Preparation method of bupleurum chinense wheat koji and application of bupleurum chinense wheat koji in preparation of yellow wine

Abstract

The invention provides a preparation method of bupleurum chinense wheat koji and application of the bupleurum chinense wheat koji in preparation of yellow rice wine, and belongs to the technical field of wine brewing and koji propagation, the preparation method of the bupleurum chinense wheat koji provided by the invention comprises the following steps: 1) mixing bupleurum chinense with water, and extracting to obtain a bupleurum chinense extracting solution; (2) infiltrating broken wheat by using the bupleurum chinense extracting solution obtained in the step (1), and then preparing the mixed material into a curved brick; 3) fermenting and drying the koji bricks to obtain bupleurum chinense wheat koji; the mass ratio of the bupleurum chinense to the water is 1: (4-6). The bupleurum chinense wheat koji prepared by infiltrating wheat with the bupleurum chinense extract is better in quality, the activity level of glucoamylase, liquefying enzyme and other enzymes in the bupleurum chinense wheat koji is improved, the fermentation time of the yellow wine can be effectively shortened, the content and variety of volatile substances in the prepared yellow wine are increased, and the fragrance is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of wine making and koji making, and in particular relates to a method for preparing northern bupleurum malt koji and application thereof in preparing yellow rice wine. Background Art

[0002] Wheat koji, as an undefined fermentation starter, is a type of koji (similar to Japanese koji) used to initiate the fermentation of rice wine. The traditional production method is to mix unautoclaved crushed wheat with water to form bricks and then let them ferment. The fungal and bacterial communities in wheat koji have different sources and play specific roles in the fermentation process. Fungi are mainly derived from the environment and drive the initial fermentation process by producing various enzymes, including glucoamylase, amylase, and protease, which break down complex molecules such as starch and protein in the raw materials into simpler compounds. Bacteria are usually derived from the raw materials and play a vital role in modifying volatile compounds, which contribute to the aroma and flavor characteristics of the final product. However, the traditional production method leads to batch-to-batch variability in quality due to non-sterile operations and reliance on experience. Differences in raw materials and brewing techniques can affect the microbial and chemical components in wheat koji, which in turn affect the sensory properties of rice wine. Therefore, further research on the volatile components and microbial dynamics of wheat koji is needed to develop standardized starter cultures that can regulate fermentation and stabilize the aroma quality of rice wine.

[0003] Bupleurum chinense DC., a perennial herb found throughout Northwest China, contains a wealth of bioactive ingredients. In traditional medicine, Bupleurum chinense DC. is widely used for its excellent medicinal value, mainly in the fields of anti-inflammatory, anti-depression, and liver protection, and has become one of the important sources of Chinese herbal medicine Bupleurum. However, the application and mechanism of Bupleurum chinense DC. in traditional rice wine brewing in northern China, especially its interaction with wheat koji, has not been studied yet. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing Bupleurum chinense malt and its application in preparing rice wine. The Bupleurum chinense malt prepared by soaking wheat with Bupleurum chinense extract has better quality, and the activity levels of enzymes such as saccharifying enzyme and liquefying enzyme in the Bupleurum chinense malt can be improved, which can effectively shorten the fermentation time of rice wine, increase the content and types of volatile substances in the prepared rice wine, and enhance the aroma.

[0005] The present invention provides a method for preparing Bupleurum chinense wheat koji, comprising the following steps:

[0006] 1) mixing Bupleurum chinense with water and extracting to obtain Bupleurum chinense extract;

[0007] 2) soaking the cracked wheat with the extract of Bupleurum chinense obtained in step 1), and then preparing the mixed materials into koji bricks;

[0008] 3) fermenting and drying the koji brick to obtain Bupleurum chinense koji;

[0009] The mass ratio of the mixture of Bupleurum chinense and water is 1:(4-6);

[0010] The mass ratio of the Bupleurum chinense extract to the cracked wheat is (9-11):50.

[0011] Preferably, the extraction temperature in step 1) is 95-105° C., and the extraction time is 20-40 min.

[0012] Preferably, the soaking temperature in step 2) is 25-35°C.

[0013] Preferably, the fermentation in step 3) is natural fermentation.

[0014] Preferably, when preparing high-temperature Bupleurum chinense wheat koji, a temperature-controlled fermentation step is further included before the natural fermentation, the temperature of the temperature-controlled fermentation is 60-70° C., and the time of the temperature-controlled fermentation is 5-8 days.

[0015] The invention provides the Bupleurum chinense wheat koji prepared by the preparation method.

[0016] The invention provides the application of the northern bupleurum maltose in preparing yellow wine.

[0017] The present invention also provides a method for preparing yellow wine, comprising the following steps:

[0018] 1) After soaking, rinsing and steaming the millet, spread it out to dry;

[0019] 2) mixing the air-dried millet with the northern bupleurum koji, placing them in a jar for fermentation, and when the fermentation temperature reaches above 30° C. to 36° C., covering them and harrowing them;

[0020] 3) separating the solid and liquid of the fermented mash, and decocting the liquid component at 80°C to 83°C for 10min to 15min to obtain liquor;

[0021] 4) The wine is blended, filtered, and then bottled and sterilized to obtain yellow wine.

[0022] Preferably, the mass ratio of millet, Bupleurum chinense malt and Bupleurum chinense extract is (20-30): (4-6): (8-10).

[0023] Preferably, the saccharification temperature during the fermentation process is 28-32° C., and the saccharification time is 40-60 h.

[0024] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses Bupleurum chinense extract to soak wheat, and ferments at two temperatures to prepare medium-temperature Bupleurum chinense koji and high-temperature Bupleurum chinense koji. The results show that both the medium-temperature Bupleurum chinense koji and the high-temperature Bupleurum chinense koji significantly improve the activities of saccharifying enzyme and liquefying enzyme, and can effectively shorten the fermentation time of rice wine; and the Bupleurum chinense koji contains a variety of bioactive substances, and the rice wine brewed using this koji has potential health effects.

[0025] The two Bupleurum chinense wheat koji provided by the present invention showed higher species richness and uniformity in the bacterial community; compared with CK, MB and HB samples showed significantly enhanced flavor characteristics in all 9 evaluation descriptions. The addition of Bupleurum chinense increased the diversity and richness of wheat koji microorganisms, especially enriched Bacillus, Staphylococcus and Aspergillus, while heat treatment was beneficial to thermophilic fungi such as Thermomyces, Rasamsonia and Thermoascus, thereby making the microbial community more stable.

[0026] The invention adopts northern bupleurum and wheat koji to ferment millet yellow wine, thereby improving the fermentation speed and wine yield, improving the safety, and enhancing the sensory quality and health care effect of the yellow wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Agarose gel electrophoresis detection diagram of the amplified ITS1 and 16SrRNA V3-V4 regions of five wheat koji samples (CK, MB1, MB2, HB1, HB2);

[0028] Figure 2 The rarefaction curve (AC) and Shannon curve (BD) of malt bacteria (left) and fungi (right);

[0029] Figure 3 is the Alpha diversity index of Aspergillus niger group;

[0030] Figure 4 PCoA diagrams of different koji bacteria (A) and fungi (B);

[0031] Figure 5 It is the dominant phylum level composition of different types of wheat koji;

[0032] Figure 6 The composition of bacteria (A) and fungi (B) at the genus level with relative abundance > 1% in different types of wheat koji;

[0033] Figure 7Representative bacterial and fungal taxa in different types of wheat koji CK, MB and HB groups; A and F are UpSet plots showing the number of shared and unique (A) bacterial and (F) fungal ASVs, and their corresponding relative abundances in different groups; B and G are LEfSe comparisons of bacterial and fungal communities at the genus level in wheat koji (LDA ≥ 4); C, D, H and I are Manhattan plots showing ASVs with relative abundance > 0.1% from MetagenomeSeq analysis; E and J circle-filled plots show differences in the taxonomic composition of representative biomarkers, where the size of the circle represents the average relative abundance of differentially abundant taxa;

[0034] Figure 8 The histograms of the distribution of volatile compounds in different types of malt koji and the pie charts of their relative contents;

[0035] Fig. 9 This is the heat map cluster analysis of 105 volatile compounds in different types of wheat koji;

[0036] Fig.10 Analysis of key aroma compounds in different types of malt koji;

[0037] Fig.11 To analyze the different volatile compounds of different types of malt koji;

[0038] Fig.12 The volatile compounds with different characteristics of different types of malt koji;

[0039] Fig.13 Correlation analysis between different types of wheat koji microbial communities and volatile organic compounds, where A and B are Procrustes analysis of the combined bacterial and fungal communities and volatile compounds; C is the co-occurrence analysis of volatile compounds and microbial taxa with an average relative abundance of >0.1% using Spearman correlation; correlations with P ≥ 0.05 and |r| < 0.9 were excluded; **, P < 0.01;

[0040] Fig.14 To predict the functions of different types of Aspergillus niger fungal communities;

[0041] Fig.15 The color photos of two kinds of rice wine MBJ and HBJ;

[0042] Fig.16 Safety test report for rice wine MBJ;

[0043] Fig.17 This is the safety test report of Huangjiu HBJ. DETAILED DESCRIPTION

[0044] The invention provides a method for preparing bupleurum chinense and wheat koji, comprising the following steps: 1) mixing bupleurum chinense with water and extracting to obtain bupleurum chinense extract; 2) soaking cracked wheat with the bupleurum chinense extract obtained in step 1), and then preparing the mixed material into koji bricks; 3) fermenting and drying the koji bricks to obtain bupleurum chinense and wheat koji; the mass ratio of the bupleurum chinense mixed with water is 1:(4-6); the mass ratio of the bupleurum chinense extract to the cracked wheat is (9-11):50.

[0045] In the present invention, Bupleurum chinense is mixed with water and extracted to obtain Bupleurum chinense extract. In the present invention, the mass ratio of Bupleurum chinense to water is 1:(4-6), preferably 1:(4.5-5.5), and more preferably 1:5; the extraction temperature is preferably 95-105°C, more preferably 98-102°C, and the extraction time is preferably 20-40min, more preferably 25-35min. In the specific implementation of the present invention, the extraction is preferably boiling extraction. The present invention performs solid-liquid separation after the extraction, and collects the liquid phase component as Bupleurum chinense extract; the solid-liquid separation is preferably performed by filtration.

[0046] After obtaining the Bupleurum chinense extract, the present invention soaks the broken wheat with the Bupleurum chinense extract, and then prepares the mixed material into koji bricks. In the present invention, the wheat is preferably crushed into a "plum petal" shape that is most suitable for fermentation to ensure that the heart is rotten but the skin is not. In the present invention, the mass ratio of the Bupleurum chinense extract to the broken wheat is (9-11):50, preferably 1:5; in the present invention, the soaking temperature is preferably 25-35°C, more preferably 28-32°C. In the specific implementation of the present invention, the amount of the Bupleurum chinense extract is preferably based on the standard of being able to be kneaded into a ball by hand without dripping. The soaking time is preferably 15-20min. After the soaking is completed, the present invention prepares the mixed material into koji bricks, and the size of the koji bricks is preferably 18cm×18cm×10cm.

[0047] After preparing the koji bricks, the koji bricks are fermented and dried to obtain northern bupleurum malt koji. In the present invention, the koji bricks are preferably fermented in a fermentation room, and further preferably, the koji bricks are separated by wheat straw and wormwood. In the present invention, the interval between two adjacent koji bricks is preferably 1 to 3 cm. Since the koji bricks are trapezoidal, the gaps on the edges also need to be filled with wheat straw and wormwood, and the thickness of wheat straw and wormwood is preferably 3 to 5 cm. In the present invention, the fermentation is preferably natural fermentation, and the time of the natural fermentation is preferably 25 to 40 days, and more preferably 30 to 35 days. After the natural fermentation is completed, the present invention is dried, and the drying is preferably carried out by sun drying, and the sun drying is preferably sun drying in the sun. The present invention has no special limitation on the time of the sun drying, and it can ensure that the moisture content of the final malt koji is less than 14%. The present invention obtains northern bupleurum malt koji after the sun drying, and the obtained northern bupleurum malt koji is medium-temperature northern bupleurum malt koji.

[0048] Furthermore, when preparing high-temperature Bupleurum humus koji, it is preferred that a temperature-controlled fermentation step is further included before the natural fermentation, and the temperature of the temperature-controlled fermentation is preferably 60-70°C, more preferably 62-68°C, and the time of the temperature-controlled fermentation is preferably 5-8 days, more preferably 6-7 days. The temperature-controlled fermentation of the present invention is preferably provided with heat by a thermostat. The present invention performs natural fermentation after the temperature-controlled fermentation, and when preparing high-temperature Bupleurum humus koji, the time of natural fermentation is preferably 23-28 days.

[0049] The present invention also provides the Bupleurum salsa koji prepared by the preparation method. The Bupleurum salsa koji provided by the present invention can significantly improve the activities of saccharifying enzyme and liquefying enzyme, effectively shorten the fermentation time of rice wine; and contains a variety of biologically active substances. The rice wine brewed using the Bupleurum salsa koji has potential health-care effects.

[0050] The invention also provides the application of the northern bupleurum maltose in preparing yellow wine.

[0051] The invention provides a method for preparing yellow rice wine, comprising the following steps: 1) soaking, rinsing and steaming millet and then airing it; 2) mixing the aired millet with the northern bupleurum malt and koji, saccharifying, and when the temperature of the product is fermented to above 30-36 DEG C, covering and harrowing; 3) separating the solid and liquid of the fermented mash, decocting the liquid component at 80-83 DEG C for 10-15 minutes to obtain liquor; 4) blending and filtering the liquor and then filling and sterilizing it to obtain the yellow rice wine.

[0052] In the present invention, millet is spread out to dry after being soaked, rinsed and steamed. The millet is preferably immersed in water for soaking, and the soaking time is preferably 12h to 72h, preferably soaked until the rice grains are loose when rubbed by hand. In the present invention, the rinsed rice is preferably rinsed with water, and the water flowing out from the bottom of the basket is preferably clear and has no white liquid, and the water is drained. In the present invention, the steamed rice is preferably placed in a steamer after the water is drained, the rice surface is smoothed, and steamed at 95 to 105°C for 25 to 35 minutes, and all steamed thoroughly. The steamed rice is preferably steamed until the rice grains are cooked but not rotten and have no hard core. After the steaming of the rice is completed, the rice is spread out to dry, preferably to 28 to 32°C.

[0053] The present invention mixes the millet, northern bupleurum malt and northern bupleurum extract after airing, mixes the mixture, puts the mixture into a vat and ferments; when the product temperature is fermented to above 30°C to 36°C, covers and starts harrowing management. In the present invention, millet, northern bupleurum malt and northern bupleurum extract are mixed (i.e., mixed with koji), and the mass ratio of the millet, northern bupleurum malt and northern bupleurum extract is (20-30): (4-6): (8-10), preferably (22-28): 5:9, and more preferably 25:5:9. The present invention puts the mixture into a vat and ferments after mixing with koji; the temperature of the mixture is preferably 28-32°C, more preferably 29-31°C, and the fermentation process includes a saccharification stage, and the time of the saccharification stage is preferably 40-60h. When the product temperature is fermented to above 30°C to 36°C, covers are pressed and a harrowing management mode is adopted. The present invention does not specifically limit the specific steps of the harrowing management, and the conventional harrowing management mode in the art can be adopted. Purpose of raking: Raking is an important part of the rice wine fermentation process. Its main purpose is to control the temperature of the fermented mash, ensure the uniformity and stability of the fermentation process, and promote the activity of fermented microorganisms, thereby improving the quality of the rice wine. Timing of raking: Raking is usually carried out in the pre-fermentation stage, and it starts when the temperature of the fermented mash rises to 30-36°C. Operation of raking: Raking includes stirring the fermented mash to promote the dissipation and uniform distribution of heat. In actual operation, manual or mechanical methods are used to turn the raking to ensure the uniformity of the fermented mash. Temperature control: The temperature of the fermented mash needs to be closely monitored during the raking process to prevent excessive temperature from inhibiting or killing the fermented microorganisms and affecting the fermentation effect of the rice wine. Frequency of raking: The frequency of raking depends on the temperature change of the fermented mash and the progress of fermentation. In the early stage of fermentation, more frequent raking may be required to control the temperature; as the fermentation progresses, the frequency of raking can be gradually reduced. Safety production specifications: During the raking process, safety production specifications must also be observed to ensure the safety of operators and the hygiene standards of rice wine production.

[0054] The present invention separates the solid and liquid of the mash after fermentation, and the liquid component is decocted at a temperature of 80°C to 83°C for 10min to 15min to obtain liquor. In the present invention, the solid-liquid separation is preferably performed by gauze filtration. In the specific implementation of the present invention, there is a circular hole near the bottom of the fermentation cylinder. The circular hole in the cylinder is blocked before the cylinder is put into fermentation, and the circular hole outside the cylinder is blocked with a special wooden wedge to prevent leakage. After the fermentation is completed, a pipe is used to connect the circular hole, and the liquor flows out and is filtered through gauze.

[0055] After obtaining the liquor, the present invention blends and filters the liquor and then fills and sterilizes it to obtain yellow wine. The present invention does not specifically limit the specific operations of blending, filtering and filling and sterilizing, and conventional operations in the art can be used. In the specific implementation process of the present invention, the specific steps of blending are as follows: First round of liquor (original liquor): liquor obtained by filtering after the first fermentation in the vat. There is a circular hole near the bottom of the lower part of the fermentation vat. Before the fermentation in the vat, bupleurum filter residue is placed at the circular hole position in the vat to block it. The circular hole outside the vat is blocked with a special wooden chip to prevent leakage. After the fermentation is completed, the circular hole is connected with a pipeline, and the liquor flows out and is filtered through gauze. Second round of liquor: After filtering the first round of liquor, add northern bupleurum extract to the vat (it must be submerged in the raw materials, 4 / 5 of the vat), and filter it by the first filtration method after 24 hours; third round of liquor: obtained by operating according to the method of the second round of liquor; the first, second and third rounds of liquor are mixed in equal proportions.

[0056] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] Example 1

[0058] A method for preparing medium-temperature Bupleurum chinense wheat koji, comprising the following steps:

[0059] a. Boil Radix Bupleuri in water for 30 minutes, with the ratio of Radix Bupleuri to water being 2kg:10L; filter the filtrate through gauze to obtain Radix Bupleuri extract;

[0060] b. Grind the wheat into the shape of "plum petals" which is most suitable for fermentation, making sure the core is rotten but the skin is not;

[0061] c. Wetting and mixing the cracked wheat with 20% of Bupleurum chinense extract for 20 minutes, wherein the specific amount of the Bupleurum chinense extract is based on the standard of kneading into a ball by hand without dripping water, the temperature of the Bupleurum chinense extract is 30° C., and the mixture is made into a brick shape of 18 cm×18 cm×10 cm;

[0062] d. Place the koji bricks in the fermentation room, with a distance of 2 cm between two adjacent koji bricks, and separate the koji bricks with wheat straw and mugwort for fermentation; ferment naturally for one month.

[0063] e. Take out the fermented koji bricks in step d and spread them out to dry until the moisture content of the final Bupleurum chinense koji is less than 14%, thereby obtaining the medium-temperature Bupleurum chinense koji.

[0064] Example 2

[0065] The preparation method of high-temperature Bupleurum chinense wheat koji comprises the following steps:

[0066] a. Boil Radix Bupleuri in water for 30 min, the ratio of Radix Bupleuri to water is 2 kg: 10 L, filter and retain the filtrate to obtain Radix Bupleuri extract;

[0067] b. Grind the wheat into the shape of "plum petals" which is most suitable for fermentation, making sure the core is rotten but the skin is not;

[0068] c. Wetting and mixing the cracked wheat with 18% of Bupleurum chinense extract for 10 min, wherein the specific amount of the Bupleurum chinense extract is based on the standard of kneading into a ball by hand without dripping water, the temperature of the Bupleurum chinense extract is 25, and the mixture is made into a brick shape of 18cm×18cm×10cm;

[0069] d. Place the koji bricks in the fermentation room, with a distance of 2 cm between two adjacent koji bricks, and separate the koji bricks with wheat straw and mugwort for fermentation. During the first 7 days of fermentation, the temperature of koji fermentation is controlled at 60-70℃ by a thermostat, and natural fermentation is subsequently adopted for a total of one month.

[0070] e. Take out the fermented koji bricks in step d and dry them in the sun until the moisture content of the final Bupleurum chinense koji is less than 14%, thereby obtaining high-temperature Bupleurum chinense koji.

[0071] Example 3

[0072] The method for producing yellow wine by medium-temperature northern bupleurum koji fermentation prepared in Example 1 comprises the following steps:

[0073] (a) Soaking rice: Soak all the millet in water for 24 hours until the rice grains fall apart when rubbed by hand.

[0074] (b) Rinse the rice: Rinse the rice with water until the water flowing out from the bottom of the basket is clear and has no white liquid, then drain the water.

[0075] (c) Steaming rice: Place the drained rice into a steamer, smooth the rice surface, and steam it thoroughly at 105°C for 30 minutes. Keep the steaming temperature for 15 to 30 minutes until the rice grains are cooked but not mushy and have no hard core.

[0076] (d) Drying the rice, mixing with koji, putting the rice into a jar, and fermenting: spread the steamed rice to dry at 30°C, add the koji and bupleurum extract that have been crushed through a 20-mesh sieve and mix evenly, the mass ratio of millet, bupleurum koji and bupleurum extract is 25:5:9, and the temperature when putting the rice into a jar is controlled at 28°C ~ 32°C; seal the jar with gauze, and after saccharification for 48 hours, cover the jar when the product temperature ferments to above 30°C ~ 36°C, and adopt the open harrow management mode.

[0077] (e) Pressing, decocting and aging: After fermentation, the mash is separated into solid and liquid, decocted at 83°C for about 15 minutes and aged in a ceramic jar for one month.

[0078] (f) Blending, filtering, filling and sterilization: The wine is blended and stirred evenly, filtered and then filled and sterilized.

[0079] Specific steps of blending:

[0080] First round of wine (original wine): the wine obtained by filtering after the first fermentation in the tank. There is a round hole near the bottom of the fermentation tank. Before fermentation in the tank, place Bupleurum filter residue in the round hole to block it. The round hole outside the tank is blocked with a special wooden chip to prevent leakage. After fermentation, a pipe is used to connect the round hole. After the wine flows out, it is filtered through gauze.

[0081] Second round of wine: After filtering the first round of wine, add Bupleurum chinense extract into the tank (it must cover the raw materials, 4 / 5 of the tank), and filter it using the first filtering method after 24 hours;

[0082] Third round wine: obtained by following the method of second round wine;

[0083] Mix the wine from rounds 1, 2, and 3 in equal proportions.

[0084] Example 4

[0085] The method for producing rice wine by fermenting the high-temperature northern bupleurum malt koji prepared in Example 2 is carried out according to the method of Example 3, except that the "medium-temperature northern bupleurum malt koji" in Example 3 is replaced by "high-temperature northern bupleurum malt koji", and the other steps are the same.

[0086] Comparative Example 1

[0087] Medium-temperature wheat koji without adding Bupleurum chinense

[0088] The preparation method is the same as that in Example 1, except that the Bupleurum chinense extract is not added and the cracked wheat is soaked with water.

[0089] Experimental Example 1

[0090] Physicochemical properties of different types of malt koji

[0091] Experimental groups:

[0092] Samples were collected from different levels of the fermentation room to account for environmental variations. Two batches of fermented koji were collected for each treatment before use. To ensure consistency and reliability, all samples were processed in triplicate, ground into 50 mesh powder under sterile conditions at the AIRTECH clean bench, and stored at -80°C.

[0093] CK: medium-temperature wheat koji without adding Bupleurum chinense (Comparative Example 1);

[0094] MB: medium-temperature wheat koji with Bupleurum chinense added (Example 1), MB1 and MB2 are samples from two batches;

[0095] HB: High-temperature malt koji with addition of Bupleurum chinense (Example 2) HB1 and HB2 are samples from two batches.

[0096] Testing methods for physical and chemical indexes of Maiqu

[0097] Water content and total titratable acidity (TTA) were determined according to QB / T 4257-2011 General Analytical Methods for Liquor. Biochemical indices were tested using test kits from Solarbio Life Sciences (Beijing, China) according to the manufacturer's protocol. Specifically, starch content assay kit (Solarbio, Cat#BC0700), saccharifying amylase assay kit (Solarbio, Cat#BC2670), α-amylase assay kit (Solarbio, Cat#BC0615) and acid protease assay kit (Solarbio, Cat#BC0290) were used for spectrophotometric determination.

[0098] Detection of microbial diversity in malt

[0099] Total DNA extraction and PCR amplification of wheat koji samples

[0100] According to use Genomic DNA was extracted from each sample according to the manufacturer's instructions of the SPIN kit. The quality of DNA extraction was assessed by 1.0% agarose gel electrophoresis, and the concentration and purity of DNA were determined by NanoDrop2000 UV-vis spectrophotometer to ensure that it met the requirements. Primers 338F (5'-ACTCCTACGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGTWTCTAAT-3') were used to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene, and primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') were used to amplify the ITS1 variable region of fungi. The PCR amplification process of the gene was as follows: initial denaturation at 98°C for 2 minutes; denaturation at 98°C for 15 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, final extension at 72°C for 5 minutes, and incubation at 10°C. A total of 25-30 cycles were performed. Finally, the library was constructed on the Novasek PE250 platform and double-end sequencing was performed. Sequencing was completed by Shanghai Paisheng Biotechnology Co., Ltd.

[0101] Sequencing sequence biological information analysis

[0102] (1) Sequence denoising

[0103] In QIIME2 software (2019.4), qiime cutadapttrim-paired was first called to remove the primer fragment of the sequence and discard the sequence of the unmatched primer; then qiime dada2 denoise-paired was used to call the DADA2 method (Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: high-resolution sample inference from illumina amplicon data [J]. Nature Methods, 2016, 13 (7): 581–583. DOI: 10.1038 / nmeth.3869) for quality control, denoising, splicing, and chimera removal. Each library was analyzed according to the above steps. After denoising all libraries, the ASVs feature sequences and ASV tables were merged, singletons ASVs were removed, and clustered with 100% similarity. Sequence length distribution statistics were used to calculate the length distribution of high-quality sequences contained in all samples using R language scripts.

[0104] (2) Species taxonomic annotation

[0105] Using QIIME2 software, the Silva database was used for the bacterial 16S rRNA gene.

[16] (Release 132, http: / / www.arb-silva.de; for fungal ITS sequences, the UNITE database was used

[17] (Release 8.0, https: / / unite.ut.ee / ) was compared with the reference sequence database and species annotation was performed.

[0106] Determination of Volatile Substances in Malt Koji

[0107] Volatile compounds were determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) in triplicate. The method of Chen et al. (Chen X, Wang L, Zhang Y-G, et al. A comparison of microbial communities and volatile compounds in wheat qu from different geographic locations [J]. LWT, 2021, 148 (111752). DOI: 10.1016 / j.lwt.2021.111752) was slightly modified.

[0108] HS-SPME conditions: The whole piece of malt koji collected was crushed and passed through a 50-mesh sieve. After being mixed evenly by quartering, 0.5 g of malt koji sample was added to a 15 mL SPME headspace bottle, and then 5 mL of saturated NaCl solution and 10 μL of internal standard (concentration of 82.07 mg / L, 2-octanol, Sigma-Aldrich, USA) were added. Then, the mixture was balanced in a water bath at 50°C for 10 min, and an aged extraction head (50 / 30 μm DVB / CAR / PDMS) was inserted into the headspace bottle for extraction and adsorption for 50 min.

[0109] GC-MS conditions: Volatile compounds were analyzed using a gas chromatography-mass spectrometry system (TRACE1310-ISQ, Thermo Fisher Scientific, San Jose, CA, United States) equipped with a TG-WAX capillary column (60 m × 0.25 mm × 0.5 μm film thickness, Thermo), and the compounds were thermally desorbed in the gas chromatograph injector at 240 °C for 5 min. The carrier gas was helium (purity 99.999%) with a flow rate of 1 mL / min. The gas chromatograph was operated at 50 °C for 10 min, then heated to 180 °C at a rate of 3 °C / min and maintained for 6 min. Splitless injection; electron ionization source; electron energy 70 eV; injection port temperature, transfer line temperature, and ion source temperature were 240 °C, 180 °C, and 250 °C, respectively; the mass scan range was 50-350 m / z.

[0110] According to the chromatographic peaks obtained by GC-MS analysis, the results with both positive and negative matching degrees > 700 were screened out, and the retention index (RI) of all detected volatile components was calculated by analyzing the retention time (RT) of C7-C24 normal alkanes. Based on the NIST14 database, the metabolites of the samples were qualitatively analyzed by mass spectrometry (absolute value of difference < 20), and the quantitative analysis was performed by comparing the peak area with the internal standard to obtain the content of the aroma components in μg kg -1 .

[0111] That is: aroma component content = aroma component peak area × internal standard content / internal standard peak area.

[0112] Data analysis

[0113] SPSS27.0 (IBM SPSS Inc., USA) was used for significance analysis, with a significance level of P<0.05 (ANOVA). Physicochemical and volatile compound data were expressed as mean ± standard deviation (n = 3). The differences in volatile metabolite profiles and microbiota were visualized using Origin 2022 and R (v.4.1.3). Adonis similarity analysis in R was used to assess the significance of PCA and PCoA among different groups. Procrustes analysis in R was used to examine the relationship between volatile flavor compounds and bacterial / fungal community composition, and Spearman correlation analysis was used for network analysis and visualization by R.

[0114] Experimental Results

[0115] The results are shown in Table 1, reflecting the physical and chemical properties of the three kinds of malt koji. The water content of malt koji is mainly determined by factors such as brewing temperature, fermentation and storage time. Lower water content can reduce the growth and metabolism of microorganisms and make malt koji easier to store. The water content of malt koji ranged from 10.02% ± 0.05 (HB1) to 12.57 ± 0.03 (CK), indicating good storage. The total titratable acidity (TTA) of the MB group was the highest (0.86-0.90 mmol / 10 g), which met the quality standard of the first-grade koji, followed by the HB group, and the CK group was the lowest. Starch, as a key energy source in the growth and metabolism of malt koji microorganisms, can measure the level of fermentation. The results showed that the starch content of the MB and HB groups was significantly lower than that of the CK group, indicating that the microbial activity of the two groups was more active. The higher enzyme activity in the malt koji of Bupleurum chinense can effectively utilize the raw materials, thereby improving the wine yield. The results showed that there were significant differences in enzyme activity between samples of different groups. Compared with the CK group, the MB and HB groups showed higher saccharifying amylase and α-amylase activities, which were 687.67-795.21 U / g and 0.56-1.32 U / g, respectively. In contrast, the acid protease activity of these groups was the lowest. The differences in microbial populations in different types may be the main reason for these differences.

[0116] Table 1 Analysis of basic physical and chemical indicators of malt koji

[0117]

[0118] Note: Different letters in the same column indicate significant differences (P<0.05).

[0119] The DNA of the malt koji samples was extracted, its quality and purity were determined, and the OD260nm / OD280nm and OD260nm / OD230nm values ​​were observed (Table 2). It can be seen that the OD260nm / OD280nm value of the sample DNA was between 1.84 and 2.02, the OD260nm / OD230nm value was between 0.95 and 1.93, and its mass concentration was between 25.07 and 249.49 ng / μl, indicating that the extracted DNA concentration was relatively pure, so that it could be used for the bacterial and fungal diversity analysis of malt koji.

[0120] Table 2 Analysis of DNA mass concentration in wheat koji

[0121]

[0122] The ITS1 and 16S rRNA V3-V4 regions of the five collected wheat koji samples (CK, MB1, MB2, HB1, HB2) were amplified, and the results were as follows: Figure 1 As shown in the figure, the PCR amplification product bands of all samples had good specificity and integrity, without other mixed bands, and the control group (CG) had no amplified bands, thus meeting the requirements for subsequent library construction.

[0123] Analysis of Maiqu sequencing samples

[0124] The extracted DNA of Maiqu samples was sequenced using the Illumina Miseq sequencing platform, and the original bacterial sequences corresponding to Maiqu CK, MB1, MB2, HB1, and HB2 samples were 113236, 84082, 80883, 95421, and 81762, respectively. After primer removal, quality filtering, denoising, splicing, and chimera removal, 71305, 59177, 55478, 61779, and 44403 high-quality reads were obtained, with an average base length of 421.24 bp, 429.93 bp, 425.75 bp, 430.47 bp, and 430.31 bp, respectively. The original fungal sequences corresponding to the Maiqu CK, MB1, MB2, HB1, and HB2 samples were 117,344, 155,796, 141,601, 128,794, and 139,550, respectively. Similarly, 95,541, 125,033, 126,003, 103,817, and 104,093 high-quality reads were obtained, with average base lengths of 267.96 bp, 257.49 bp, 261.31 bp, 256.77 bp, and 271.85 bp, respectively.

[0125] Combined with the sequencing depth estimation, it shows that ( Figure 2 ), with the increase of sequencing depth, the number of species found also increased, and the Shannon curve of each sample has entered a plateau period, indicating that the sequencing depth is large enough to obtain the diversity characteristics of most bacteria and fungi in the sample. It can be seen that although new bacterial phylotypes may be discovered with the increase of sequencing amount, the microbial diversity of the sample no longer changes with the increase of sequencing amount, indicating that the sequencing amount is sufficient and reasonable. Therefore, the sequencing amount of koji bacteria and fungi in this study meets the requirements of subsequent bioinformatics analysis.

[0126] Analysis of Microbial Diversity in Wheat Koji

[0127] Analysis of α-diversity of microorganisms in wheat koji

[0128] Shannon, Chaol, and Simpson indices are alpha diversity indices commonly used to describe species diversity in microbial communities. The larger the Shannon index, the higher the species diversity in the community, and vice versa; the larger the Chao1 index, the higher the species richness of the community. Coverage reflects the coverage of low-abundance species in the sample by sequencing; the higher the coverage rate, the lower the probability that the sequence in the sample has not been measured; if the coverage rate is greater than 97%, it proves that the sampling is reasonable. By calculating the diversity index of the samples, it was found that the Goods coverage values ​​of all samples exceeded 99%, which means that the sequencing data has a high coverage of the species. Figure 3 As shown in the figure, different types of malt koji have obvious differences in α-diversity index. The order of species richness of bacteria is HB>MB>CK, and the order of species richness of fungi is MB>CK>HB. However, due to the influence of the dominance of specific species, the two diversity indices show different trends. Nevertheless, HB shows higher species richness and uniformity in the bacterial community. These differences may be related to the koji making process, production raw materials and surrounding environment of the wine company, or the dominant bacteria replace non-functional species during the production and fermentation process.

[0129] Analysis on β-diversity of koji microorganisms

[0130] To evaluate the differences in microbial community structure among different samples, principal coordinates analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) were performed using the Bray-Curtis distance matrix. Figure 4 The results showed that the bacterial and fungal microbial community structures of different wheat koji samples were significantly different, and the fungi were larger than the bacteria (F 细 =8.632, F 真 =16.308; P = 0.001), and the proportion of the total variation explained by the model was 61.63% and 76.1%, respectively (R 2 细 =0.59, R 2 真 =0.731), indicating that there are significant differences in the microbial communities between different samples. The PCoA score graph shows that there is no overlap between the groups, indicating that the similarity between the MB and HB microbiota is minimal. At the same time, the bacterial communities in the MB samples have higher dispersion, while the bacterial communities in the HB samples are more tightly clustered, indicating that the microbial communities in the HB group have greater similarity. The confidence ellipse also confirms that the fungal community in MB varies more than that in HB. The changes in bacterial and fungal microbial communities in different batches of MB koji may be due to the empirical amount of Bupleurum chinense added. Heat treatment can reduce the heterogeneity of the microbial communities of different batches of Bupleurum chinense koji.

[0131] Analysis of the microbial community structure of wheat koji

[0132] According to the results of species taxonomy analysis, the number of species of bacteria and fungi in each sample at different levels was counted, and the results are shown in Table 3. The 16SrRNA sequences obtained were annotated to 15 phyla and 175 genera, while the ASVs obtained from the ITS sequences were annotated to 4 phyla and 75 genera, among which the phyla and genera with an average relative content of >1.0% were defined as dominant phyla and genera. This study further analyzed the bacterial and fungal groups in the five wheat koji at the dominant phylum and genus levels.

[0133] Table 3 Bacterial and fungal sequencing quality evaluation and ASV biological annotation classification

[0134]

[0135]

[0136] Phylum-level analysis of microbial composition

[0137] The bacterial phyla detected in the samples were Firmicutes, Actinobacteria, Proteobacteria, Cyanobacteria, Bacteroides, Deinococcus-Thermus, Verrucomicrobia, Gemmatimonadetes, Chloroflexi, etc. At the fungal phylum level, a total of 4 fungal phyla were detected, namely Ascomycota, Basidiomycota, Mucoromycota and Rozellomycota.

[0138] like Figure 5As shown in the results, both treatments caused significant changes in bacterial community structure at the phylum level, while the effects on fungal phyla were relatively small. Among the 175 bacterial ASVs identified, most of them belonged to the phyla Firmicutes, Actinobacteria, and Proteobacteria. The 75 fungal ASVs were dominated by Ascomycota, Mucor, and Basidiomycota. These dominant phyla were common in various wheat koji, among which Actinobacteria was the dominant phylum in CK samples (MRA: 65.63%), but decreased significantly in MB and HB samples, which were 3.58% and 0.57%, respectively. The number of Proteobacteria also decreased, but the relative abundance in HB (5.10%) was higher than that in MB (1.15%). In contrast, Firmicutes increased with the addition of Bupleurum chinense and became the dominant phylum in MB and HB, reaching 95.16% in MB. In all wheat koji samples, the dominant fungal phylum was Ascomycota, with a relative abundance ranging from 89.32% to 99.92%. Although other dominant bacterial phyla were also distributed in different wheat koji samples, their relative abundances were different.

[0139] Genus-level analysis of microbial composition

[0140] Dominant genera, defined as genera with a mean relative abundance above 1%, showed variability across samples, even within the same treatment group ( Figure 6 At the genus level, there were 12 bacterial genera with a relative abundance greater than 1% in the five samples, including Bacillus, Staphylococcus, Saccharopolyspora, Kroppenstedtia, Weissella, Pantoea, Pediococcus, Corynebacterium, Pseudonocardia, Enterobacter, Kosakonia, and Enterococcus. The main fungal genera with a relative abundance greater than 1% in the samples were Aspergillus, Thermomyces, Rasamsonia, Thermoascus, Microascus, Rhizopus, Pichia, Lichtheimia, Blumeria,

[0141] Wallemia, Alternaria, and Monascus. Bacillus was the most abundant bacterial genus in wheat koji, especially in MB and HB samples, with its abundance ranging from 1.37% to 92.12%. Staphylococcus was the second most abundant bacterial genus, ranging from 0% to 75.18%, mainly appearing in CK and MB samples. Other dominant bacterial genera varied depending on the treatment methods. In terms of fungal communities, Aspergillus was found in all samples, especially in CK and MB samples, accounting for 19.49±0.81% and 63.18±21.27% of the total fungal sequences, respectively. The analysis results showed that there were significant differences in the composition of dominant fungal genera between MB and HB samples.

[0142] Representative bacteria and fungi in different types of malt koji

[0143] The Venn diagram showed that there were 55 bacterial ASVs in all wheat koji types, accounting for 20.91%, 5.80% and 4.75% of the total bacterial communities in CK, MB and HB, respectively ( Figure 7 A in MRA). More unique bacterial ASVs were found in MB (787, 82.93%) and HB (989, 85.41%) compared with CK (155 ASVs, MRA: 58.94%). For fungi, 37 ASVs were shared among the wheat types, accounting for 22.08%, 5.35%, and 11.64% of the fungal communities in CK, MB, and HB, respectively ( Figure 7 F in Figure 3). The MB group showed the most unique fungal ASVs (269, 84.59%), the CK group had 38, and the HB group had 115. The differences in community composition were consistent with the diversity index.

[0144] The linear discriminant analysis effect size (LEfSe) was used to identify the microbial community taxa with a relative abundance greater than 0.1% and statistically significant in the three wheat kojis. At the bacterial genus level, CK enriched biomarkers including Saccharopolyspora, Pantoea, Klebsiella, Pseudoncardiaceae, and Enterobacter. MB enriched Staphylococcus, Brachybacterium, Lactiplantibacillus, and Corynebacterium, and HB enriched Bacillus, Kroppenstedtia, Weissella, Pediococcus, Kosakonia, and Enterococcus. Figure 7In the fungal biomarkers. At the fungal genus level, CK was characterized by Thermomyces and Wallemia, MB was characterized by Aspergillus, Microascus, Rhizopus, and Alternaria, and HB was characterized by Rasamsonia, Pichia, Thermoascus, and Rhizomucor. Figure 7 G in.

[0145] To reduce potential bias due to data sparsity, metagenomeSeq was used to compare ASVs with relative abundance greater than 0.1% in sample groups, and the results were presented in the form of Manhattan plots. The horizontal axis classifies ASVs according to taxonomic name (from phylum to genus); the vertical axis represents -log10 (adjusted P-value). The more significant the difference, the higher the position on the y-axis. Each dot or circle in the figure represents an ASV, and its size is proportional to the relative abundance (log2 (CPM / n), where CPM = copies per million, n = sample number. Dashed lines are used to separate significant differences, colored dots or rings represent significant points, and gray circles represent insignificant points. Colored solid points represent ASVs with significant increases within the group. The color of the point is used to identify the phylum-level taxonomic group, which is marked at the bottom of the figure. The top 10 genera with significantly increased scores are highlighted with a gray background and marked at the top of the figure.

[0146] In terms of bacterial composition, MB was enriched in seven core genera compared with CK, among which Bacillus and Staphylococcus represented the majority of ASVs ( Figure 7 C in Figure 2). Compared with MB, HB was enriched in seven core genera, and Bacillus remained dominant ( Figure 7 Although Bacillus and Weissneri were significantly upregulated in both MB and HB, different ASVs were identified between the two groups ( Figure 7 In addition, Weissella and Lactobacillus were present in MB and HB, but not in CK. Corynebacterium, Brachybacteriu, and Brevibacterium were unique to MB, while Kroppenstedtia and Pediococcus were unique to HB ( Figure 7 For fungal composition, differences between CK, MB, and HB were mainly observed within the phylum Ascomycota. Compared with CK, MB was enriched in seven core genera, mainly concentrated in Aspergillus ( Figure 7 For details on the differences, see Figure 7J in the CK. For example, Aspergillus, Alternaria, Blumeria, Microascus, Lichtheimia, and Rhizopus were more abundant in MB but less abundant in HB, with Microascus only present in MB. Rhizomucor was less abundant in MB but more abundant in HB. Thermomyces was absent in MB but prevalent in CK and HB. Pichia and Thermoascus were not detected in CK but showed obvious ASV in MB and HB.

[0147] Analysis of Volatile Substances in Malt Koji

[0148] In order to explore the differences in volatile flavor components of different types of malt koji, the present invention uses headspace solid-phase microextraction (HS-SPME) combined with gas chromatography / mass spectrometry (GC-MS) to identify and analyze the aroma components and relative contents of malt koji.

[0149] A total of 105 volatile substances were identified in the malt koji samples, including 17 alcohols, 11 aldehydes, 6 acids, 9 esters, 12 ketones, 8 phenols, 7 pyrazines, 18 terpenes, 6 alkanes, 2 lactones and 9 other substances; 61, 61, 51, 80 and 50 aroma components were identified in CK, MB1, MB2, HB1 and HB2 respectively, and the contents of aroma components from high to low were CK (2606.99 μg / kg), MB1 (15534.49 μg / kg), MB2 (12789.72 μg / kg), HB1 (13063.17 μg / kg) and HB2 (89717.01 μg / kg). The concentrations and odor activity values ​​(OAVs) of different volatile compounds are shown in Table 4. The types of compounds detected in all samples are mainly alcohols, aldehydes, ketones, esters and terpenes, among which alcohols and terpenes account for the largest proportion; and the content and proportion of different types of compounds vary among different malt koji samples. The aroma component with the highest content is pyrazine, accounting for 9.3% to 83.94% of the relative content of volatiles, followed by alcohols (2.27% to 37.58%), phenols (3.71% to 19.98%) and terpenes (2.43 to 19.70%). It is worth noting that the volatiles produced by MB and HB samples are about five times that of CK samples. Alcohols dominate the CK and MB samples, while pyrazines are particularly prominent in HB samples ( Figure 8 ).

[0150] Heat map cluster analysis further revealed the specific changes in the distribution of volatiles in the samples ( Fig. 9 ), providing a deeper insight into the metabolic changes during the production of malt koji. The types and contents of volatile flavor substances in different types of malt koji vary greatly, but there are 20 common volatile components in the three types of malt koji, namely, n-pentanol, 1-octen-3-ol, n-octanol, 1-nonanol, benzyl alcohol, β-phenylethanol, n-hexanal, trans-2-octenal, n-hexanoic acid, ethyl hexanoate, 2-octanone, methyl heptenone, acetophenone, 2,6-di-tert-butyl-p-cresol (BHT), 2,3,5,6-tetramethylpyrazine, 1,8-cineole, camphor, 4-terpineol, α-terpineol, and n-tetradecane.

[0151] Analysis of key aroma compounds in malt koji

[0152] The content of aroma components cannot be used as a basis for determining the aroma characteristics of samples, so the odor activity value (OAV) is used to evaluate the specific contribution of each compound to the overall aroma. Aroma components with higher OAVs indicate their important contribution to the aroma characteristics and overall flavor of the sample. Volatile compounds with concentrations exceeding their perception thresholds play an important role in the characteristic aroma of the sample. It is generally believed that components with OAV < 1 contribute less to the overall aroma of the sample, while volatile compounds with OAV ≥ 1 are key aroma compounds for analyzing samples. When OAV ≥ 10, it is believed that the aroma component contributes greatly to the overall aroma of tea. Based on the thresholds and property descriptions of aroma components reported in the literature, the OAV of malt koji aroma components was calculated.

[0153] The results showed that there were 46 aroma components with OAV greater than 1 (Table 4), and it is believed that these aroma substances may play an important role in the aroma characteristics of malt koji. Among them, the OAVs of the volatile compounds 1-octen-3-ol, n-hexanal, trans-2-octenal, ethyl hexanoate, 1,8-cineole, and linalool common to the three malt koji were basically higher than 10, and it is believed that these aroma substances may be the characteristic aroma compounds in traditional malt koji in Qingyang area. Based on the principal component analysis (PCA) and hierarchical clustering ( Fig.10 Figures A and B in Figure 3 show that there are significant differences in the aroma profiles of malt koji produced by different processes, especially along the PC1 axis (P < 0.001). These differences are further highlighted in the radar plots ( Fig.10 C), where MB and HB samples exhibited significantly enhanced flavor characteristics in all 9 evaluated descriptors compared to CK. In particular, the HB group showed an increase in caramel characteristics relative to MB, while other aroma characteristics were significantly reduced.

[0154] Analysis of Differential Volatile Compounds in Malt Koji

[0155] To further measure the contribution of each volatile compound to different malt koji, 105 aroma components were used as dependent variables and different types of malt koji were used as independent variables. OPLS-DA was used to effectively distinguish the three types of malt koji samples ( Fig.11 The results show that the cumulative explanation rate of the model is R 2 =0.994, model cumulative prediction rate Q 2 =0.987. To verify the validity of the model, 200 permutation tests were performed on the model. Fig.11 The results in B show that R 2 =0.155, Q2=-0.8, no overfitting phenomenon occurred, indicating that the detected volatile compounds can be used as independent variables to better separate and predict different malt koji samples. In order to further understand the volatiles that distinguish different malt koji, the VIP values ​​of all dependent variables were calculated, and the Duncan test of one-way analysis of variance (ANOVA) was used to screen 35 candidate differential compounds with VIP>1 and P<0.05 ( Fig.11 C).

[0156] Table 4 Identification and relative content of volatile organic compounds in malt koji based on HS-SPME-GC-MS analysis

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Characteristic differences in volatile compounds of malt koji

[0163] The variable projection importance (VIP) of orthogonal partial least squares discriminant analysis (OPLS-DA) was used to determine the candidate differential volatiles among different malt koji samples, and the differential compounds were further screened by analyzing the aroma activity value (OAV). Based on the results of VIP>1 and OAV≥1, 18 substances were finally screened out, including 1-Nonanol, Phenylethyl Alcohol, 2-Methylbutanal, Isovaleric acid, 2-Octanone, Guaiacol, 2,3-Dimethylpyrazine, 5-Ethyl-2,3-dimethylpyrazine, 2,3,5,6-Tetramethylpyrazine, Camphor, α-Terpineol, etc. (some of them are shown in Table 1). Fig.12 ). These compounds were identified as the key volatile compounds that distinguish different malt kojis. Together, they constitute the characteristic aroma quality of malt koji samples and play an important role in distinguishing the aroma characteristics of different malt kojis.

[0164] Among the 18 key substances screened, most are volatile compounds with green, floral or fruity aromas, which usually emit pleasant aromas. In contrast, 2,3-dimethylpyrazine, 2,3,5,6-tetramethylpyrazine, and 5-ethyl-2,3-dimethyl-pyrazine have unique nutty and caramelized aromas, which are particularly prominent in the HB group. From dimethylpyrazine to trimethylpyrazine, the odor also changes from nutty to earthy and / or roasted. When the 5-position of the nitrogen six-membered heterocyclic ring of pyrazine substances is connected to an ethyl group, the odor threshold gradually increases. In addition, the contents of β-phenylethanol, 2-methylbutyraldehyde, and isovaleric acid in the HB group were significantly higher than those in the MB group. Previous research results have shown that β-phenylethanol is one of the key components for evaluating the quality of rice wine. This experiment found that the content of β-phenylethanol in wheat koji was significantly increased after adding Bupleurum chinense and heat treatment, which may provide a new way to improve the quality of rice wine.

[0165] Correlation between microbial communities and volatile compounds

[0166] Procrustes analysis was performed to assess the common trends between microbial community structure and volatile metabolites. Fig.13As shown in Figures A and B, both bacterial and fungal communities showed significant correlations with flavor compounds (P < 0.05), highlighting the key role of these microorganisms in the formation of these compounds. The covariance between fungi and volatile metabolites was lower than that between bacteria and volatile metabolites, indicating that fungi play a more prominent role in the production of volatile flavor compounds.

[0167] To further elucidate the relationship between microbial communities and volatile metabolites, Spearman correlation analysis found 44 significant correlations (|r|>0.9, P<0.05), involving 21 bacterial and 23 fungal interactions with volatile metabolites ( Fig.13 C in ). The correlation network showed that one genus could affect multiple compounds, while one compound could be affected by several genera. Among bacteria, Enterococcus was positively correlated with phenol and 2,3,5-trimethylpyrazine, but negatively correlated with 1-heptanol, 1-octanol, and 1,8-cineole. Pantoea and Klebsiella were also negatively correlated with 1,8-cineole. Kroppenstedtia and Pediococcus were positively correlated with 2-octen-1-ol, benzyl methyl ketone, and 4,5-dimethyloxazole. Among fungi, Microascus was positively correlated with a variety of volatile compounds, especially 1-pentanol, 1-octen-3-ol, hexanoic acid, ethyl hexanoate, and methyl isohexenone, which were significantly correlated. Rasamsonia was positively correlated with benzyl alcohol, 2,3-dimethylpyrazine, TTMP, and 4-terpineol. Thermoascus was positively correlated with 2-octanone. In addition, Alternaria, Monascus, and Rhizomucor showed different correlations with a variety of volatile compounds. For example, Monascus was positively correlated with 2-octanone but negatively correlated with 2-methyldecane. Some volatile compounds, such as phenol, 2,3,5-trimethylpyrazine, and piperitone, were jointly affected by bacterial and fungal genera. 2,3,5-Trimethylpyrazine was strongly positively correlated with Enterococcus and significantly negatively correlated with Alternaria. The above results showed that there were 18 positive correlations between fungi and volatile compounds, which exceeded the 12 of bacteria, which was consistent with the results of Procrustes analysis.

[0168] Fungal function prediction

[0169] Based on the MetaCyc pathway database, PICRUSt2 was used to predict the functional spectrum of fungi in the wheat region. Most of the predicted functional genes were related to biosynthetic pathways (39.66-45.05%), production of precursor metabolites and energy (32.72-37.75%), degradation / utilization / assimilation (13.91-16.32%), cellular processes (5.10-5.42%), and glycan pathways (1.18-1.41%). Fig.14 A).

[0170] Fig.14 Figure B shows the 2-level classification comparison of the functional prediction of wheat koji fungal community. The MB group showed higher activity in many key metabolic pathways, highlighting its potential as an excellent fermentation agent for enhancing the aroma characteristics of wheat, which is a significant improvement over traditional fermentation technology. The HB group showed strong performance in amino acid degradation, which plays a key role in the production of aroma compounds in wheat koji. Bray-Curtis PCoA analysis based on the 3-level pathway is shown in Figure 2. Fig.14 As shown in C. Adonis test showed that the functional maps of wheat koji fungi under different treatments were significantly different (R 2 =0.6027, P = 0.002), which may be attributed to the changes in the fungal community composition.

[0171] Experimental Example 2

[0172] Properties of the yellow wine prepared in Examples 3 and 4

[0173] The yellow wine prepared in Example 3 (MBJ) and Example 4 (HBJ) meets the quality standards of yellow wine products. The color also shows a satisfactory color. In addition, no ethyl carbamate was detected in the products of Example 3 and Example 4. This result shows that the methods of Examples 3 and 4 not only improve the sensory quality of yellow wine, but also significantly improve the safety of the product.

[0174] Physical and chemical indicators of rice wine

[0175] Table 5 Physical and chemical indicators of rice wine prepared in Examples 3 and 4

[0176]

[0177] The analysis results of this study revealed differences in key physical and chemical indicators between the two rice wines. The MBJ sample had higher alcohol content, total acid, volatile acid, fructose and glycerol content, while the HBJ sample was more prominent in reducing sugar, glucose and pH value. These differences may have an impact on the flavor, taste and stability of rice wine, providing a scientific basis for further optimizing the production and quality of rice wine.

[0178] Physical and chemical indicators of rice wine

[0179] Table 6 Pyrazine content of rice wine prepared in Examples 3 and 4

[0180]

[0181] Chinese rice wine contains a wealth of aromatic substances, including a variety of trace components that are beneficial to health, such as pyrazine compounds, aromatic compounds, and terpenes. As an important flavor and health factor in Chinese rice wine, pyrazine compounds, with their extremely low aroma threshold, have built a unique flavor system for Chinese rice wine, and are also beneficial to human health. This study revealed significant differences in pyrazines between the two types of rice wine: only 2,3-dimethylpyrazine and 2,3,5,6-tetramethylpyrazine (Tetramethylpyrazine, TTMP) were detected in the MBJ sample; while the HBJ sample was particularly prominent in the content of pyrazines, especially the content of 2,3,5,6-tetramethylpyrazine, which was more than 10 times higher than that of the MBJ sample. This compound was originally an active pharmaceutical ingredient extracted from the traditional Chinese medicine Chuanxiong, also known as Chuanxiongzine. It is not only the core compound of the flavor of Chinese rice wine, but also has important pharmacological effects. It can dilate blood vessels, inhibit platelet aggregation, improve microcirculation, and increase coronary blood and cerebral blood flow, thereby effectively preventing the occurrence of cardiovascular and cerebrovascular diseases, ischemic stroke, cancer, diabetes and many other diseases.

[0182] Yellow wine color

[0183] Color presentation is one of the important indicators of rice wine quality, and is of great significance for improving the overall sensory experience and market competitiveness of the product. Fig.15 ), not only adds visual appeal to rice wine, but also reflects its inherent chemical composition and the fine regulation of brewing technology.

[0184] Safety index of rice wine

[0185] Ethyl carbamate (EC) is a substance that may be produced in fermented foods and alcoholic beverages, and it poses a potential hazard to human health. In recent years, EC has also become one of the safety indicators of rice wine. After testing two types of rice wine, no ethyl carbamate was found in them. This discovery provides an important guarantee for the safety of rice wine, and also allows consumers to feel more at ease when enjoying this traditional drink.

[0186] It can be seen from the above embodiments that the Bupleurum chinense malt provided by the present invention can significantly improve the activities of saccharifying enzyme and liquefying enzyme, and can effectively shorten the fermentation time of rice wine; the present invention uses Bupleurum chinense malt to ferment millet rice wine, which improves the fermentation speed and wine yield, improves safety, and enhances the sensory quality and health care effect of rice wine.

[0187] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Bupleurum chinense malt, characterized in that: The following steps are involved: 1) mixing Bupleurum chinense with water and extracting to obtain Bupleurum chinense extract; 2) soaking the cracked wheat with the extract of Bupleurum chinense obtained in step 1), and then preparing the mixed materials into koji bricks; 3) fermenting and drying the koji brick to obtain Bupleurum chinense koji; The mass ratio of the mixture of Bupleurum chinense and water is 1:(4-6); The mass ratio of the Bupleurum chinense extract to the cracked wheat is (9-11):

50.

2. The preparation method according to claim 1, characterized in that: Step 1) The extraction temperature is 95-105° C. and the extraction time is 20-40 min.

3. The preparation method according to claim 1 or 2, characterized in that: The soaking temperature in step 2) is 25-35°C.

4. The preparation method according to claim 3, characterized in that: The fermentation in step 3) is natural fermentation.

5. The preparation method according to claim 4, characterized in that: When preparing high-temperature Bupleurum chinense wheat koji, a temperature-controlled fermentation step is also included before the natural fermentation. The temperature of the temperature-controlled fermentation is 60-70° C. and the time of the temperature-controlled fermentation is 5-8 days.

6. The Bupleurum chinense koji prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the Bupleurum chinense and wheat koji described in claim 6 in preparing yellow rice wine.

8. A method for preparing yellow rice wine, characterized in that: The following steps are involved: 1) After soaking, rinsing and steaming the millet, spread it out to dry; 2) mixing the millet after airing, the northern bupleurum malt koji and the northern bupleurum extract according to claim 6, and putting them into a vat for fermentation; when the product temperature reaches above 30° C. to 36° C., covering and harrowing are performed; 3) separating the solid and liquid of the fermented mash, and decocting the liquid component at 80°C to 83°C for 10min to 15min to obtain liquor; 4) The wine is blended, filtered, and then bottled and sterilized to obtain yellow wine.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the millet, northern bupleurum malt and northern bupleurum extract is (20-30): (4-6): (8-10).

10. The preparation method according to claim 8, characterized in that: The saccharification temperature during the fermentation process is 28-32° C., and the saccharification time is 40-60 hours.

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