Carbon koji and preparation method thereof as well as wine brewing method and carbon koji wine based on carbon koji

Abstract

The invention provides carbon koji and a preparation method thereof as well as a wine brewing method and carbon koji wine based on the carbon koji, and belongs to the technical field of wine brewing. The preparation method comprises the following steps: firstly, mixing a starchy raw material with carbon, then cooking, then adding seed distiller's yeast for fermentation to obtain white spirit and carbon distiller's yeast at the same time, and the obtained carbon distiller's yeast can be used as the seed distiller's yeast for next-round wine making. In the application, the charcoal is bamboo charcoal, so that the obtained charcoal koji is the full-bamboo charcoal koji, and the white spirit obtained by brewing the wine by using the full-bamboo charcoal koji is the full-bamboo charcoal koji wine. By means of the technical scheme, koji making is carried out while wine making is carried out, repeated preparation of the medicinal koji can be avoided, and convenience and rapidness are achieved. In addition, the obtained carbon koji liquor is high in yield and good in quality.

Description

Technical Field

[0001] The present application relates to the field of winemaking technology, and in particular to a carbonized koji, a preparation method thereof, a winemaking method based thereon, and carbonized koji wine. Background Art

[0002] Yao Xiaoqu, also known as Jiu Baiyao or Jiuqu pills, is made from indica rice powder with Chinese herbal powder and koji seed. The Chinese herbal powder is made from a combination of three different Chinese medicinal herbs that have different effects on brewing bacteria: (1) aromatic herbs that attract natural beneficial bacteria, such as cinnamon, ginger, and cardamom; (2) herbs that provide nutrients and growth factors to brewing bacteria and promote their reproduction, such as Polygonum hydropiper, Prunella vulgaris, and Bamboo leaves; and (3) herbs that inhibit the growth of other bacteria and act as antibiotics, such as mulberry leaves, Kaempferia galanga, and Nardostachys grandiflora.

[0003] The preparation process for Yao Xiaoqu is complex: First, various Chinese medicinal herbs are combined and boiled into a decoction or ground into a powder. Then, they are mixed with indica rice flour in appropriate proportions to form dumplings (yuanxiao-sized balls). The balls are then coated with koji powder and spread on a straw-lined bamboo winnowing basket to ferment for about 48 hours. Once the balls are covered with white mycelium, they are sun-dried and then crushed for use.

[0004] The preparation of medicinal koji is also affected by season and weather, and its quality is unstable. Summary of the Invention

[0005] In order to solve at least one of the above technical problems, the technical solution adopted in this application is as follows.

[0006] The first aspect of the present application provides a winemaking method comprising the following steps: S1, mixing a starchy raw material and charcoal in a weight ratio of 10-15:1 to obtain a charcoal-raw material mixture; S2, steaming or boiling the charcoal-raw material mixture and cooling it to 30-35°C; S3, adding seed distiller's yeast in an amount of 0.5-2% of the mass of the starchy raw material for fermentation to obtain liquor.

[0007] In some embodiments of the present application, the winemaking method further comprises the following steps: obtaining charcoal from the fermented grains during and / or after fermentation, drying and crushing it to obtain charcoal koji. Thus, the winemaking method can achieve simultaneous winemaking and koji making.

[0008] In some embodiments of the present application, the starchy raw material is mixed with the charcoal after soaking. It is worth noting that in order to ensure that the charcoal can fully absorb the organic solution in the raw material during cooking, the charcoal cannot be soaked and absorbed with the raw material.

[0009] In some embodiments of the present application, in step S3, the seed koji is a medicinal koji, and thus the obtained liquor is a medicinal koji liquor, and the preparation of the carbonized koji is completed for the first time. The preparation method of the medicinal koji comprises: Mix rice flour, koji powder and traditional Chinese medicine powder to make koji embryo, and culture it at a temperature of 28-32℃ and a humidity of 70-80% until the mycelium covers the surface of the koji embryo; continue to culture it at a temperature of 20-35℃ and a humidity of 60-70% until the koji embryo becomes hard; and culture it at 35-40℃ and a humidity below 50% until the color of the mycelium changes.

[0010] In other embodiments of the present application, in step S3, the seed koji is the carbonized koji obtained in the previous round of brewing, and the liquor obtained therefrom is carbonized koji liquor, and / or the carbonized koji obtained in the current brewing can be used as the seed koji for the next round of brewing, thereby realizing the circulation of koji.

[0011] In some embodiments of the present application, the charcoal is bamboo charcoal. The charcoal koji obtained is a pure bamboo charcoal wine koji, and the charcoal wine obtained by further brewing using the pure bamboo charcoal wine koji as a seed wine koji is a pure bamboo charcoal wine koji wine.

[0012] In some embodiments of the present application, the density of the bamboo charcoal is not less than 0.9 g / cm 3 The density of bamboo charcoal is too small and its adsorption capacity is weak, so the quality of the bamboo charcoal koji prepared is poor, and it seriously affects the yield and quality of the full bamboo charcoal koji wine.

[0013] In some embodiments of the present application, the size of the bamboo charcoal is not less than 3 cm 3 The size of bamboo charcoal is too small to be easily recycled, which seriously affects the recycling amount of bamboo charcoal wine yeast.

[0014] In some embodiments of the present application, the starchy raw material is a cereal starchy raw material and / or a potato starchy raw material.

[0015] In some possible implementation plans of the present application, the cereal starchy raw materials are one or more of rice, corn, sorghum, barley, wheat, highland barley, buckwheat and oats; the tuber starchy raw materials are one or more of sweet potato, cassava, potato and yam.

[0016] In some specific embodiments of the present application, the starchy raw material is sorghum.

[0017] The second aspect of the present application provides liquor prepared by any of the brewing methods described in the first aspect of the present application.

[0018] During the brewing process, the seed koji added can be the original medicinal koji, and the liquor obtained therefrom is medicinal koji liquor; or it can be the charcoal koji obtained from the previous brewing, and the liquor obtained therefrom is charcoal koji liquor. In this application, charcoal koji liquor has the same quality as medicinal koji liquor.

[0019] The third aspect of the present application provides charcoal koji prepared by any of the brewing methods described in the first aspect of the present application.

[0020] During the winemaking process of preparing carbonized koji, the seed koji added can be the original medicinal koji or the carbonized koji obtained from a previous winemaking process. When the seed koji added is the medicinal koji, the carbonized koji is prepared for the first time. When the seed koji added is the carbonized koji, the carbonized koji is prepared after one or more cycles.

[0021] In some embodiments of the present application, the carbonized koji can continue to be used as a seed koji for wine making and carbonized koji can be obtained again at the same time.

[0022] Compared with the prior art, this application has the following beneficial effects: By utilizing the technical solution of the present application, koji can be prepared simultaneously with winemaking, thus avoiding repeated preparation of medicinal koji, which is convenient and rapid. Moreover, the obtained liquor (medicinal koji liquor or charcoal koji liquor) has high yield and good quality.

[0023] After multiple cycles, the microbial structure of the carbonized koji of the present application will not change significantly, and will not affect the yield and quality of the wine.

[0024] The method of the present application has simple steps, easily obtainable raw materials, and does not require complicated operations.

[0025] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and advantages of the exemplary embodiments of the present application will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present application are shown in an illustrative and non-limiting manner, in which: Figure 1 A schematic diagram of the process of preparing medicinal koji to prepare charcoal koji and medicinal koji wine is shown, wherein the charcoal koji is recycled to make wine to obtain charcoal koji wine; Figure 2The electrophoresis detection diagram of DNA extracted from traditional Chinese medicine Xiaoqu and charcoal qu in Example 3 of the present application is shown. 1 is the traditional Chinese medicine Xiaoqu; 2 is the charcoal qu; P1 to P3 are blank controls of the extraction reagents; M is the marker (DL9000), and the bands from top to bottom are 9000bp, 5000bp, 3000bp, 2000bp, 1000bp, and 500bp, respectively; Figure 3 Shown is an electrophoresis detection diagram of the sequencing library in Example 3 of the present application; Figure 4 The figure shows the rank-abundance diagram of microorganisms in Example 3 of the present application, where the horizontal axis number "50" represents the OTU ranked 50th in terms of abundance in the sample; Figure 5 The Venn diagram of the OTUs of charcoal koji and medicinal koji in Example 3 of the present application is shown; Figure 6 The histogram of species distribution at the genus level of charcoal koji and medicinal koji in Example 3 of the present application is shown; Figure 7 Shown is a heat map of species distribution at the genus level for Charcoal Qu and Yao Xiao Qu in Example 3 of the present application; Figure 8 The combined analysis diagram of the genus-level sample clustering tree and histogram in Example 3 of the present application is shown. On the left is the hierarchical clustering analysis (Bray-Curtis algorithm) based on community composition between samples, and on the right is the community structure histogram of the samples. DETAILED DESCRIPTION

[0027] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, particularly for definitions of relevant terms in the art disclosed therein. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0028] The numerical ranges in this application are approximate, so unless otherwise stated, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For ranges containing values ​​less than 1 or containing fractions greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For ranges containing single-digit numbers less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0029] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination thereof.

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer and more understandable, the present application is further described in detail below in conjunction with the embodiments.

[0031] The following examples are provided herein to illustrate preferred embodiments of the present application. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application and, therefore, can be considered preferred embodiments of the present application. However, those skilled in the art will appreciate, based on this specification, that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present application.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the disclosures and materials cited therein are hereby incorporated by reference.

[0033] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0035] refer to Figure 1 In Example 1 of the present application, rice flour, seed koji powder, and Chinese medicinal powder are first used to prepare medicinal koji through mixing and inoculation, koji embryo making, early culture (molding), intermediate culture (airing and mold formation), late culture (dry fire), after-ripening and drying; then, sorghum is soaked and mixed with bamboo charcoal and steamed, and after cooling, the medicinal koji is added as seed wine koji for fermentation and winemaking, and the bamboo charcoal is used as a medium for extracting the bacteria contained in the seed wine koji, and sorghum wine (white wine) and charcoal koji are obtained at the same time; when making wine later, the charcoal koji will be used as the seed wine koji, so that the medicinal koji only needs to be prepared once, and the koji can be made at the same time during the winemaking process.

[0036] Example 1 Preparation of Medicinal Xiaoqu 1. Raw material preparation and processing Rice Flour Processing: Grind and sieve the indica rice (40-60 mesh) to obtain fine rice flour. Place the fine rice flour in a steamer and steam over high heat for 20-30 minutes to sterilize and gelatinize. Once steamed thoroughly, remove from the steamer and let cool to room temperature (approximately 30°C). Ensure there are no lumps.

[0037] Processing of seed koji: Grind the seed koji into fine powder and set aside.

[0038] Processing of Chinese medicinal materials: The selected Chinese medicinal materials (polygonum odoratum, cinnamon bark, ginger, cardamom, selfheal, bamboo leaves, mulberry leaves, galangal and spikenard) are dried and crushed into fine powder. The polygonum odoratum powder, selfheal powder and bamboo leaves powder are mixed in equal weight proportions to obtain nutritious medicinal powder. The cinnamon bark powder, ginger powder and cardamom powder are mixed in equal weight proportions to obtain fragrant medicinal powder. The mulberry leaf powder, galangal powder and spikenard powder are mixed in equal proportions to obtain antibacterial medicinal powder. The nutritious medicinal powder, fragrant medicinal powder and antibacterial medicinal powder are mixed in a ratio of 3:1:1 to obtain Chinese medicinal powder.

[0039] 2. Mixing and inoculation Pour the cooled rice noodles into a clean basin, sprinkle the crushed koji powder evenly on the rice noodles at 1% of the dry weight of the rice noodles, and then sprinkle the Chinese medicinal powder evenly at 10-12% of the dry weight of the rice noodles.

[0040] Slowly add clean cold boiled water (water temperature close to room temperature), stirring quickly and evenly while adding. The amount of water added should be 35%-45% of the dry weight of the rice noodles.

[0041] 3. Molding (making koji) Pass the mixed wet material through a coarse sieve (10-20 mesh) to make it into loose, uniform granules. Form the wet material into small balls with a diameter of about 1.5-2.5 cm.

[0042] 4. Preliminary culture (mold preparation) Place the formed koji pieces evenly and loosely on a bamboo plate covered with a layer of clean straw, leaving appropriate gaps (about 1-2 cm) between the pieces to ensure air circulation.

[0043] Move the koji tray into a warm, humid and clean koji room (temperature controlled at 28~32℃; relative humidity maintained at 70~80%).

[0044] During static culture, the mycelium begins to grow and covers the surface of the embryo in about 48 hours, forming a "white coat".

[0045] 5. Mid-term culture (drying and mold-drying) When the surface of the koji embryo is covered with white mold, the humidity in the koji room is reduced (60~70%) and ventilation is carried out, and the temperature is controlled at 20~35℃ to control excessive growth of hyphae and promote the growth of hyphae into the interior of the koji embryo. After 1~2 days, the koji embryo becomes hard and solid.

[0046] 6. Late cultivation (dry fire) Continue to increase ventilation and further reduce humidity (to below 50%). Remove coverings. Keep the product temperature between 35 and 40°C and allow it to gradually decrease naturally.

[0047] Turn the koji embryos regularly to ensure even drying. After 2-3 days, the koji embryos will become noticeably drier, harder, and lighter. The color of the mycelium will change from white to slightly yellowish or light gray.

[0048] 7. Ripening and drying When the koji embryos are basically dry and the product temperature drops to near room temperature, stack the koji embryos and place them at room temperature for 3-7 days for ripening.

[0049] Spread the ripened Xiaoqu out in the sun to dry it until its moisture content drops below 12%. Place the thoroughly dried Xiaoqu in a clean, dry, well-sealed container and store it in a cool, dry, well-ventilated place away from light.

[0050] Example 2 Preparation of carbon koji 1. Preparation of bamboo charcoal Choose bamboo charcoal made by dry distillation at 800-1200 degrees. Choose charcoal fragments or pieces (not less than 3cm) with a length of 2-15cm and a width of 2-5cm. 3 The density of the selected bamboo charcoal should be greater than 0.9g / cm 3 It is best if it can enter the water without floating, floating or sinking.

[0051] 2. Preparation of Carbonized Koji (1) Soaking and moistening grain Place the sorghum in a container and add water to soak for about 24 hours.

[0052] (2) Steaming and gelatinization The purpose of steaming is to thoroughly gelatinize the sorghum starch, kill bacteria, and volatilize unpleasant odors. In this embodiment, the soaked sorghum is mixed with bamboo charcoal (10% of the dry weight of the sorghum), washed together, and steamed in a steamer until the sorghum is cooked thoroughly and gelatinous.

[0053] (3) Spreading cold koji The steamed sorghum-bamboo charcoal was taken out and spread on a drying bed, stirred and cooled quickly, cooled to 30-35°C, and mixed with the medicinal koji prepared in Example 1 at 1% of the dry weight of the sorghum.

[0054] (4) Fermentation The fermentation is carried out in open vats for 24-48 hours before being sealed in a winery at a temperature of 20-25°C and a humidity of 70-80%. During fermentation, the vats are turned and stirred 1-2 times. During stirring, bamboo charcoal is removed and sun-dried. The temperature of the mash is monitored throughout the entire fermentation process.

[0055] (5) Take the wine and collect the koji After fermentation is complete after about 7 to 20 days, the remaining bamboo charcoal in the mash is removed and distilled to produce sorghum liquor (also known as Yaoxiaoqu liquor). The bamboo charcoal is then dried and crushed to create charcoal koji, completing both koji making and winemaking simultaneously.

[0056] In order to verify the advantages of the above brewing method, the inventors also conducted the following comparisons: ①Omit bamboo charcoal, and the rest of the steps are the same; ② Add bamboo charcoal after steaming the sorghum raw material. The remaining steps and parameters are the same.

[0057] After the winemaking process was complete, the yields of the three winemaking methods were compared. 100 mL of each sample was taken and the pH was measured at room temperature using a Reye's pHSj-4F. The alcohol content was determined according to "GB5009.225-2016 Determination of Ethanol Concentration in Wines," the total acid content was determined according to "GB12456-2021 Determination of Total Acidity in Foods," and the total ester content was determined according to "GB / T10345-2022 Liquor Analysis Method." The results are shown in Table 1. Table 1: Effects of bamboo charcoal on wine yield and quality

[0058] As shown in Table 1, when 50 kg of sorghum raw materials are fermented and brewed using traditional technology (without adding bamboo charcoal), less than 30 kg of 53-proof liquor can be produced, and the flavor substances (total esters) are only about 0.5 g / L, which is of relatively poor quality.

[0059] If bamboo charcoal is added after the raw materials are steamed, 50 kg of sorghum raw materials can produce less than 30 kg of 53-proof liquor. Although the total ester content is slightly increased, it is only 0.64 g / L, and the flavor is still lacking.

[0060] Conversely, adding bamboo charcoal to the raw materials before steaming them yielded over 32kg of 53% alcohol liquor from the same 50kg of sorghum, significantly increasing yield. The total ester content was also significantly higher, reaching around 0.83g / L.

[0061] The inventors further analyzed that steaming bamboo charcoal with sorghum, thanks to its thermal conductivity and permeability, as well as the far-infrared radiation it releases, allows for even heating of the sorghum, allowing the starch to cook thoroughly and gelatinize, resulting in a high starch conversion rate and, consequently, a higher liquor yield. Furthermore, it causes the sorghum husk to crack and puff, thereby stimulating the release of the sorghum's flavor compounds and enhancing the wine's aroma. Furthermore, the far-infrared radiation released by bamboo charcoal has heat-insulating and heat-resistant properties, providing a superior warming effect, maintaining a stable temperature between 20 and 25°C during fermentation, thus enhancing both yield and quality. Finally, due to its fine and porous structure, bamboo charcoal not only serves as a medium for extracting microorganisms during fermentation, but also as a "nest" for brewing bacteria. After brewing, the bamboo charcoal can be removed from the mash and crushed to create charcoal koji, which can be used as seed koji for subsequent brewing. Furthermore, the total amount of charcoal koji produced is approximately 10 times the amount of the medicinal koji added, enabling larger-scale brewing.

[0062] Example 3 Analysis of the consistency of microbial structures of carbonized koji and medicinal koji In order to verify that there is no difference in the microbial composition between charcoal koji and medicinal koji, the inventors took samples of charcoal koji and medicinal koji respectively and performed microbiome analysis. The specific steps are as follows: 1. Genomic DNA Extraction The genomic DNA was extracted using proteinase K lysis method, and 3 μL was taken for 1.2% agarose gel electrophoresis. The results were as follows: Figure 1 shown.

[0063] 2. PCR Primer Design According to the requirements of Illumina high-throughput sequencing, bidirectional sequencing was performed and a fusion primer of "5' adapter-barcode-sequencing primer-specific primer-3'" was designed, wherein the specific primer targeted the target region.

[0064] Forward primer structure: 5'-AATGATACGGCGACCACCGAGATCTACAC-barcodeF2-TCTTTCCCTACACGACGCTCTTCCGATCT-barcodeF1-specific primer-3' Reverse primer structure: 5'-CAAGCAGAAGACGGCATACGAGAT-barcodeR2-GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-barcodeR1-specific primer-3' The specific primer sequences are shown in Table 2: Table 2: Specific primer sequences

[0065] 3. PCR Amplification A two-step PCR amplification method was used to construct the library. First, the target fragment was amplified using specific primers (described below as inner primers), the target fragment was recovered by gel recovery, and then the recovered product was used as a template for secondary PCR amplification (described below as amplification using outer primers). The purpose was to add the adapters, sequencing primers, and barcodes required for Illumina platform sequencing to both ends of the target fragment.

[0066] Inner primer forward: 5'-TTCCCTACACGACGCTCTTCCGATCT-barcodeF1-specific primer-3' Inner primer reverse: 5'-GAGTTCCTTGGCACCCGAGAATTCCA-barcodeR1-specific primer-3' Outer primer forward: 5'-AATGATACGGCGACCACCGAGATCTACAC-barcodeF2-TCTTTCCCTACACGACGCTC-3' Outer primer reverse: 5'-CAAGCAGAAGACGGCATACGAGAT-barcodeR2-GTGACTGGAGTTCCTTGGCACCCGAGA-3' (1) Single PCR amplification Amplification system: 5× Buffer 10 μL, dNTP (10 mM) 1 μL, Phusion High-Fidelity DNA Polymerase 1 U, inner primer (10 μM) 1 μL each of forward and reverse directions, DNA template 5 ng~50 ng, make up to 50 μL with ddH2O.

[0067] Amplification program: 94°C for 2 min; 94°C for 30 s, annealing temperature for 30 s, 72°C for 30 s, 22 cycles, 72°C for 5 min, and then incubation at 10°C. The annealing temperature for 16S V4-V5 amplification was 55°C, and the annealing temperature for ITS1 amplification was 50°C.

[0068] Amplification was completed on an ABI9700 PCR instrument. All PCR products were recovered using an AxyPrep DNA gel recovery kit and quantified using an FTC-3000™ real-time PCR instrument. Samples were mixed at an equimolar ratio and then subjected to secondary PCR amplification.

[0069] (2) Secondary PCR amplification Amplification system: 5× Buffer 8μL, dNTP (10mM) 1μL, Phusion high-fidelity DNA polymerase 0.8U, outer primers (10μM) forward and reverse 1μL each, DNA template 5μL, add ddH2O to 40μL.

[0070] Amplification program: 94°C for 2 min; 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, 8 cycles, 72°C for 5 min, and then incubation at 10°C.

[0071] PCR product was taken and 3 μL PCR product was tested by 1.2% agarose gel electrophoresis. Figure 2 As shown, the secondary PCR amplification results in a single band of moderate brightness. The PCR amplification product was electrophoresed on a 2% agarose gel and recovered using the AxyPrep DNA Gel Recovery Kit from AXYGEN.

[0072] 4. Sequencing and Data Quality Control Sequencing was performed using the Illumina sequencing platform. The resulting PE reads were first distinguished from each sample by barcode. Sequence quality control was performed using Trimmomatic (version 0.38) software, employing a window-based low-quality removal method. The following procedures were used: within a 50-bp window, if the average quality score within the window was less than 20, the trailing bases were truncated from the window, filtering reads shorter than 50 bp after quality control. Sequencing adapters and primers were processed using cutadapt (version 1.16). FLASH (version 1.2.11) software was used to splice paired reads into a single sequence based on overlap between PE reads. The minimum overlap length was 10 bp, and the maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2 to filter out non-compliant sequences.

[0073] 5. OTU Cluster Analysis The software USEARCH was used to cluster the spliced ​​sequences into OTUs. The main process is as follows: (1) Use UPARSE to cluster at 97% similarity to obtain the representative sequence of OTU; (2) UCHIME was used to remove chimeras generated by PCR amplification from OTU representative sequences. 16S and ITS chimeras were removed by comparing them with existing chimera databases. The 16S chimera database was golddatabase (v20110519), and the ITS chimera database was UNITE (v20140703).

[0074] (3) Use the usearch_global method to align all sequences back to the OTU representative sequences and obtain the abundance statistics of each sample in each OTU.

[0075] (4) After obtaining the OTU representative sequence, the OTU representative sequence was compared with the database using the mothur software for species annotation, and the confidence threshold was set to 0.6.

[0076] (5) Filter the annotation results: remove OTUs without annotation results; remove species whose annotation results do not belong to the analysis project, for example, if the OTU is annotated with Archaea, remove it.

[0077] 6. Species Analysis 6.1 Abundance level map Rank-abundance curve is a way to analyze diversity. By counting the number of sequences contained in each OTU in a single sample, OTUs are sorted from large to small according to their abundance (the number of sequences contained). Then, a graph is drawn with OTU rank as the horizontal axis and the relative abundance of each OTU (taking the log10 logarithm) as the vertical axis. The results are as follows: Figure 4 shown.

[0078] Depend on Figure 4 It can be seen that the OTU levels of charcoal koji and Yaoxiaoqu are very consistent with the relative abundance curves of each OTU.

[0079] 6.2 Venn Diagram The Venn diagram can count the number of common and unique OTUs in Yao Xiaoqu and Tanqu, and can more intuitively show the similarity and overlap of the OTU number composition of the two samples. In this example, the OTU sample with a similarity level of 97% was selected for plotting, as shown in the figure below. Figure 5 shown.

[0080] Depend on Figure 5 It can be seen that the common OTUs of charcoal koji and Yaoxiaoqu account for about 83% (63 / 76), indicating that the species information of the two is very similar.

[0081] 6.3 Species Information Analysis Using statistical analysis methods, we observed the community structure of samples at different taxonomic levels. We compared the community structure analysis of two samples to observe their changes. Figure 6 As shown in Figure 2, the community structures of the two samples at the genus level were basically the same.

[0082] Furthermore, a heatmap can use color changes to reflect the data information in a two-dimensional matrix or table. It can intuitively represent the size of the data value with a defined color depth. In this embodiment, a heatmap is used to cluster the similarity of species abundance at the genus level between two samples, such as Figure 7 As shown. Figure 7 It can be seen that there is little difference in species between the two samples at the genus level.

[0083] 6.4 Beta diversity based on species information Bray-Curtis distance is a commonly used indicator to reflect the difference between two communities. The value of Bray-Curtis distance is between 0 and 1. The larger the value, the greater the difference between the samples. At the genus level, the inventors conducted a hierarchical cluster analysis based on species information. Figure 8 shown.

[0084] Depend on Figure 8 It can be seen that the difference between charcoal koji and medicinal koji is small, indicating that there is no difference between the two microbial communities.

[0085] The results of this embodiment show that using bamboo charcoal as a carrier to recover brewing bacteria not only absorbs the organic matter of the raw materials required by the microorganisms, but also contains the minerals required for microbial growth, maintaining a good nutritional balance, thereby maintaining the stability of the microbial structure of the koji, and providing a solid guarantee for the stability of the yield and quality of the koji wine obtained by adding all-charcoal koji to brew wine.

[0086] Example 4: Winemaking using charcoal koji The carbon koji prepared in Example 2 was rehydrated and activated for 10 minutes.

[0087] The brewing process is the same as that in the embodiment, except that charcoal koji is used instead of medicinal koji, and the amount used is also 1% of the raw material amount, and the brewing is carried out using the same temperature and humidity conditions.

[0088] By using bamboo charcoal and charcoal koji to brew wine, charcoal koji wine and charcoal koji can be obtained at the same time, which can be used again for brewing wine, thus realizing the recycling of charcoal koji.

[0089] The yield and quality of wine produced by using charcoal koji and medicinal koji were further improved. Moreover, the yield and quality of wine did not change after multiple cycles of charcoal koji, as shown in Table 3: Table 3: Effect of koji on wine yield and quality

[0090] As shown in Table 3, as long as the quality of the first batch of koji is maintained, the same koji can be used repeatedly for subsequent brewing. Brewing different batches of wine under the same temperature and humidity conditions will not reduce yield, nor will the taste and flavor change. The inventors have used koji that has been recycled over 3,000 times to maintain stable yield and quality, exceeding expectations.

[0091] Example 5 Effects of Different Bamboo Charcoals on Yeast Recovery By adding bamboo charcoal to the raw materials, the present invention can recycle the charcoal to prepare koji at the end of brewing, allowing for the next round of brewing, once again achieving simultaneous brewing and koji making. Therefore, ensuring effective recycling requires that the bamboo charcoal can carry sufficient brewing bacteria and the required organic matter, minerals, etc. To this end, this example explores the effects of using different types of charcoal on adsorption. The following three types of charcoal were prepared: (1) Bamboo charcoal prepared in the same manner as in Example 2; (2) Bamboo charcoal was prepared using the same method as in Example 2, but bamboo charcoal with a lower density (floating in water) was selected; (3) Hardwood charcoal, the size of which is comparable to the bamboo charcoal selected in Example 2.

[0092] The three types of charcoal were used to brew wine with the medicinal koji prepared in Example 1 according to the method of Example 2. The increase rate of the koji weight relative to the weight of the added charcoal was calculated.

[0093] Increase rate = (weight of koji - weight of added charcoal) / weight of added charcoal × 100% The results showed that when using the first type of charcoal, the increase rate reached about 40%, indicating that the first type of charcoal is easier to absorb raw organic solution, trace elements, minerals, brewing microorganisms, etc., and has a higher recovery rate; when using the second type of charcoal, the increase rate was only about 20%, indicating that the low-density bamboo charcoal has a weak adsorption capacity and it is difficult to achieve the purpose of recovering brewing bacteria; when using the third type of charcoal, the increase rate was only about 15%, the adsorption capacity was even weaker, and it was even more difficult to achieve the purpose of recovering brewing bacteria.

[0094] In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A winemaking method, characterized in that: The following steps are involved: S1, mixing a starchy raw material and charcoal in a mass ratio of 10-15:1 to obtain a charcoal-raw material mixture; S2, steaming or boiling the charcoal-raw material mixture and cooling it to 30-35°C; S3, adding seed distiller's yeast in an amount of 0.5-2% of the mass of the starchy raw material for fermentation to obtain liquor.

2. The brewing method according to claim 1, characterized in that Further comprising the steps of: The charcoal in the fermented grains is obtained during the fermentation process and / or after the fermentation is completed, and is dried and crushed to obtain charcoal koji.

3. The winemaking method according to claim 2, characterized in that: In step S3, the seed koji is the medicinal koji or the charcoal koji, and the obtained liquor is the medicinal koji liquor or the charcoal koji liquor.

4. The brewing method according to any one of claims 1 to 3, characterized in that: The charcoal is bamboo charcoal.

5. The brewing method according to claim 4, characterized in that: The density of the bamboo charcoal is not less than 0.9g / cm 3 .

6. The winemaking method according to claim 4, characterized in that: The size of the bamboo charcoal is not less than 3cm 3 .

7. The brewing method according to any one of claims 1 to 3, characterized in that: The starchy raw material is a cereal starchy raw material and / or a potato starchy raw material.

8. The winemaking method according to claim 7, characterized in that: The cereal starch raw materials are one or more of rice, corn, sorghum, barley, wheat, highland barley, buckwheat and oats; the potato starch raw materials are one or more of sweet potato, cassava, potato and yam.

9. Liquor prepared by the brewing method according to any one of claims 1 to 8.

10. Carbonized koji prepared by the brewing method according to claim 2 or 3.

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