Evaluation method of Maotai-flavor liquor spreading, airing and yeast mixing process based on microbial diversity and application of evaluation method

Abstract

The invention belongs to the technical field of microorganisms, and relates to a microbial diversity-based evaluation method of a Maotai-flavor liquor spreading, airing and yeast mixing process and application of the microbial diversity-based evaluation method. According to the method disclosed by the invention, a relationship is established between stacking fermentation and a yeast spreading and mixing process at the front end of stacking fermentation, and fermented grain microorganisms in a stacking fermentation link are associated with fermented grain microorganisms after spreading and mixing yeast, so that the method for evaluating different yeast spreading and mixing processes based on microbial diversity is formed; and optimization of a yeast spreading, airing and mixing process in the actual production of the Maotai-flavor liquor is facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a method for evaluating a fermentation process of Maotai-flavor liquor based on microbial diversity and application thereof. BACKGROUND

[0002] The brewing process of Maotai-flavor liquor follows the mode of "one-year cycle, twice of grain input, nine times of cooking, eight times of fermentation, and seven times of liquor collection". After each distillation and liquor collection, the out-drum fermented grains (grain mash) are taken out, spread on the airing hall, mixed with high-temperature Daqu after being aired to a suitable temperature, and then subjected to stacking fermentation and pit fermentation before entering the next round of distillation and liquor collection. The spreading and mixing process is a key front-end process of stacking fermentation, and its process parameters such as operation mode, temperature, and thickness will affect the microorganisms in the stacking fermented grains, especially after the spreading and mixing, the thickness and temperature of the fermented grains will affect the distribution of microorganisms in the Daqu. However, the existing related technologies mainly focus on the invention and creation of mechanical equipment in the spreading and mixing process, aiming to solve the problems of high labor cost, high labor intensity, and long time consumption in the traditional brewing industry, ignoring the influence of the spreading and mixing process on the microorganisms in the brewing process of Maotai-flavor liquor. In addition, in the actual production process, the evaluation of the spreading and mixing process mainly depends on the sense of smell and temperature, and the reference basis is single. Moreover, there is no clear process parameter for the thickness and temperature of the mixed fermented grains, and the differences in manual operation make it difficult to evaluate the operation quality of the spreading process among different teams, which further affects the stacking fermentation process. SUMMARY

[0003] In some embodiments, the present application provides a method for evaluating the spreading and mixing process of fermented grains, which comprises the following steps: (1) obtaining microbial sequence information of fermented grains at different time nodes in the liquor production process; (2) performing OTU clustering on the microbial sequence information to obtain microbial OTU data, wherein the OTU data comprises the genus classification of microorganisms and the total relative abundance of microorganisms of the genus; (3) based on the genus classification of microorganisms, obtaining common microorganisms of the fermented grains at the different time nodes and the total relative abundance of the common microorganisms at the different time nodes; (4) obtaining the evaluation result of the spreading and mixing process of fermented grains according to the total relative abundance of the common microorganisms at the different time nodes.

[0004] In some embodiments, the fermented grains at the different time nodes are selected from at least two kinds of fermented grains at the time points of after spreading and mixing, after stacking, entering the pit, and the middle period of stacking in the liquor production process.

[0005] In some embodiments, the higher the total relative abundance, the more conducive the spreading and mixing process of fermented grains to ethanol production.

[0006] In some embodiments, the step (4) is performed according to the sum of the relative abundances of the common microorganisms at the different time nodes.

[0007] In some embodiments, the microbial sequence information is obtained by extracting DNA of the fermented grains and sequencing.

[0008] In some embodiments, the microorganisms comprise bacteria and / or fungi.

[0009] In some embodiments, the sequencing region of the bacteria is selected from 16S rRNA gene In some embodiments, the sequencing region of the fungi is selected from ITS sequence.

[0010] In some embodiments, the sequencing region of the bacteria is selected from 16S rRNA gene V3-V4 region.

[0011] In some embodiments, the sequencing region of the fungi is selected from ITS1-ITS2 region.

[0012] In some embodiments, the microorganisms comprise Thermoascus 、 Aspergillus 、 Byssochlamys 、 Thermomyces 、 Microascus 、 Rhizopus 、 Hyphopichia 、 Pichia 、 Geosmithia 、 Virgibacillus 、 Kroppenstedtia 、 Oceanobacillus 、 Bacillus 、 Scopulibacillus 、 Lactobacillus 、 Lederbergia 、 Issatchenkia 、 Saccharopolyspora 、 Anaerosalibacter and Lactiplantibacillus .

[0013] In some embodiments, the microorganisms comprise Thermoascus 、 Aspergillus 、 Byssochlamys 、 Thermomyces 、 Pichia 、 Virgibacillus 、 Kroppenstedtia 、 Oceanobacillus 、 Bacillus 、 Scopulibacillus 、 Lactobacillus 、 Issatchenkia and Saccharopolyspora .

[0014] In some embodiments, the microorganisms comprise Thermoascus, Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus ) Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora .

[0015] In some embodiments, the total relative abundance of microorganisms of each genus in step (2) is greater than 0.5%.

[0016] In some embodiments, the total relative abundance of microorganisms of each genus in step (2) is greater than 0.75%.

[0017] In some embodiments, the total relative abundance of microorganisms of each genus in step (2) is greater than 0.85%.

[0018] In some embodiments, the total relative abundance of microorganisms of each genus in step (2) is greater than 1%.

[0019] In some embodiments, the spread and mixed fermented grains after spreading and mixing are fermented grains when spreading and mixing is completed and the fermented grains are prepared for collection.

[0020] In some embodiments, the fully stacked fermented grains are fermented grains when the spread and mixed fermented grains are collected into a saccharification stack.

[0021] In some embodiments, the pit-entered fermented grains are fermented grains when the saccharification stack is heated to the required temperature for pit entry during the stacking fermentation process.

[0022] In some embodiments, the mid-stacking fermented grains are fermented grains at an intermediate time node between the fully stacked fermented grains and the pit-entered fermented grains.

[0023] In some embodiments, the liquor is baijiu.

[0024] In some embodiments, the liquor is Jiangxiang Baijiu.

[0025] In some embodiments, the temperature of the spread and mixed fermented grains after spreading and mixing is 15-30°C.

[0026] In some embodiments, the temperature of the spread and mixed fermented grains after spreading and mixing is 17-25°C.

[0027] In some embodiments, the method for evaluating the temperature of the spreaded and mixed fermented grains comprises the following steps: based on the common microorganisms, combining the common microorganisms that are significantly correlated with the temperature of the complete heap to obtain a combination of common microorganisms; and obtaining an evaluation result of the temperature of the spreaded and mixed fermented grains according to the relative abundance of the combination of common microorganisms.

[0028] In some embodiments, the method for evaluating the temperature of the spreaded and mixed fermented grains further comprises: obtaining an evaluation result of the temperature of the spreaded and mixed fermented grains according to the increase or decrease of the relative abundance of the combination of common microorganisms.

[0029] In some embodiments, the temperature of the complete heap is the temperature of the fermented grains in the complete heap.

[0030] In some embodiments, the fermented grains in the complete heap are the fermented grains when the spreaded and mixed fermented grains are gathered into a saccharification heap.

[0031] In some embodiments, the significant correlation refers to performing a Pearson correlation analysis on the relative abundance of the common microorganisms and the temperature of the complete heap, and the correlation coefficient is greater than 0.8 and the p value is less than 0.05.

[0032] In some embodiments, the significant correlation refers to performing a Pearson correlation analysis on the relative abundance of the common microorganisms and the temperature of the complete heap, and the correlation coefficient is greater than 0.9 and the p value is less than 0.05.

[0033] In some embodiments, the significant correlation refers to performing a Pearson correlation analysis on the relative abundance of the common microorganisms and the temperature of the complete heap, and the correlation coefficient is greater than 0.9 and the p value is less than 0.01.

[0034] In some embodiments, the combination of common microorganisms is selected from bacteria and / or fungi.

[0035] In some embodiments, the combination of common microorganisms comprises Thermoascus 、 Aspergillus 、 Bacillus Byssochlamys 、 Thermomyces 、 Microascus 、 Rhizopus 、 Hyphopichia 、 Pichia 、 Geosmithia 、 Virgibacillus 、 Kroppenstedtia 、 Oceanobacillus 、 Bacillus 、 Scopulibacillus 、 Lactobacillus 、 Lederbergia 、 Issatchenkia 、 Saccharopolyspora 、 Anaerosalibacter and Lactiplantibacillus .

[0036] In some embodiments, the common microbial combination comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora .

[0037] In some embodiments, the common microbial combination comprises Thermoascus , Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus、 Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora .

[0038] In some embodiments, the common microbial combination comprises Kroppenstedtia , Pichia , Thermoascus and Thermomyces .

[0039] In some embodiments, the common microbial combination comprises Kroppenstedtia , Pichia , Thermoascus and Thermomyces .

[0040] In some embodiments, the total relative abundance of microorganisms of each genus in the common microorganisms is greater than 0.5%.

[0041] In some embodiments, the total relative abundance of microorganisms of each genus in the common microorganisms is greater than 0.75%.

[0042] In some embodiments, the total relative abundance of microorganisms of each genus in the common microorganisms is greater than 0.85%.

[0043] In some embodiments, the total relative abundance of microorganisms of each genus in the common microorganisms is greater than 1%. In some embodiments, the thickness of the spread and aired kojied fermented grains is 2.0-5.0 cm.

[0044] In some embodiments, the thickness of the spread and aired kojied fermented grains is 3.0-4.5 cm.

[0045] In some embodiments, the thickness evaluation method of the spreaded and mixed fermented grains comprises the following steps: calculating an alpha diversity index based on the microbial OTU data, and obtaining an evaluation result of the thickness of the spreaded and mixed fermented grains according to the alpha diversity index.

[0046] In some embodiments, the alpha diversity index is selected from a shannon diversity index, a chao index, or an evenness.

[0047] In some embodiments, the alpha diversity index is selected from a chao index.

[0048] In some embodiments, the microorganism is selected from bacteria and / or fungi.

[0049] In some embodiments, the formula of the chao index is: ; wherein, S chao1 is the estimated number of OTUs; S obs is the actual number of detected OTUs; n1 is the number of OTUs containing only one sequence; and n2 is the number of OTUs containing only two sequences.

[0050] In some embodiments, the present application provides an evaluation system for a spreaded and mixed fermented grains process, which comprises the following modules: a data acquisition module for acquiring microbial sequence information of fermented grains at different time nodes and temperature data of completed fermented grains; a data processing module for processing genus classification of microorganisms, total relative abundance of microorganisms of the genus, and an alpha diversity index based on the microbial sequence information; an optimization processing module for obtaining common microorganisms of the fermented grains at different time nodes based on the genus classification of microorganisms obtained by the data processing module, and for combining common microorganisms significantly related to the temperature of completed fermented grains based on the temperature data of completed fermented grains and the common microorganisms, to obtain a common microorganism combination; a result output module for evaluating the spreaded and mixed fermented grains process based on the common microorganisms or the common microorganism combination, in combination with the total relative abundance of microorganisms of the genus or the alpha diversity index, and outputting an evaluation result.

[0051] In some embodiments, the microbial sequence information is obtained by extracting DNA of fermented grains and sequencing.

[0052] In some embodiments, the microorganism comprises bacteria and / or fungi.

[0053] In some embodiments, the sequenced region of the bacteria is selected from the 16S rRNA gene In some embodiments, the sequenced region of the fungus is selected from the ITS sequence.

[0054] In some embodiments, the sequenced region of the bacteria is selected from the 16S rRNA gene V3-V4 region.

[0055] In some embodiments, the sequenced region of the fungus is selected from the ITS1-ITS2 region.

[0056] In some embodiments, the microorganism comprises Kroppenstedtia , Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Microascus , Rhizopus , Hyphopichia , Pichia , Geosmithia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Lederbergia , Issatchenkia , Saccharopolyspora and Anaerosalibacter .

[0057] In some embodiments, the microorganism comprises Lactiplantibacillus , Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus and Issatchenkia .

[0058] In some embodiments, the microorganism comprises Saccharopolyspora , Thermoascus , Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia ) Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus and Issatchenkia .

[0059] In some embodiments, in the data processing module, the total relative abundance of microorganisms of each genus is greater than 0.5%.

[0060] In some embodiments, in the data processing module, the total relative abundance of microorganisms of each genus is greater than 0.75%.

[0061] In some embodiments, in the data processing module, the total relative abundance of microorganisms of each genus is greater than 0.85%.

[0062] In some embodiments, in the data processing module, the total relative abundance of microorganisms of each genus is greater than 1%. In some embodiments, the different time nodes are selected from at least two of the following: post-spreading and mixing, complete stacking, pit entry, and mid-heap.

[0063] In some embodiments, the post-spreading and mixing lees are the lees at the end of spreading and mixing, and ready for stacking.

[0064] In some embodiments, the complete stacking lees are the lees when the post-spreading and mixing lees are stacked into a saccharification heap.

[0065] In some embodiments, the pit entry lees are the lees when the saccharification heap is heated to the required temperature for pit entry during heap fermentation.

[0066] In some embodiments, the mid-heap lees are the lees at an intermediate time node between complete stacking and pit entry.

[0067] In some embodiments, the complete stacking temperature is the temperature of the lees at complete stacking.

[0068] In some embodiments, the significant correlation means that the relative abundance of the common microorganism and the complete stacking temperature are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.8, and the p value is less than 0.05.

[0069] In some embodiments, the significant correlation means that the relative abundance of the common microorganism and the complete stacking temperature are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.9, and the p value is less than 0.05.

[0070] In some embodiments, the significant correlation means that the relative abundance of the common microorganism and the complete stacking temperature are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.9, and the p value is less than 0.01.

[0071] In some embodiments, the alpha diversity index is selected from the shannon diversity index, the chao index, or the evenness.

[0072] In some embodiments, the alpha diversity index is selected from the chao index.

[0073] In some embodiments, the liquor is Baijiu.

[0074] In some embodiments, the liquor is Jiangxiang Baijiu.

[0075] In some embodiments, the spreading and airing and koji mixing process is selected from a spreading and airing and koji mixing method, a spreading and airing and koji mixing temperature, or a spreading and airing and koji mixing thickness.

[0076] In some embodiments, the spreading and airing and koji mixing temperature is the temperature at the end of spreading and airing and koji mixing of the fermented grains, and before the preparation of the heap.

[0077] In some embodiments, the spreading and airing and koji mixing thickness is the thickness after the spreading and airing and koji mixing of the fermented grains.

[0078] In some embodiments, the present application provides the use of the evaluation method or the evaluation system in the field of liquor making.

[0079] In some embodiments, the present application links the piling fermentation to the spreading and airing process at the front end, and utilizes the correlation between the microorganisms in the piling fermentation link and the microorganisms in the fermented grains after spreading and airing, thereby forming an evaluation method for different spreading and airing and koji mixing methods based on microbial diversity, which datafies the empirical data and evaluates the spreading and airing and koji mixing process, and is conducive to the optimization of the spreading and airing and koji mixing process in the actual production of Jiangxiang Baijiu.

[0080] In some embodiments, the evaluation results of the spreading and airing and koji mixing process obtained by the evaluation method of the present application are consistent with the results of the ethanol content of the pit cellar fermented grains under the corresponding spreading and airing and koji mixing method, and the accuracy is very high, which is conducive to the selection of more suitable spreading and airing and koji mixing methods by the person skilled in the art in the actual production link, and effectively improves the ethanol yield in the subsequent liquor production link.

[0081] In some embodiments, the application includes the application in evaluating or selecting the spreading and airing and koji mixing method.

[0082] In some embodiments, the application includes the application in evaluating or selecting the spreading and airing and koji mixing temperature.

[0083] In some embodiments, the application includes the application in evaluating or selecting the spreading and airing and koji mixing thickness.

[0084] In some embodiments, the liquor is Baijiu.

[0085] In some embodiments, the liquor is Jiangxiang Baijiu.

[0086] In some embodiments, the application includes the application in improving the liquor yield. BRIEF DESCRIPTION OF DRAWINGS

[0087] Saccharopolyspora Schematic diagram for sampling fermented grains.

[0088] Figure 1 The relationship between the total relative abundance of shared microbes (%) and the ethanol content of the pit-finished distiller's grains (g / L).

[0089] Figure 2 The relationship between the total relative abundance of shared core microbes (%) and the ethanol content of the pit-finished distiller's grains (g / L).

[0090] Figure 3 The relationship between the number of OTUs in the distiller's grains after spreading and airing and after the pile is completed. DETAILED DESCRIPTION

[0091] The technical solutions of the present application are further illustrated below by specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0092] In this article, the professional terms in the field are explained as follows: Spreading and airing and mixing with koji: the process of uniformly spreading the steamed distiller's grains in a spreading and airing site, turning and piling into rows, when the temperature of the distiller's grains uniformly decreases to about 30℃, sprinkling an appropriate amount of koji powder, uniformly mixing, and collecting into a pile.

[0093] Relative abundance: in a sample, the proportion of the number of DNA sequences of a certain microorganism (such as bacteria or fungi) to the total number of DNA sequences of all microorganisms in the sample. This proportion is usually expressed in percentage. In this article, the relative abundance of bacteria is the proportion of the number of DNA sequences of a certain bacterial genus to the total number of DNA sequences of all bacterial genera in the sample. In this article, the relative abundance of fungi is the proportion of the number of DNA sequences of a certain fungal genus to the total number of DNA sequences of all fungal genera in the sample.

[0094] R 2 : coefficient of determination, a statistical indicator to measure the goodness of fit of a linear regression model, between 0 and 1, the closer the value is to 1, the better the model fits the data, i.e. the stronger the explanatory power of the model.

[0095] OTU: Operational Taxonomic Unit, classification operation unit, in phylogenetic or population genetic studies, a certain classification unit (strain, species, genus, group, etc.) is artificially set with the same mark for ease of analysis. In microbial diversity analysis, all sequences are divided into OTUs according to different similarity levels.

[0096] OTU clustering: a technique used in microbial diversity research, by classifying similar sequences into the same OTU to simplify data analysis and species annotation.

[0097] Alpha diversity index: an important indicator for measuring species diversity within a specific area or ecosystem, mainly including the following common indices: Shannon index, Chao index, Simpson index, and evenness, etc.

[0098] Shannon diversity index: also known as Shannon index or Shannon-Wiener index, a commonly used indicator for measuring biodiversity. The greater the value of Shannon diversity index, the higher the diversity and evenness of species in the community.

[0099] Chao index: also known as Chao1 index, which can estimate the total number of species that may exist in the community based on the existing species data, i.e., species richness. The greater the Chao index, the higher the richness of the community.

[0100] Evenness: also known as Shannon evenness or Shannon’s evenness or evenness or shannon even, an index used to reflect the uniformity of the distribution of species individuals in the community, with a value between 0 and 1. The closer to 1, the higher the evenness of species in the community, i.e., the relative proportion of each species composition is basically equal.

[0101] In this article, "fermented grains" refers to an important intermediate product in the process of liquor making, mainly composed of high-temperature Daqu, sorghum, and microbial cells and metabolic products produced during fermentation.

[0102] In this article, the common core microorganism refers to the microorganism that is common in the fermented grains after spreading, airing, and mixing, and has a relative abundance greater than 1% during the stacking process (complete stacking, mid-stacking, and pit stacking).

[0103] In this article, the spreading, airing, and mixing temperature refers to the temperature of the fermented grains after spreading, airing, and mixing, and before stacking.

[0104] In this article, the complete stacking temperature refers to the average temperature of the three sites: the upper center, middle center, and lower center of the saccharification stack when the fermented grains after spreading, airing, and mixing are stacked.

[0105] In this article, the spreading, airing, and mixing thickness refers to the thickness of the fermented grains after spreading, airing, and mixing.

[0106] In this paper, the time node of spreading and mixing is the time node of spreading and mixing after spreading and airing and evenly mixing after scattering koji powder, preparing for stacking; the time node of stacking is the time node of stacking after spreading and mixing, stacking the fermented grains into saccharifying stacks; the time node of pit entry is the time node of pit entry required by the saccharifying stacks in the stacking fermentation process to rise to the temperature; the time node of mid-stage stacking is the time node of mid-stage stacking from stacking to pit entry. If the saccharifying stacks from stacking to pit entry experience 5 days in total, the stacking is the 0th day, and the pit entry is the 5th day, then the 3rd day is the mid-stage stacking. In this paper, the term "fermented grains at different time nodes" refers to the fermented grains at the time nodes of spreading and mixing, stacking, pit entry or mid-stage stacking.

[0107] In this paper, the interval range of temperature or thickness includes the left end point value and does not include the right end point value. For example, the range of 17-20°C includes 17°C and does not include 20°C; the range of 3.0-3.5 cm includes 3.0 cm and does not include 3.5 cm.

[0108] Example 1: Evaluation of spreading and mixing method 1. Sample acquisition and information extraction 1.1 Fermented grains sample acquisition Fermented grains in the production process of Maotai-flavor liquor were selected, which were taken from the time nodes of spreading and mixing, stacking, mid-stage stacking and pit entry. The fermented grains at the time nodes of stacking, mid-stage stacking and pit entry were mixed samples taken from the surface layer and the upper, middle and lower layers of the saccharifying stacks (sampling points 1-6); the fermented grains after spreading and mixing were mixed samples taken from three different positions after spreading and mixing (sampling points 7-9), and the sampling points are shown in Figure 4 .

[0109] In this paper, the time node of spreading and mixing is the time node of spreading and mixing after spreading and airing and evenly mixing after scattering koji powder, preparing for stacking; the time node of stacking is the time node of stacking after spreading and mixing, stacking the fermented grains into saccharifying stacks; the time node of pit entry is the time node of pit entry required by the saccharifying stacks in the stacking fermentation process to rise to the temperature; the time node of mid-stage stacking is the time node of mid-stage stacking from stacking to pit entry. If the saccharifying stacks from stacking to pit entry experience 5 days in total, the stacking is the 0th day, and the pit entry is the 5th day, then the 3rd day is the mid-stage stacking.

[0110] Fermented grains after pit entry for detecting ethanol content were obtained by sampling from the upper, middle and lower layers of the pit.

[0111] 1.2 Information extraction (1) Microbial information: The total DNA of the fermented grains sample was extracted using the E.Z.N.A.™ Mag-Bind Soil DNA Kit kit. The microbial data was obtained using the Illumina MiSeq sequencing platform. The V3-V4 region of the 16S rRNA gene was amplified and sequenced for bacteria using the universal primers 341 F (5'-CCTACGGGNGGCWGCAG-3', SEQ ID NO: 1) and 805 R (5'-GACTACHVGGGTATCTAATCC-3', SEQ ID NO: 2) (Reference: Qin Y, Fan B, Mei L. Comparison of bacterial community composition and diversity in Wanfeng Lake during wet and dry seasons [J]. Journal of Dalian Ocean University, 2023, 38(03): 414-422. DOI: 10.16535 / j.cnki.dlhyxb.2022-276.). The ITS1-ITS2 region was amplified and sequenced for fungi using the universal primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 3) and primer ITS2 R (5'-GCTGCGTTCTTCATCGATGC-3', SEQ ID NO: 4). OTU clustering analysis was performed based on sequence similarity (97%), and the OTU data of the fermented grains were obtained (Reference: Bjørnsgaard Aas A, Davey ML, Kauserud H. ITS all right mama: investigating the formation of chimeric sequences in the ITS2 region by DNA metabarcoding analyses of fungal mock communities of different complexities. Mol Ecol Resour. 2017 Jul;17(4):730-741. doi: 10.1111 / 1755-0998.12622. Epub 2016 Nov 24. PMID: 27775220.).

[0112] At the genus classification level, the relative abundance of bacteria and the relative abundance of fungi were calculated. The calculation method of the relative abundance of bacteria / fungi is as follows: ; (2) The ethanol content of the fermented grains was detected by a biosensor.

[0113] 2. Evaluation method based on common microorganisms 2.1 Screening of common microorganisms The "spreading and airing stirring method ①" specifically refers to the mechanization of the whole process of spreading and airing stirring. The fermented grains after being discharged from the distiller's still are placed on a mechanical conveying belt for spreading and airing, stirring, and stacking. The "spreading and airing stirring method ②" specifically refers to the fermented grains after being discharged from the distiller's still being placed on an airing hall, and the mechanical spreading and airing, stirring, and manual stacking. The "spreading and airing stirring method ③" specifically refers to the fermented grains after being discharged from the distiller's still being placed on an airing hall, and the whole process of spreading and airing, stirring, and stacking being manually performed. The spreading and airing stirring temperature of the three spreading and airing stirring methods is 18-20°C, and the spreading and airing stirring thickness is 3.5-4.5 cm.

[0114] Among them, the common microorganisms in the spread and stirred after fermentation grains and the stacking process under the spread and stirred method ① are 18 kinds (see Table 1), and the common microorganisms in the spread and stirred after fermentation grains and the stacking process under the spread and stirred method ② and the spread and stirred method ③ are 13 kinds (see Tables 2 and 3).

[0115] Table 1 Relative abundance (%) of common microorganisms in spread and stirred after fermentation grains and stacking process under spread and stirred method ①

[0116] Table 2 Relative abundance (%) of common microorganisms in spread and stirred after fermentation grains and stacking process under spread and stirred method ②

[0117] Table 3 Relative abundance (%) of common microorganisms in spread and stirred after fermentation grains and stacking process under spread and stirred method ③

[0118] 2.2 Evaluation results based on common microorganisms The common microorganisms in the spread and stirred after fermentation grains and the stacking process were screened out, and the sum of the relative abundance of the four time nodes of spread and stirred after fermentation, complete stacking, mid-stacking, and pit entry was compared (see Table 4).

[0119] The results show that under different spread and stirred methods, the sum of the relative abundance of the common microorganisms in the spread and stirred after fermentation grains and the stacking process is ranked from high to low as follows: spread and stirred method ③ > spread and stirred method ① > spread and stirred method ②, that is, under this evaluation method, the spread and stirred method ③ is the best, followed by the spread and stirred method ① and the spread and stirred method ②.

[0120] Table 4 Evaluation results based on the relative abundance (%) of common microorganisms

[0121] 2.3 Verification of evaluation results based on common microorganisms To further verify the rationality of this evaluation method, a regression analysis was performed on the sum of the relative abundance of common microorganisms under each spreading and mixing method and the corresponding ethanol content of the fermented mash. The results are as follows: Figure 1 As shown, R 2 The value was 0.3336, indicating a low degree of fit. Furthermore, the results of this evaluation method were inconsistent with the order of ethanol content in the fermented mash (see Table 5), suggesting that the method needs further optimization.

[0122] Table 5. Comparison of evaluation results based on common microorganisms and ethanol content (g / L) of fermented mash under different spreading and mixing methods.

[0123] 3. Evaluation methods based on shared core microorganisms 3.1 Screening of common core microorganisms To further optimize the evaluation method, the evaluation method was based on the sum of the relative abundance of the core microorganisms (relative abundance > 1%) shared between the fermented mash and the mash during the stacking process under different spreading and mixing methods. Microorganisms with a relative abundance greater than 1% at four time points—after spreading and mixing, at the end of stacking, during the middle of stacking, and at the bottom of the pit—were selected as the core microorganisms. The screening results for different spreading and mixing methods are shown in Tables 6-8.

[0124] Table 6. Core microorganisms shared between the spread-and-mixed koji and the fermented mash from the stacking process under the spreading-and-mixing method ①.

[0125]

[0126] Table 7. Core microorganisms shared between the spread-and-mixed mash and the fermented mash from the stacking process under the second treatment method ②.

[0127] Table 8. Core microorganisms shared between the spread-and-mixed mash and the piled mash under the third treatment method of spreading and mixing.

[0128] 3.2 Evaluation results based on shared core microorganisms The relative abundance of core microorganisms in the fermented mash after spreading and mixing and during the stacking process under different spreading and mixing methods was compared (see Table 9).

[0129] The results show that under different spreading and airing mixing methods, the total relative abundance of common core microorganisms in the pile after spreading and airing mixing and the stacking process is ranked from high to low as follows: spreading and airing mixing method ③ > spreading and airing mixing method ② > spreading and airing mixing method ①. Different from the evaluation results based on common microorganisms, the results of this evaluation method are as follows: spreading and airing mixing method ③ is the best, followed by spreading and airing mixing method ② and spreading and airing mixing method ①.

[0130] Table 9 Evaluation results based on common core microorganisms

[0131] 3.3 Verification of evaluation results based on common core microorganisms To further verify the rationality of the evaluation method, the total relative abundance of common core microorganisms under each spreading and airing mixing method is regressed with the corresponding ethanol content of the pit-leaved fermented grains, and the results are shown in Table 10. Figure 2 R 2 is 0.9687, the fitting degree is high, and the results of the evaluation method are consistent with the order of the ethanol content of the pit-leaved fermented grains (see Table 10).

[0132] Therefore, the optimal evaluation method of spreading and airing mixing method is the evaluation method based on common core microorganisms, and the preferred spreading and airing mixing method is spreading and airing mixing method ③.

[0133] Table 10 Comparison of evaluation results based on common core microorganisms and ethanol content (g / L) of pit-leaved fermented grains under different spreading and airing mixing methods

[0134] Example 2: Evaluation of spreading and airing mixing temperature Based on the preferred spreading and airing mixing method ③ of Example 1 and the screened common core microorganisms, the relative abundance of the common core microorganisms under the spreading and airing mixing temperature, the complete stacking temperature and the complete stacking time of the spreading and airing mixing method ③ of Example 1 is collected. The experimental steps for obtaining the relative abundance are the same as those of Example 1.

[0135] The temperature is detected by a temperature measuring rod. The spreading and airing mixing temperature is the average value of the temperature of the fermented grains at sampling points 7-9 (see Figure 3 ) after spreading and airing mixing and before stacking.

[0136] The complete stacking temperature is the average value of the temperature of the fermented grains at the upper center, middle center and lower center of the saccharification pile (sampling points 1, 3 and 5, see Figure 1 ) when the fermented grains after spreading and airing mixing are stacked.

[0137] The relationship between the complete stacking temperature and the relative abundance of the common core microorganisms at the complete stacking time and the spreading and airing mixing temperature is established by Pearson correlation analysis.

[0138] The results are shown in Table 11. The temperature of spreading and airing and mixing is significantly correlated with the temperature of complete stacking, and the temperature of complete stacking is significantly correlated with the relative abundance of Figure 1 , Kroppenstedtia , Pichia or Thermoascus .

[0139] Table 11 Correlation coefficient of the temperature of complete stacking with the common core microorganisms and the temperature of spreading and airing and mixing

[0140] ** The correlation is significant at the level of 0.01 (two-tailed); * The correlation is significant at the level of 0.05 (two-tailed).

[0141] In this example, different spreading and airing and mixing temperatures (17 ℃-20 ℃, 20 ℃-23 ℃, and 23 ℃-25 ℃) under the mode of spreading and airing and mixing ③ were collected, as well as the corresponding ethanol content of pit bottom Daqu, the ethanol content of complete stacking Daqu, the relative abundance of four microorganisms after spreading and airing and mixing and at the time of complete stacking. The ethanol content of Daqu was detected by a biosensor, and the accumulated ethanol content during the stacking process was the ethanol content of pit bottom Daqu minus the ethanol content of complete stacking Daqu. The sampling points of pit bottom Daqu and complete stacking Daqu were the same as in Example 1. The remaining experimental steps were the same as in Example 1. Thermomyces Kroppenstedtia Pichia and Thermoascus

[0142] In this example, the Thermomyces , Kroppenstedtia , Pichia and Thermoascus were arranged and combined, and the relative abundance changes of microorganisms from after spreading and airing and mixing to the time of complete stacking were used to evaluate the temperature of spreading and airing and mixing (relative abundance change = relative abundance of Daqu microorganisms at the time of complete stacking minus relative abundance of Daqu microorganisms after spreading and airing and mixing). The less the relative abundance change of microorganisms decreased or the more the relative abundance change of microorganisms increased, the better the temperature of spreading and airing and mixing. The specific results are shown in Table 12. Table 12 Evaluation results based on the relative abundance changes of core microorganisms

[0143]

[0144]

[0145] Based on the evaluation results of the above evaluation method, it can be seen that the sum of the relative abundance changes of the common core microorganisms ( Thermomyces and Kroppenstedtia ) in the evaluation method has the highest fitting degree with the accumulation of ethanol content of Daqu during the stacking process. ​​​

[0146] When the temperature of spreading and mixing is 20-23℃, Thermomyces The sum of the relative abundance of Kroppenstedtia is the smallest, and the accumulated ethanol content of the pile is the highest. Therefore, 20-23℃ is the most suitable temperature for spreading and mixing of method ③.

[0147] Example 3: Evaluation of the thickness of spreading and mixing 1. Evaluation method Based on the microbial information of spreading and mixing method ③ extracted in Example 1, this example uses linear regression analysis to fit the OTU number of the starter after spreading and mixing with the OTU number of the pile at the end of the fermentation process. The results show that the diversity of the starter microorganisms after spreading and mixing is significantly positively correlated with the diversity of the microorganisms at the end of the fermentation process (see Thermomyces ). Therefore, this example uses the diversity of the starter after spreading and mixing as the condition for evaluating the thickness of spreading and mixing.

[0148] This example collects different spreading and mixing thicknesses (3.0-3.5 cm, 3.5-4.0 cm, 4.0-4.5 cm) under spreading and mixing method ③, as well as the corresponding microbial information, ethanol content of the starter in the cellar, and ethanol content of the pile at the end of the fermentation process. The thickness of the starter after spreading and mixing is the average of the thickness of the starter at sampling points 7-9 (see Figure 4 Figure 1 ), the experimental steps for obtaining microbial information are the same as in Example 1, and the experimental steps for obtaining the ethanol content of the starter are the same as in Example 2. The ethanol content of the starter is detected by a biosensor, and the accumulated ethanol content of the starter during the pile-up process is the ethanol content of the starter in the cellar minus the ethanol content of the pile at the end of the fermentation process. The sampling points of the starter in the cellar and the pile at the end of the fermentation process are the same as in Example 1.

[0149] The microbial diversity of the starter after spreading and mixing is evaluated by selecting the Shannon diversity index, Chao index, and evenness. The Shannon diversity index is one of the indices used to estimate the microbial diversity of a sample. The larger the Shannon diversity index, the higher the community diversity. The calculation formula is as follows: ; Where S obs = the actual number of OTUs detected; n i = the number of sequences contained in the i-th OTU; N = the total number of individuals, which is the total number of sequences in this case.

[0150] The Chao index is an index that estimates the number of OTUs in the community using the chao1 algorithm, which is commonly used in ecology to estimate the total number of species. The calculation formula is as follows: ; Among them, S chao1 =Estimated number of OTUs; S obs = The actual number of OTUs detected; n1 = the number of OTUs containing only one sequence; n2 = the number of OTUs containing only two sequences.

[0151] Evenness is an index used to reflect how evenly the number of individuals of a species is distributed within a community. The calculation formula is as follows: ; in, =The Shannon diversity index obtained from actual calculation; =In(S), which is the maximum species diversity index, where S is the total number of OTUs actually detected.

[0152] 2. Optimal Evaluation Method for Diversity Index 2.1 Evaluation Method Based on Shannon Diversity Index In this embodiment, the collected thickness of the spread and mixed koji was divided into three intervals: 3.0~3.5 cm, 3.5~4.0 cm, and 4.0~4.5 cm. The Shannon diversity index of the mash at each spreading thickness is shown in Table 13. The higher the Shannon diversity index value, the better the diversity. In this embodiment, the final evaluation index was determined by the ethanol content accumulated during the accumulation of mash at different spreading and mixed koji thicknesses.

[0153] The results are shown in Table 13. Based on the Shannon diversity index, the preferred order of different spreading and mixing koji thicknesses is 3.0~3.5 cm < 3.5~4.0 cm < 4.0~4.5 cm, with the preferred thickness being 4.0~4.5 cm. However, since the accumulated ethanol content at the spreading and mixing koji thickness of 4.0~4.5 cm is 3.72 g / L, which is the lowest among the three thicknesses, this evaluation method is not suitable for evaluating the spreading and mixing koji thickness.

[0154] Table 13 Ethanol content accumulation during mash stacking under different spreading and mixing koji thicknesses

[0155] 2.2 Evaluation Method Based on Uniformity In this embodiment, the collected thickness of the spread-and-mixed mash was divided into three intervals: 3.0~3.5 cm, 3.5~4.0 cm, and 4.0~4.5 cm. The uniformity of the mash at each spreading thickness is shown in Table 14. The higher the uniformity value, the more uniform the species distribution. In this embodiment, the final evaluation index was determined by the ethanol content accumulated during the accumulation of the mash at different spreading and mixing thicknesses.

[0156] The results are shown in Table 14. Based on the uniformity, the preferred order of different spreading and airing fermentation thicknesses is 3.0-3.5 cm < 3.5-4.0 cm < 4.0-4.5 cm, and the preferred thickness is 4.0-4.5 cm; but the accumulated ethanol content under the 4.0-4.5 cm spreading and airing fermentation thickness is 3.72 g / L, which is the lowest among the three thicknesses, i.e., this evaluation method is not suitable for evaluating the spreading and airing fermentation thickness.

[0157] Table 14 Accumulation of ethanol content during the accumulation process of the fermented grains under different spreading and airing fermentation thicknesses

[0158] 2.3 Evaluation method based on Chao index In this example, the collected spreading and airing fermentation thicknesses are divided into three intervals, i.e., 3.0-3.5 cm, 3.5-4.0 cm, and 4.0-4.5 cm; the Chao indexes of the fermented grains under different spreading thicknesses are shown in Table 15, and the larger the Chao index value, the higher the abundance. In this example, the final evaluation index is determined by the accumulated ethanol content during the accumulation process of the fermented grains under different spreading and airing fermentation thicknesses.

[0159] The results are shown in Table 15. Based on the Chao index, the preferred order of different spreading and airing fermentation thicknesses is 3.0-3.5 cm > 3.5-4.0 cm > 4.0-4.5 cm, and the preferred thickness is 3.0-3.5 cm, under which the accumulated ethanol content is 6.35 g / L, which is the highest among the three thicknesses, and the evaluation results based on the Chao index are consistent with the order of the accumulated ethanol content. This method is suitable for evaluating the spreading and airing fermentation thickness, and the most suitable spreading and airing fermentation thickness under the spreading and airing fermentation method III is 3.0-3.5 cm.

[0160] Table 15 Accumulation of ethanol content during the accumulation process of the fermented grains under different spreading and airing fermentation thicknesses

[0161]

[0162] The above examples are only used to further illustrate and describe the technical solutions of the present application, so that those skilled in the art can more accurately understand the inventive concept and operation scheme of the present application, and are not further limited to the present application. Improvements made by those skilled in the art on the basis of the above examples, which do not have outstanding substantial features and are not significant progress, are all within the protection scope of the present application.

Claims

1. An evaluation method of a daoyao spreading and mixing process, characterized in that, The evaluation method comprises the following steps: (1) obtaining microbial sequence information of the dregs at different time nodes in the wine production process; (2) OTU clustering of the microbial sequence information to obtain microbial OTU data, wherein the OTU data comprises genus classification of the microorganisms and total relative abundance of the microorganisms of the genus; (3) based on the genus classification of the microorganisms, obtaining common microorganisms of the dregs at the different time nodes and a sum of relative abundances of the common microorganisms at the different time nodes; (4) obtaining an evaluation result of the dregs spreading, airing and koji mixing process according to the sum of relative abundances of the common microorganisms at the different time nodes; The dregs at the different time nodes are selected from at least two kinds of dregs after spreading, airing and koji mixing, after complete stacking, after entering the cellar and during the middle period of stacking in the wine production process.

2. The evaluation method according to claim 1, characterized by, The higher the sum of relative abundances, the more conducive the dregs spreading, airing and koji mixing process is to ethanol production; Preferably, in step (4), the evaluation result of the dregs spreading, airing and koji mixing process is obtained according to the sum of relative abundances of the common microorganisms at the different time nodes; Preferably, the microbial sequence information is obtained by extracting DNA of the dregs and sequencing; Preferably, the microorganisms comprise bacteria and / or fungi; Preferably, the sequencing region of the bacteria is selected from 16S rRNA genes; Preferably, the sequencing region of the fungi is selected from ITS sequences; Preferably, the sequencing region of the bacteria is selected from 16S rRNA gene V3-V4 region; Preferably, the sequencing region of the fungi is selected from ITS1-ITS2 region; Preferably, the consensus microbe comprises at least one of Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Microascus , Rhizopus , Hyphopichia , Pichia , Geosmithia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Lederbergia , Issatchenkia , Saccharopolyspora , Anaerosalibacter and Lactiplantibacillus . Preferably, the consensus microbe comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Pichia, Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, the microorganism comprises Thermoascus , Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, in the step (2), the total relative abundance of the microorganisms of each genus is greater than 0.5%; Preferably, in the step (2), the total relative abundance of the microorganisms of each genus is greater than 0.75%; Preferably, in the step (2), the total relative abundance of the microorganisms of each genus is greater than 0.85%; Preferably, in the step (2), the total relative abundance of the microorganisms of each genus is greater than 1%.

3. The evaluation method according to claim 1, wherein The spread and mixed koji after spreading is the distiller's grains when the spreading and mixing are finished and the pile is prepared; Preferably, the finished pile of distiller's grains is the distiller's grains when the spread and mixed koji is gathered into a saccharification pile; Preferably, the down-pit distiller's grains is the distiller's grains when the saccharification pile is heated to the required temperature for entering the pit during the stacking fermentation process; Preferably, the mid-pile distiller's grains is the distiller's grains at the intermediate time node between the finished pile and the down-pit; Preferably, the liquor is baijiu; Preferably, the liquor is Jiangxiang Baijiu.

4. The evaluation method according to claim 3, characterized by, The temperature of the spread and mixed koji after spreading is 15-30℃; Preferably, the temperature of the spread and mixed koji after spreading is 17-25℃; Preferably, the temperature evaluation method of the spread and mixed koji after spreading comprises the following steps: based on the common microorganisms, combining the common microorganisms that are significantly correlated with the finished pile temperature to obtain a combination of common microorganisms; and according to the relative abundance of the combination of common microorganisms, obtaining the evaluation result of the temperature of the spread and mixed koji after spreading; Preferably, the temperature evaluation method of the spread and mixed koji after spreading further comprises: according to the increase or decrease of the relative abundance of the combination of common microorganisms, obtaining the evaluation result of the temperature of the spread and mixed koji after spreading.

5. The evaluation method according to claim 4, characterized by, The finished pile temperature is the temperature of the distiller's grains of the finished pile; Preferably, the finished pile of distiller's grains is the distiller's grains when the spread and mixed koji is gathered into a saccharification pile; Preferably, the significant correlation refers to performing Pearson correlation analysis on the relative abundance of the common microorganisms and the finished pile temperature, and the correlation coefficient is >0.8 and the p value is less than 0.05; Preferably, the significant correlation refers to performing Pearson correlation analysis on the relative abundance of the common microorganisms and the finished pile temperature, and the correlation coefficient is >0.9 and the p value is less than 0.05; Preferably, the significant correlation refers to performing Pearson correlation analysis on the relative abundance of the common microorganisms and the finished pile temperature, and the correlation coefficient is >0.9 and the p value is less than 0.01; Preferably, the combination of common microorganisms is selected from bacteria and / or fungi; Preferably, the consensus microbiome combination comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Microascus , Rhizopus , Hyphopichia , Pichia , Geosmithia、Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Lederbergia , Issatchenkia , Saccharopolyspora , Anaerosalibacter and Lactiplantibacillus . Preferably, the common microbial combination comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, the consensus microbiome combination comprises Thermoascus , Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, the consensus microbiome combination comprises Kroppenstedtia , Pichia , Thermoascus and Thermomyces at least one of Preferably, the consortium of microorganisms comprises Kroppenstedtia , Pichia , Thermoascus and Thermomyces at least two of Preferably, the total relative abundance of the microorganisms of each genus in the common microorganisms is greater than 0.5%; Preferably, the total relative abundance of the microorganisms of each genus in the common microorganisms is greater than 0.75%; Preferably, the total relative abundance of the microorganisms of each genus in the common microorganisms is greater than 0.85%; Preferably, the total relative abundance of the microorganisms of each genus in the common microorganisms is greater than 1%.

6. The evaluation method according to claim 3, wherein The thickness of the spread and aired mixed koji after the spread and airing is 2.0-5.0 cm; Preferably, the thickness of the spread and aired mixed koji after the spread and airing is 3.0-4.5 cm; Preferably, the thickness evaluation method of the spread and aired mixed koji after the spread and airing comprises the following steps: based on the microorganism OTU data, calculating an alpha diversity index, and obtaining an evaluation result of the thickness of the spread and aired mixed koji after the spread and airing according to the alpha diversity index.

7. The evaluation method according to claim 6, wherein The alpha diversity index is selected from a shannon diversity index, a chao index or an evenness; Preferably, the alpha diversity index is selected from a chao index; Preferably, the microorganism is selected from bacteria and / or fungi; Preferably, the calculation formula of the chao index is: ； where S chao1 is the estimated number of OTUs; S obs is the actual number of OTUs detected; n1 is the number of OTUs containing only one sequence; n2 is the number of OTUs containing only two sequences.

8. An evaluation system of a liquor spreading and mixing process, characterized in that, The system comprises the following modules: A data acquisition module: acquiring microorganism sequence information of distiller's grains and temperature data of complete heap of distiller's grains at different time nodes; A data processing module: based on the microorganism sequence information, processing to obtain genus classification of microorganisms, total relative abundance of microorganisms of the genus and an alpha diversity index; The optimization processing module obtains common microorganisms of the different time nodes of the fermented grains based on the genus classification of the microorganisms obtained by the data processing module; and combines common microorganisms that are significantly related to the temperature of the fermented grains based on the temperature data of the fermented grains and the common microorganisms, to obtain a combination of common microorganisms. The result output module evaluates the spreading and airing and mixing process based on the common microorganisms or the combination of common microorganisms, in combination with the total relative abundance of the microorganisms of the genus or the alpha diversity index, and outputs an evaluation result.

9. The evaluation system of claim 8, wherein, The microbial sequence information is obtained by extracting DNA of the fermented grains and sequencing; Preferably, the microorganisms include bacteria and / or fungi; Preferably, the sequencing region of the bacteria is selected from a 16S rRNA gene; Preferably, the sequencing region of the fungi is selected from an ITS sequence; Preferably, the sequencing region of the bacteria is selected from a V3-V4 region of a 16S rRNA gene; Preferably, the sequencing region of the fungi is selected from an ITS1-ITS2 region; Preferably, the microorganism comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Microascus , Rhizopus , Hyphopichia , Pichia , Geosmithia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Lederbergia , Issatchenkia , Saccharopolyspora , Anaerosalibacter and Lactiplantibacillus . Preferably, the microorganism comprises Thermoascus , Aspergillus , Byssochlamys , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, the microorganism comprises Thermoascus , Aspergillus , Thermomyces , Pichia , Virgibacillus , Kroppenstedtia , Oceanobacillus , Bacillus , Scopulibacillus , Lactobacillus , Issatchenkia and Saccharopolyspora at least one of Preferably, in the data processing module, the total relative abundance of the microorganisms of each genus is greater than 0.5%; Preferably, in the data processing module, the total relative abundance of the microorganisms of each genus is greater than 0.75%; Preferably, in the data processing module, the total relative abundance of the microorganisms of each genus is greater than 0.85%; Preferably, in the data processing module, the total relative abundance of the microorganisms of each genus is greater than 1%. Preferably, the different time nodes are selected from at least two of the following: after spreading and mixing, after stacking, after entering pit and during stacking of the fermentation process; Preferably, the after spreading and mixing is the liquor at the end of spreading and mixing, ready for stacking; Preferably, the after stacking is the liquor at the end of stacking; Preferably, the after entering pit is the liquor at the end of the fermentation process when the temperature of the fermentation process reaches the temperature required for entering pit; Preferably, the during stacking is the liquor at the time between the end of stacking and the end of the fermentation process; Preferably, the temperature of the after stacking is the temperature of the liquor at the end of stacking; Preferably, the significant correlation means that the relative abundance of the common microorganism and the temperature of the after stacking are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.8, and the p value is less than 0.05; Preferably, the significant correlation means that the relative abundance of the common microorganism and the temperature of the after stacking are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.9, and the p value is less than 0.05; Preferably, the significant correlation means that the relative abundance of the common microorganism and the temperature of the after stacking are subjected to Pearson correlation analysis, and the correlation coefficient is greater than 0.9, and the p value is less than 0.01; Preferably, the alpha diversity index is selected from shannon diversity index, chao index or evenness; Preferably, the alpha diversity index is selected from chao index; Preferably, the liquor is Baijiu; Preferably, the liquor is Jiangxiang Baijiu; Preferably, the spreading and mixing process is selected from spreading and mixing mode, spreading and mixing temperature or spreading and mixing thickness; Preferably, the spreading and mixing temperature is the temperature of the liquor at the end of spreading and mixing, ready for stacking; Preferably, the spreading and mixing thickness is the thickness of the liquor at the end of spreading and mixing.

10. Use of the evaluation method of any one of claims 1-7 or the evaluation system of any one of claims 8-9 in the field of liquor making; Preferably, the use includes use in evaluating or selecting spreading and mixing mode; Preferably, the use includes use in evaluating or selecting spreading and mixing temperature; Preferably, the use includes use in evaluating or selecting spreading and mixing thickness; Preferably, the liquor is Baijiu; Preferably, the liquor is Jiangxiang Baijiu; Preferably, the use includes use in improving liquor yield.

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