%, and 18.8%, respectively, while the total production of higher alcohols in *Issa mesosoma orientalis* increased slightly (by 2%). Gene expression analysis showed that both higher alcohol synthesis pathways in *Schizophyllum commune* and *Saccharomyces cerevisiae* were significantly inhibited; the BAT2 gene in *Wickham's aberrant* was significantly downregulated, but the ILV2 and ILV3 genes were significantly upregulated, indicating that its higher alcohol synthesis mainly depends on the Ehrlich pathway, and the upregulation of the sugar metabolism pathway could not offset the inhibitory effect of the amino acid breakdown pathway; all three genes in *Issa mesosoma orientalis* were significantly upregulated, and its production increase was not significant, which may be related to the metabolic lag effect caused by competition for carbon and nitrogen sources by lactic acid bacteria or acid production.

[0076] The present invention has been illustrated through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An absolute quantitative method for the core yeast strain in Baijiu brewing, characterized in that, The method includes the following steps: (1) Obtain pure culture of core yeast and establish a standard correspondence between the absorbance of the culture at 600 nm wavelength and the concentration of live cells; take culture with known live cell concentration and perform 10-fold serial dilution, extract genomic DNA from each gradient culture as a standard template, and use specific primer pairs for the higher alcohol synthesis gene of core yeast for qPCR amplification to obtain the fluorescence quantitative Ct value, and establish a linear standard curve between the logarithm of different yeast live cell concentrations and the Ct value. (2) Extract genomic DNA from the microorganisms in the sample to be tested; (3) Using the specific primer pair described in step (1), qPCR amplification is performed with the genomic DNA extracted in step (2) as a template to obtain the fluorescence quantitative Ct value of the sample to be tested. Substitute it into the corresponding standard curve established in step (1) to calculate the absolute content of the target core yeast in the sample to be tested.

2. The absolute quantification method as described in claim 1, characterized in that, The core yeast strain is selected from one or more of the following: Schizosaccharomyces cerevisiae, Saccharomyces cerevisiae, Isaac's orientalis, and Wickham's abnormal yeast.

3. The absolute quantitative method as described in claim 1 or 2, characterized in that, The liquor is brewed using a small-batch, light-aroma baijiu.

4. The absolute quantification method as described in claim 1 or 2, characterized in that, The key genes for higher alcohol synthesis are selected from one or more of the BAT2, ILV2, and ILV3 genes.

5. The absolute quantification method as described in claim 1 or 2, characterized in that, The nucleotide sequences of the specific primer pairs are selected from any pair shown in SEQ ID NO:1 to SEQ ID NO:

24.

6. The absolute quantification method as described in claim 1 or 2, characterized in that, When used for the detection of *Schizosaccharomyces cerevisiae*, *Saccharomyces cerevisiae*, and *Wickhamia lanceolata*, the specific primer pair is a primer pair targeting the ILV3 gene; when used for the detection of *Issa mesasura*, the specific primer pair is a primer pair targeting the ILV2 gene.

7. The absolute quantification method as described in claim 1 or 2, characterized in that, The annealing temperature for the qPCR amplification is 60±5℃.

8. The absolute quantification method as described in claim 1 or 2, characterized in that, The samples to be tested include samples from the koji-making process or the brewing process.

9. A method for analyzing the absolute content of core yeast and its higher alcohol synthesis metabolism function, characterized in that, The method includes the following steps: (1) The absolute content of each core yeast in the sample to be tested is obtained by using the absolute quantification method described in any one of claims 1-8; (2) Extract total RNA from microorganisms in the sample to be tested and reverse transcribe it to synthesize cDNA; (3) Using the specific primer pair described in claim 1, qPCR amplification was performed with the cDNA synthesized in step (2) as a template to obtain transcriptional level data of key genes for higher alcohol synthesis in each core yeast. (4) The absolute content data obtained in step (1) and the gene transcription level data obtained in step (3) were correlated to analyze the correlation between the absolute quantification of the core yeast and the function of higher alcohol synthesis metabolism.

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