Novel lactobacillus plantarum fortified yeast and application of novel lactobacillus plantarum fortified yeast in improvement of quality of vinegar yeast

Abstract

The invention discloses novel lactobacillus plantarum fortified koji and application thereof in improving the quality of vinegar koji. The invention provides a method for preparing plant lactobacillus fortified yeast. The method comprises the following steps: (A1) mixing barley and peas, and sterilizing to obtain a base material; (A2) inoculating plant lactobacillus into the base material, adding water, and stirring to obtain a mixture; (A3) preparing vinegar koji by using the mixture, and obtaining the strengthened koji when the moisture content of finished koji reaches 12-15% by mass; the lactobacillus plantarum is the lactobacillus plantarum 9600 ACCC (ACCC 64531), and the lactobacillus plantarum is the lactobacillus plantarum 9600 ACCC 64531, the lactobacillus plantarum, the lactobacillus plantarum, the lactobacillus plantarum, the lactobacillus plantarum and the lactobacillus plantarum. Compared with traditional koji, the prepared enhanced koji has the advantages that the enzyme activities of acid protease, xylanase, cellulase and pectinase of the vinegar koji are effectively improved, so that the aroma production quality of the vinegar koji and the utilization rate of base materials are improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a novel Lactobacillus plantarum-enhanced koji and its application in improving the quality of vinegar koji. Background Technology

[0002] Shanxi vinegar koji is typically made from a 7:3 ratio of barley and peas as a base material, fermented naturally over a period of 1-3 months. Strict control of temperature, humidity, and microorganisms is required. Due to the fermentation action of the koji microorganisms, the temperature in the koji room and the koji itself usually rises. This is accompanied by the formation of beneficial bacteria, the generation of biological enzymes, the effective degradation of the base material, and a decrease in water content. The applicant discovered significant differences in the growth performance and enzyme production capacity of different koji microbial strains, leading to variations in the saccharification power, aroma, and raw material utilization of the koji, thus reducing its quality and causing incalculable losses to the brewing industry.

[0003] To improve the quality of Shanxi vinegar koji, based on the inheritance of traditional fermentation technology, microbial technology is used to optimize and innovate the koji. The resulting enhanced koji not only improves the product, but also helps to save grain consumption and reduce production costs, thereby enhancing the flavor characteristics of Shanxi vinegar koji. Summary of the Invention

[0004] The purpose of this invention is to provide a novel Lactobacillus plantarum-enhanced koji and its application in improving the quality of vinegar koji.

[0005] In a first aspect, the present invention claims a method for preparing Lactobacillus plantarum-enhanced broth.

[0006] The method for preparing *Lactobacillus plantarum* enhanced koji claimed in this invention may include the following steps:

[0007] (A1) Barley and peas are mixed and sterilized to obtain the substrate; (A2) Inoculate the base material with Lactobacillus plantarum, add water, and stir evenly to obtain a mixture; (A3) The vinegar koji is prepared using the mixture according to the traditional process. When the moisture content of the koji reaches 12-15% by mass, the plant lactobacillus-enhanced koji is obtained. The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

[0008] Furthermore, in (A1), when preparing the base material, the mass ratio of barley to peas can be 7:3, and the barley is pulverized to 40 mesh.

[0009] Further, in (A2), inoculating the substrate with *Lactobacillus plantarum* means inoculating 10 *Lactobacillus plantarum* per gram of the substrate. 4 The plant lactobacillus mentioned in CFU.

[0010] Further, in (A2), the amount of water added is such that the water content of the mixture is 40% by mass.

[0011] Furthermore, in (A3), the preparation of vinegar koji using the mixture according to traditional processes may include the following stages: mold application, mold drying, damp firing, high-temperature firing, post-firing, and koji formation.

[0012] In some embodiments of the present invention, the "molding" refers to the initial stage of koji making. After the koji blanks are arranged in the room, beneficial microorganisms are promoted to grow on the surface of the koji blanks by covering them with reed mats and closing the windows, forming white spot-like "raw skin". This process lasts for 2-4 days, and the room temperature is maintained at 20-42°C, which helps to retain the moisture of the koji blanks and start microbial metabolism.

[0013] In some embodiments of the present invention, the mold-drying process involves opening windows for ventilation or adjusting the covering to evaporate the surface moisture of the koji blank, thereby lowering the product temperature and preventing the koji skin from becoming too thick or the internal moisture from stagnating. This ensures that the inside of the koji blank is loose and breathable. This process lasts for 5-7 days, with the koji blank being turned over once every day. The room temperature is maintained at 16-40°C. After that, the windows are closed to retain moisture and the koji blank is then ignited.

[0014] In some embodiments of the present invention, the "moistening" process involves raising the temperature of the koji blank to 45-48°C by closing doors and windows and turning the koji, and increasing the day-night temperature difference to 20-25°C, thereby promoting microbial reproduction and enhancing enzyme activity, while simultaneously removing moisture. This process lasts for 3-5 days, creating conditions for subsequent microbial colonization.

[0015] In some embodiments of the present invention, the "high fire" refers to the period when the temperature of the koji cake is stable at 44-46℃, with a day-night temperature difference of 25-30℃, lasting for 3-5 days. The koji cake is turned over once every day. During this stage, heat-resistant microorganisms dominate metabolism, and the mycelium grows inward and further dehydrates, forming the basic structure of the koji cake.

[0016] In some embodiments of the present invention, the post-fire is the final stage of koji making. This process lasts for 5-7 days. By gradually cooling to 20-30°C, the moisture content of the koji core is reduced to 12-15% (by mass) to prevent mold or water accumulation, and finally dry and mature koji blocks are formed.

[0017] In some embodiments of the present invention, the fermentation process involves maintaining the fermentation temperature at 15-30℃ for 1-3 months, after which the activity of microorganisms and various enzymes tends to stabilize.

[0018] In some embodiments of the present invention, the process of preparing vinegar koji using the mixture according to conventional techniques in (A3) takes 60 days.

[0019] Secondly, the present invention claims protection for the *Lactobacillus plantarum* enhanced broth prepared using the method described in the first aspect above.

[0020] Thirdly, the present invention claims protection for the use of the *Lactobacillus plantarum*-enhanced koji described in the second aspect above in the production of vinegar (such as edible vinegar).

[0021] Fourthly, the present invention claims protection for the use of *Lactobacillus plantarum* in the preparation of fortified koji having at least one of the following properties (B1)-(B4): (B1) Increased activity of acidic protease; (B2) Increased xylanase activity; (B3) Increased cellulase activity; (B4) Increased pectinase activity.

[0022] The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

[0023] The enhanced vinegar starter is the *Lactobacillus plantarum* enhanced starter prepared using the method described in the first aspect above.

[0024] In this invention, the increased activity of acidic protease, xylanase, cellulase, and pectinase are all relative to traditional vinegar koji. The traditional vinegar koji is prepared according to conventional processes, and the only difference from the method described in the first aspect above is that step (A2) does not include the step of inoculating with *Lactobacillus plantarum*.

[0025] Fifthly, the present invention claims protection for the use of *Lactobacillus plantarum* in the preparation of fortified koji having at least one of the following properties (C1)-(C2): (C1) Improved utilization rate of vinegar koji base material; (C2) Improved quality of vinegar starter.

[0026] The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

[0027] Furthermore, the quality of the vinegar koji includes both apparent quality (such as improved appearance and aroma) and intrinsic quality (such as increased activity of acidic protease, xylanase, cellulase and pectinase).

[0028] The enhanced vinegar starter is the *Lactobacillus plantarum* enhanced starter prepared using the method described in the first aspect above.

[0029] In this invention, the improved utilization rate of the vinegar koji base material and / or the improved quality of the vinegar koji are both relative to traditional vinegar koji. The traditional vinegar koji is prepared according to traditional processes, and the only difference from the method described in the first aspect above is that step (A2) does not include the step of inoculating with *Lactobacillus plantarum*.

[0030] The present invention has the following beneficial effects: This invention utilizes the addition of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )9600 (ACCC64531) regulates the composition of the vinegar koji microbial community and combines it with traditional fermentation processes to prepare enhanced koji, effectively improving the quality of vinegar koji. Compared with traditional vinegar koji fermentation processes, by regulating the vinegar koji microbial community through the *Lactobacillus plantarum* isolated and screened by the applicant, the enzyme activities of acidic protease, xylanase, cellulase, and pectinase in the vinegar koji are effectively increased, thereby improving the aroma quality of the vinegar koji and the utilization rate of the base material. Attached Figure Description

[0031] Figure 1 Lactobacillus plantarum in Example 1 ( Lactiplantibacillus plantarum Colony morphology on agar plates (9600). Left side: front view; right side: back view.

[0032] Figure 2 Lactobacillus plantarum in Example 1 ( Lactiplantibacillus plantarum )9600 phylogenetic tree.

[0033] Figure 3 This refers to the koji-making process in Example 2.

[0034] Figure 4 The figures show the appearance and cross-sectional views of the enhanced curve (LAB) and the conventional curve (CK) in Example 2 on days 5, 15, 28, and 60. In the figures, the first row from top to bottom represents the enhanced curve, and the second row represents the conventional curve.

[0035] Figure 5 The acidic protease activity of the enhanced koji (LAB) and the conventional koji (CK) after 60 days of curing in Example 3 is shown. Different capital letters indicate significant differences (P<0.05).

[0036] Figure 6 The xylanase activity of the enhanced koji (LAB) and the conventional koji (CK) after 60 days of curing in Example 3 is shown. Different capital letters indicate significant differences (P<0.05).

[0037] Figure 7The cellulase activity of the enhanced koji (LAB) and the conventional koji (CK) after 60 days of curing in Example 3 is shown. Different capital letters indicate significant differences (P<0.05).

[0038] Figure 8 The pectinase activity of the enhanced koji (LAB) and the conventional koji (CK) after 60 days of fermentation in Example 3 is measured. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Lactobacillus plantarum in the following examples ( Lactiplantibacillus plantarum Strain 9600 has been deposited with the Agricultural Culture Collection of China (ACCC, address: Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing, 100081, China) on the application date of this application, with accession number ACCC 64531. The collection date is January 14, 2026. From the date of collection, the public can obtain this strain from the Agricultural Culture Collection of China. ACCC has a dedicated website at http: / / www.accc.org.cn, where the public can directly order the strain online.

[0042] Example 1, Lactobacillus plantarum ( Lactiplantibacillus plantarum Separation and identification of 9600 This embodiment is based on the previous isolation and screening of microorganisms for vinegar koji, which yielded an excellent strain of *Lactobacillus plantarum*. Precise identification was carried out based on colony morphology and molecular tag sequence to explore the feasibility of *Lactobacillus plantarum*-enhanced koji.

[0043] The *Lactobacillus plantarum* used in this embodiment is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum )9600, which is obtained by separating vinegar koji from Yugen Daqu Co., Ltd.

[0044] Strain strain 9600, stored at -80℃, was incubated on MRS solid medium at 37℃ for 2 days. The colony morphology of 9600 was observed, such as...Figure 1 As shown, the colonies have regular edges, are translucent, and have a smooth, moist surface, and are preliminarily identified as lactobacilli.

[0045] To accurately identify strain 9600, colony PCR was used, employing universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for bacterial 16S rDNA, and primers pheS-F (5'-CCGTGAAGAACTGGAACA-3') and pheS-R (5'-CCTAACCCAAAGGCAAAA-3') for the pheS gene. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2. The purity and size of the PCR amplification products were detected by 1.2% agarose gel electrophoresis. PCR products with the correct amplification length were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0046] Table 1. PCR Reaction System

[0047] Table 2. PCR Amplification Procedure

[0048] The partial sequences of the 16S rRNA and pheS gene of strain 9600 are shown in SEQ ID NO:1 and SEQ ID NO:2. Based on the BLAST results, strain 9600 is related to *Lactobacillus plantarum* (…). Lactiplantibacillus The type strain of *Lactobacillus plantarum* Lactiplantibacillus plantarum The 16S rRNA and pheS gene sequences of DSM 20174 showed similarities of 98.31% and 98.76%, respectively. Phylogenetic analysis of the 16S rRNA+pheS gene sequences of strain 9600 and type strains of *Lactobacillus plantarum* was performed using the PAUP maximum parsimony method. Figure 2 As shown, the constructed phylogenetic tree indicates that strain 9600 is related to the type strain of *Lactobacillus plantarum*. Lactiplantibacillus plantarum DSM 20174 is listed in the same branch.

[0049] Based on the combined colony morphology, 16S rRNA, and pheS gene sequence, strain 9600 can be identified as *Lactobacillus plantarum* (Lactobacillus plantarum). Lactiplantibacillus plantarum ( ), and was deposited on January 14, 2026 at the Agricultural Microbiology Center of the China Microbial Culture Collection Committee, No. 12, Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, with accession number ACCC 65431.

[0050] Example 2, Lactobacillus plantarum (Lactiplantibacillus plantarum 9600 Enhanced Curve Test This embodiment focuses on *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The enhanced curd test, in this example, used *Lactobacillus plantarum* (Lactobacillus plantarum) Lactiplantibacillus plantarum )9600, the inoculum was obtained through LB liquid fermentation, with 1×10 per gram of vinegar koji base material. 4 The CFU inoculum is added to the vinegar koji base material, and the koji fermentation and key indicator evaluation are carried out according to the traditional koji-making process.

[0051] The specific preparation method of the enhanced curing material is as follows: (1) Inoculum preparation: Lactobacillus plantarum stored at -80℃ ( Lactiplantibacillus plantarum After activation on plates, 9600 was inoculated onto LB medium and cultured at 30°C and 180 rpm for 3 days, resulting in a biomass of 5.1 × 10⁻⁶. 8 cfu / mL, diluted with sterile water to 10. 7 The cfu / mL value represents the inoculum of strain 9600.

[0052] (2) Preparation of vinegar starter base: Barley and peas are mixed in a mass ratio of 7:3 and crushed to 40 mesh.

[0053] (3) Preparation of enhanced koji: The strain 9600 inoculum prepared in step (1) is used at a concentration of 1×10⁻⁶ per gram of vinegar koji base material. 4 The CFU inoculum is added to the vinegar koji base obtained in step (2), and water is added to a total moisture content of 40% (by mass). Then, the koji is fermented according to the traditional koji-making process. Specifically, the preparation of vinegar koji according to the traditional process includes the following stages: Mold growth: In the initial stage of koji making, after the koji blanks are arranged in the room, operations such as covering them with reed mats and closing the windows promote the growth of beneficial microorganisms on the surface of the koji blanks, forming white spot-like "raw coats". This process lasts for 2-4 days, with the room temperature maintained at 20-42℃, which helps to retain the moisture of the koji blanks and initiate microbial metabolism.

[0054] Mold drying: By opening windows for ventilation or adjusting the covering, the surface moisture of the koji cake evaporates, the temperature of the product is lowered, and the skin of the koji cake is prevented from being too thick or the internal moisture is retained, thus ensuring that the inside of the koji cake is loose and breathable. This process lasts for 5-7 days, with the koji cake turned over once every day. The room temperature is maintained at 16-40℃. After that, the windows are closed to keep the koji cake moist and then ignited.

[0055] Tide-fire: This is the heating stage. The temperature of the koji is raised to 45-48℃ by closing doors and windows and turning the koji, and the temperature difference between day and night is increased to 20-25℃. This promotes the reproduction of microorganisms and enhances enzyme activity, while removing moisture. This process lasts for 3-5 days, creating conditions for subsequent microbial colonization.

[0056] High-temperature stage: This stage is when the temperature of the koji cake is stabilized at 44-46℃, with a day-night temperature difference of 25-30℃, lasting for 3-5 days. The koji cake is turned over once every other day. During this stage, heat-resistant microorganisms dominate metabolism, and the mycelium grows inward and further dehydrates, forming the basic structure of the koji cake.

[0057] Post-fire: This is the final stage of koji making. This process lasts 5-7 days. By gradually cooling to 20-30℃, the moisture content of the koji core is reduced to 12-15% (by weight) to prevent mold or water accumulation, and finally, dry and mature koji blocks are formed. Preparation of koji: Keep koji at 15-30℃ for 1-3 months. After koji is prepared, the activity of microorganisms and various enzymes tends to stabilize.

[0058] Figure 3 This describes the general process for preparing vinegar koji.

[0059] The experiment also included a traditional koji as a control. The only difference between the traditional koji preparation method and the above-mentioned enhanced koji preparation method is that no microorganisms are artificially added to the vinegar koji base.

[0060] (4) Sensory evaluation of the enhanced koji: One piece of enhanced koji was randomly sampled at 5 days (mold removal), 15 days (high heat), 28 days (high heat), and 60 days (finished koji). The pieces were physically broken, and the sensory evaluation was as follows: Figure 4 As shown, referring to QB / T 4257-2011, a sensory evaluation was conducted by 10 professionals on the color and microbial growth of the reinforced surface and cross-section.

[0061] The results are shown in Table 3. It can be seen that the addition of *Lactobacillus plantarum* (…) Lactiplantibacillus plantarum The enhanced 9600 koji is superior to the traditional koji in appearance, aroma, and cross-section.

[0062] Table 3. Sensory evaluations of the enhanced (LAB) and classic (CK) pieces on days 5, 15, 28, and 60.

[0063] Note: Different letters represent P Significant difference at the <0.05 level.

[0064] The results showed that compared with traditional vinegar koji, the addition of *Lactobacillus plantarum* (…) Lactiplantibacillus plantarum 9600 helps to accelerate mold growth, form a red or yellow core in the inner layer, and enhance the appearance, aroma, and cross-sectional index of the koji, thus comprehensively improving the sensory index of the vinegar koji.

[0065] Example 3, Lactobacillus plantarum ( Lactiplantibacillus plantarum ) 9600 Enhanced Curve Key Enzyme Activity Assay This embodiment focuses on *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The key enzyme activity assay of 9600 enhanced curd was conducted to discover *Lactobacillus plantarum* (Lactobacillus plantarum). Lactiplantibacillus plantarum ) 9600 Enhanced Curve Mechanism.

[0066] (1) Preparation of reagents Acetic acid-sodium acetate buffer solution (20 mmol / L, pH 4.6), glucose standard solution (0.00-3.50 mg / mL), L-tyrosine standard solution (100 μg / mL), DNS reagent, soluble starch solution (20 g / L, pH 4.6), xylan solution (20 g / L, pH 4.6), sodium carboxymethyl cellulose solution (10 g / L, pH 4.6), polygalacturonic acid solution (2 g / L, pH 4.6), casein solution (10 g / L, pH 4.6), sodium carbonate solution (0.4 mol / L), trichloroacetic acid solution (0.4 mol / L), hydrochloric acid solution (0.1 mol / L), and sodium hydroxide solution (0.5 mol / L).

[0067] (2) Preparation of crude enzyme solution for enhanced koji In Example 2, the fortified koji was pulverized to 20 mesh using a traditional Chinese medicine processing machine. 1 g of the sample was weighed and placed in 9 mL of pre-cooled 20 mmol / mL acetate-sodium acetate buffer (pH 4.6). The mixture was shaken at 180 r / min for 1 h at 4°C, centrifuged at 8000 r / min for 10 min at 4°C, filtered through a 0.45 μm filter, and brought to a final volume of 10 mL to obtain the fortified koji crude enzyme solution. The traditional koji obtained in Example 2 was used as a control.

[0068] (3) Enzyme activity assay The activities of acidic protease, xylanase, cellulase, and pectinase in the crude enzyme solutions of the enhanced and conventional koji produced in Example 2 were determined and calculated according to the following standards: GB / T23874-2009 (Light Industry Standard of the People's Republic of China), GB 5009.9-2016 (National Standard of the People's Republic of China), GB 1886.174-2024 (National Standard of the People's Republic of China), and SB / T 10317-1999 (Professional Standard of the People's Republic of China, Method for Determination of Protease Activity). Three replicates were set up for each enzyme activity reaction, and the results were taken as mean ± standard deviation.

[0069] Depend on Figure 5 As shown, compared with traditional vinegar koji, the *Lactobacillus plantarum* (Lactobacillus plantarum) prepared in this invention... Lactiplantibacillus plantarum The acidic protease activity of the 9600-enhanced vinegar koji increased by 9.41%.

[0070] Depend on Figure 6 As shown, compared with traditional vinegar koji, the *Lactobacillus plantarum* (Lactobacillus plantarum) prepared in this invention... Lactiplantibacillus plantarum The xylanase activity of the 9600-enhanced acetic acid koji was increased by 81.55%.

[0071] Depend on Figure 7 As shown, compared with traditional vinegar koji, the *Lactobacillus plantarum* (Lactobacillus plantarum) prepared in this invention... Lactiplantibacillus plantarum The cellulase activity of the 9600-enhanced vinegar koji was increased by 21.92%.

[0072] Depend on Figure 8 As shown, compared with traditional vinegar koji, the *Lactobacillus plantarum* (Lactobacillus plantarum) prepared in this invention... Lactiplantibacillus plantarum The pectinase activity of the 9600-enhanced vinegar koji was increased by 4.04%.

[0073] Comprehensive analysis of multiple enzyme activities in the enhanced curing agent shows that: the *Lactobacillus plantarum* prepared in this invention (… Lactiplantibacillus plantarum The 9600 enhanced vinegar koji improves the activity of acidic protease, xylanase, cellulase and pectinase, effectively increasing the utilization rate of protein, hemicellulose, cellulose and pectin in the vinegar koji base.

[0074] In summary, this invention utilizes *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The preparation of 9600 fortified vinegar koji effectively improves the appearance, aroma, and cross-section of the vinegar koji, and increases the activity of acidic protease, xylanase, cellulase, and pectinase in the vinegar koji. This effectively improves the quality, aroma, and utilization rate of the base material of the vinegar koji. Compared with the traditional koji-making process, the targeted shaping of the vinegar koji microbiome is more conducive to improving the quality of the vinegar koji.

[0075] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing *Lactobacillus plantarum*-enhanced koji, comprising the following steps: (A1) Barley and peas are mixed and sterilized to obtain the substrate; (A2) Inoculate the base material with Lactobacillus plantarum, add water, and stir to obtain a mixture; (A3) Prepare vinegar koji using the mixture, and obtain the enhanced koji when the moisture content of the koji reaches 12-15% by mass. The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillusplantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

2. The method according to claim 1, characterized in that: In (A1), when preparing the base material, the mass ratio of barley to peas is 7:3, and the material is pulverized to 40 mesh.

3. The method according to claim 1 or 2, characterized in that: In (A2), inoculating the substrate with *Lactobacillus plantarum* means inoculating 10 *Lactobacillus plantarum* per gram of the substrate. 4 The plant lactobacillus mentioned in CFU.

4. The method according to any one of claims 1-3, characterized in that: In (A2), the amount of water added is such that the water content of the mixture is 40% by mass.

5. The method according to any one of claims 1-4, characterized in that: In (A3), the preparation of vinegar koji using the mixture includes the following stages: mold growth, mold drying, moisture treatment, high heat treatment, post-heat treatment, and koji formation.

6. The method according to claim 5, characterized in that: The process of applying mold includes: arranging the koji blanks prepared using the mixture into a room, adding a covering, and continuing for 2-4 days while maintaining the room temperature at 20-42℃, resulting in white spots forming on the surface of the koji blanks; and / or The process of drying the mold includes: evaporating the surface moisture of the koji blank by opening windows for ventilation or adjusting the covering to lower the product temperature, continuing for 5-7 days, turning the koji blank once every day, and maintaining the room temperature at 16-40℃. and / or The "moistening" process includes: raising the temperature of the koji blanks to 45-48°C by closing doors and windows and turning the koji, and increasing the day-night temperature difference to 20-25°C for 3-5 days; and / or The intense heat includes maintaining the temperature of the koji blanks at 44-46℃, with a day-night temperature difference of 25-30℃, for 3-5 days, and turning the koji blanks once every day. and / or The post-heating process includes: gradually cooling to 20-30℃ for 5-7 days to reduce the moisture content of the core to 12-15% by mass, thus forming a dry and mature koji block; and / or The process of curing the koji involves maintaining the koji at a temperature of 15-30℃ for 1-3 months.

7. The *Lactobacillus plantarum* enhanced broth prepared using the method described in any one of claims 1-6.

8. The application of the *Lactobacillus plantarum*-enhanced koji according to claim 7 in vinegar production.

9. Application of *Lactobacillus plantarum* in the preparation of fortified vinegar koji with at least one of the following properties (B1)-(B4): (B1) Increased activity of acidic protease; (B2) Increased xylanase activity; (B3) Increased cellulase activity; (B4) Increased pectinase activity; The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillusplantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

10. Application of *Lactobacillus plantarum* in the preparation of fortified vinegar koji with at least one of the following properties (C1)-(C2): (C1) Improved utilization rate of vinegar koji base material; (C2) Improved quality of vinegar starter; The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillusplantarum )9600, whose accession number at the China Agricultural Microbial Culture Collection Center is ACCC 64531.

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