A method for preparing selenium-rich aromatic ester old vinegar by using selenium-rich indigenous yeast and active nano-selenium-chitosan compound

Abstract

This invention belongs to the field of vinegar fermentation technology and provides a method for preparing selenium-enriched aged vinegar by combining high-selenium-enriched indigenous yeast with active nano-selenium-chitosan complex. The method uses selenium-enriched sorghum, wheat, wheat bran, and rice husks as raw materials, and uses selenium-enriched soybeans, wheat, and wheat bran as saccharification and fermentation agents. Solid-state alcoholic fermentation and solid-state acetic acid fermentation are carried out using a fermentation agent made from the indigenous brewing yeast CGMCC 34906, the source of aged vinegar. After fermentation, the mixture is smoked and leached to obtain new leached vinegar. Active nano-selenium-chitosan complex is added to the new leached vinegar, and the mixture is then aged, filtered, sterilized, and bottled to obtain selenium-enriched aged vinegar. The vinegar is clear and bright, with a rich and harmonious ester aroma, possessing the unique smoky and aged fragrance of aged vinegar; the sour taste is mellow, rich and long-lasting; total selenium ≥ 0.23 mg / L, total acid ≥ 5.50 g / 100 mL, amino acid nitrogen ≥ 0.30 g / 100 mL, non-volatile acid ≥ 1.80 g / 100 mL, total ester ≥ 3.50 g / 100 mL, and rich in organic acids and volatile aromas.

Description

Technical Field

[0001] This invention belongs to the field of vinegar fermentation technology, specifically relating to a method for preparing selenium-enriched aged vinegar by combining high-selenium-enriched indigenous yeast with active nano-selenium-chitosan complex. Background Technology

[0002] Selenium, as one of the essential trace elements for the human body, has important physiological functions such as antioxidation and immune regulation. Many diseases, including Keshan disease, Kashin-Beck disease, and some cardiovascular diseases, are closely related to selenium deficiency. Due to the scarcity of naturally abundant selenium resources, dietary selenium intake is generally low. Inorganic selenium compounds are highly toxic, and direct ingestion poses health risks. Nano-selenium and organic selenium (especially nano-selenium) have been proven to have significantly lower toxicity than inorganic selenium and are easily absorbed by the body through daily diet, making them safe and reliable forms of selenium supplementation. Yangquan City in Shanxi Province, a major selenium-rich area, possesses high-quality natural selenium-rich soil resources. The sorghum, wheat, bran, and rice husks grown there are naturally enriched with selenium, providing a geographical advantage for obtaining selenium-rich raw materials. Developing selenium-enriched functional foods based on these local selenium-rich crops can fully utilize the advantages of local resources and achieve the natural integration and efficient utilization of selenium.

[0003] Shanxi aged vinegar, a traditional Chinese condiment, is made primarily from sorghum and wheat bran, with rice husks and millet husks as auxiliary ingredients. It uses a starter culture made from barley and peas as a saccharification and fermentation agent. After alcoholic fermentation, it undergoes solid-state acetic acid fermentation, followed by smoking and aging processes. Combining selenium with the traditional brewing techniques of Shanxi aged vinegar allows for convenient selenium supplementation through everyday vinegar consumption, while also enriching the health benefits of aged vinegar. This aligns with current consumer demands for healthy eating, making the development of selenium-enriched vinegar an important direction for the functional upgrading of vinegar, and related technological exploration and product development are receiving increasing attention.

[0004] The invention patent "Production Method of Selenium-Enriched Vinegar" (application number: CN 101397536 B) uses selenium-enriched grains such as selenium-enriched rice, selenium-enriched wheat, selenium-enriched bran, and selenium-enriched rice husks as raw materials, and produces selenium-enriched vinegar through scientific ingredient formulation, alcoholic fermentation, and acetic acid fermentation.

[0005] The invention patent "A production method of selenium-enriched edible vinegar" (application number: CN 107475062 A) involves adding inorganic selenium to the vinegar brewing process, and utilizing its own fermentation bacteria under suitable conditions to convert the inorganic selenium into selenoamino acids that are easily absorbed by the human body, thus forming edible vinegar rich in organic selenium.

[0006] The invention patent "A method for preparing selenium-enriched vinegar" (application number: CN 104109622 B) uses biotransformation technology to convert inorganic selenium in vinegar-making raw materials into organic selenium by adding selenium-enriched yeast, and then further degrades and completely dissolves it in vinegar liquid to obtain selenium-enriched vinegar.

[0007] In summary, existing selenium-enriched vinegar technologies and products mostly rely on selenium-enriched raw materials, directly adding inorganic selenium or exogenous selenium-enriched yeast, and are prepared through alcoholic and acetic acid fermentation. However, there are significant limitations. The selenium content of selenium-enriched raw materials fluctuates greatly due to the growing environment, and the large amount of water used in the vinegar leaching process dilutes the selenium concentration, making it difficult to standardize the selenium content of the product. Inorganic selenium is highly toxic, and improper control of its addition can easily lead to excessive residues. Furthermore, the selenium conversion capacity of naturally fermenting bacteria in the brewing system is limited, and there is a lack of targeted screening and optimization. The added exogenous strains are not well-suited to the specific microenvironment of brewing, their metabolic activity is limited, and the selenium conversion efficiency and alcohol and ester production performance cannot be fully utilized. They are also prone to competitive inhibition with native microorganisms, disrupting the balance of the fermentation community. Summary of the Invention

[0008] This invention provides a method for preparing selenium-enriched aged vinegar using a combination of high-selenium-enriched indigenous yeast and an active nano-selenium-chitosan complex. The method uses local selenium-enriched sorghum, wheat, wheat bran, and rice husks from Yangquan as raw materials, combined with selenium-enriched soybeans, wheat, and wheat bran-based selenium-enriched koji as a saccharification and fermentation agent, supplemented with sodium selenite. Inoculated with indigenous brewing yeast selected from aged vinegar mash, known for its strong selenium-enriching ability and high alcohol and ester production, the process involves solid-state alcoholic fermentation and solid-state acetic acid fermentation in earthen pits. Newly extracted vinegar is obtained through fumigation and leaching, achieving selenium enrichment throughout the brewing process. By adding selenium-enriched raw materials and superior indigenous selenium-enriched yeast, the bioconversion efficiency and accumulation of selenium are significantly improved, while simultaneously promoting the formation of flavor compounds such as esters. Based on the newly extracted vinegar obtained from fermentation, an active nano-selenium-chitosan complex is added. The protective and stabilizing effect of chitosan reduces selenium oxidation and loss, resulting in a final product rich in organic acids and volatile aromas. This method effectively solves the problems of insufficient selenium content, low conversion efficiency, poor strain compatibility, and monotonous flavor in existing selenium-enriched vinegars.

[0009] This invention is achieved by the following technical solution: a method for preparing selenium-enriched aged vinegar using a combination of high-quality indigenous yeast and active nano-selenium-chitosan complex. The method uses selenium-enriched sorghum, wheat, wheat bran, and rice husks as raw materials, and uses selenium-enriched soybeans, wheat, and wheat bran as saccharification and fermentation agents. Solid-state alcoholic fermentation and solid-state acetic acid fermentation are carried out using a fermentation agent made from the indigenous brewing yeast CGMCC 34906, the source of aged vinegar. After fermentation, the mixture is smoked and leached to obtain new leached vinegar. An active nano-selenium-chitosan complex is added to the new leached vinegar, and the mixture is then aged, filtered, sterilized, and bottled to obtain selenium-enriched aged vinegar.

[0010] Further, the specific steps include: Selenium-enriched sorghum and selenium-enriched wheat are crushed to 50-60 mesh and mixed with selenium-enriched bran, rice husks, and rice husks in a mass ratio of 2.5:1:2:2:0.19. Water equal to 140% of the sorghum mass is added and mixed thoroughly. The mixture is allowed to soak for 24 hours. After soaking, the mixture is steamed for 90 minutes, then cooled to 28-32°C. Saccharifying enzyme (0.1% of the sorghum mass) and sodium selenite (0.01%-0.02% of the sorghum mass) are added, along with selenium-enriched bran koji (40% of the sorghum mass) and brewing yeast CGMCC 34906 (0.1-0.5% of the sorghum mass). Water equal to 100% of the sorghum mass is added, and the mixture is mixed thoroughly. The mixture is then placed in an alcohol fermentation tank for solid-state fermentation for 8-20 days, achieving an alcohol content of 8-10%. The alcoholic fermentation was stopped at %vol to obtain the mash. The fermented mash was then divided into acetic acid fermentation tanks, and then 10% of the mash was added to the fermented mash. The fermentation was carried out for 11 to 15 days until the total acid content was 5 to 6 g / 100 mL. The acetic acid fermentation was then stopped. After smoking the mash and leaching the vinegar, new leached vinegar was obtained. Then, active nano-selenium-chitosan complex with a particle size of 50 to 100 nm and a mass-volume ratio of 1% to 2% of the new leached vinegar were added. The mixture was then sun-dried and aged for one year. After filtration, sterilization and bottling, the selenium-enriched aged vinegar was obtained.

[0011] The method for preparing selenium-enriched wheat bran koji is as follows: Selenium-enriched soybeans and selenium-enriched wheat are mixed and crushed at a mass ratio of 1:1. Ten times the mass of soybeans' wheat bran and ten times the mass of soybeans' water are added and mixed evenly to form a mixture. Simultaneously, a nylon bag containing 25% of the soybeans' mass of dried wheat bran and the mixture are steamed together for 40 minutes. After steaming, the mixture is spread out and cooled to 40℃ to form koji material. The sterilized 25% wheat bran in the nylon bag is mixed with 0.3% of the soybeans' mass of Aspergillus oryzae CGMCC 42103 koji seed, and then inoculated into the cooled koji material and mixed evenly. This mixture is then placed in a koji bed, and the room temperature and product temperature are controlled at 32–35℃ throughout the process. The mixture is turned over every 12 hours, for a total of two times. During the first turning, the state of the koji material shows no significant change; during the second turning, white mycelium can be seen growing. After culturing for 36 hours, the mixture is divided into blocks, and ventilation continues for about two days until the koji material matures. It is then dried and stored for later use.

[0012] The preparation method of Aspergillus oryzae CGMCC 42103 spore suspension is as follows: Transfer 4 mL of Aspergillus oryzae CGMCC 42103 spore suspension (10 7 ~10 8(CFU / mL) was injected into a 250 mL Erlenmeyer flask containing 30 g of wheat bran and soybean flour culture medium. The flask was placed in a 30℃ incubator, and the flask was tapped every 12 hours. After 72 hours of incubation, the flask was dried in a 40℃ constant temperature drying oven for 3 hours to obtain Aspergillus oryzae inoculum. The preparation method of wheat bran and soybean flour culture medium was as follows: soybeans were crushed into flour-like powder, and wheat bran and soybean flour were mixed evenly at a mass ratio of 9:1. Water was added at 0.5 times the dry weight of the culture medium and mixed. The mixture was then sterilized in a high-pressure steam sterilizer at 0.1 MPa and 121℃ for 30 minutes. After sterilization, the raw materials were allowed to cool to about 40℃.

[0013] The Aspergillus oryzae CGMCC 42103 is an excellent indigenous strain isolated and screened from Shanxi aged vinegar starter. It has high acid protease-glutaminase activity. After being cultured in soybean bran flour medium for 3 days, its acid protease activity and glutaminase activity were 630.25 U / g and 7.03 U / g, respectively. It also has a rich enzyme system including amylase, β-glucosidase, aminopeptidase, cellulase and xylanase. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

[0014] The preparation method of the selenium-enriched Saccharomyces cerevisiae CGMCC 34906 fermentation agent is as follows: Activated selenium-enriched Saccharomyces cerevisiae CGMCC 34906 is inoculated into PDA liquid culture medium at an inoculation rate of 3%, and then statically cultured at 30 ℃ for 24 h until the cell concentration reaches 10. 9 CFU / mL; collect bacterial cells by centrifugation at 8000 r / min for 10 min, wash the bacterial cells twice with sterile physiological saline, add 3 times the amount of cell mass protectant, and freeze-dry under vacuum to obtain bacterial powder, which is the selenium-enriched brewer's yeast starter, with a viable count ≥10. 10 CFU / g.

[0015] The protective agent formulation is as follows (w / v): 12% maltodextrin, 1.5% sodium caseinate, 6% whey powder, 2.5% lactose, 4% glucose, 0.3% sodium citrate, 0.2% ascorbic acid, 0.1% Tween 80, and the remainder is water.

[0016] The selenium-enriched brewing yeast CGMCC 34906 is an excellent indigenous strain isolated and screened from Shanxi aged vinegar mash. It has a strong selenium enrichment capacity. In selenium-enriched malt extract medium, with an initial selenium concentration of 16 μg / mL and an inoculum amount of 4%, the selenium enrichment capacity reaches 3000 μg / g dry cells after 48 h of shake-flask fermentation at 30℃. It also has excellent fermentation characteristics with high alcohol and ester production. In sorghum juice medium, the alcohol production is 7.85% vol and the ester production is 5.72 g / 100 mL. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

[0017] The preparation method of the active nano-selenium-chitosan complex is as follows: Take a chitosan solution with a mass concentration of 5-15 mg / mL, add 40-60 mmol / L sodium selenite solution to it, and then add an ascorbic acid solution with a concentration of 60-100 mmol / L dropwise to the mixture. The volume ratio of the chitosan solution, sodium selenite solution, and ascorbic acid solution is 1:1:1. Stir continuously during the dropwise addition to obtain a primary active nano-selenium-chitosan complex. Transfer the primary complex into a dialysis bag and dialyze it in sufficient deionized water for 2-3 days. After dialysis, adjust the volume with deionized water to 10 times the initial volume of the chitosan solution. By adjusting the concentration, ensure the uniform dispersion of the composite particles and accurately control the particle size range of the product to finally obtain a high-purity active nano-selenium-chitosan complex with a particle size of 50-100 nm.

[0018] The prepared selenium-enriched aged vinegar is clear and bright, with a rich and harmonious ester aroma, possessing both the smoky and aged fragrance unique to aged vinegar; it has a mellow and long-lasting sour taste; total selenium ≥ 0.23 mg / L, total acid ≥ 5.50 g / 100 mL, amino acid nitrogen ≥ 0.30 g / 100 mL, non-volatile acid ≥ 1.80 g / 100 mL, total ester ≥ 3.50 g / 100 mL, and is rich in organic acids and volatile aromas.

[0019] Compared with the prior art, the present invention has the following advantages: 1. The sorghum, wheat, bran, rice husks, and soybeans used in this invention are all sourced from land grown in Yangquan City, a major selenium-rich area in Shanxi Province. Their total selenium contents are 0.137 μg / g, 0.312 μg / g, 0.156 μg / g, 0.454 μg / g, and 4.775 μg / g, respectively. This provides a natural foundation for the selenium-rich nature of the product from the source. By relying on the selection of raw materials from local high-quality selenium-rich resources, the quality and selenium stability of the product are strictly controlled from the source. This also opens up a path for the efficient transformation of Yangquan's selenium-rich agricultural resources and lays the foundation for the research and development of high-value-added, high-quality products.

[0020] 2. The selenium-enriched yeast used in this invention is the indigenous and superior brewing yeast CGMCC 34906, which was screened from Shanxi aged vinegar mash in the laboratory. In selenium-enriched malt extract medium, its selenium enrichment capacity reaches 3000 μg / g dry cells. It also has excellent fermentation characteristics with high alcohol and ester production. In sorghum juice medium, its alcohol production is 7.85% vol and its ester production is 5.72 g / 100 mL. Compared with conventional commercial Angel yeast, it has significant advantages in fermentation adaptability, selenium conversion efficiency and accumulation of flavor substances in the product, and can be better adapted to the vinegar fermentation system.

[0021] 3. This invention innovatively uses protein-fortified selenium-enriched bran koji as a saccharification and fermentation agent. Selenium-enriched soybeans and selenium-enriched wheat are mixed and ground in a 1:1 ratio, providing a sufficient nitrogen source for the growth and metabolism of Aspergillus oryzae CGMCC 42103. Compared with traditional bran koji that only relies on the nitrogenous substances in the bran itself, the high-quality plant protein in selenium-enriched soybeans can be efficiently utilized by Aspergillus oryzae, significantly improving the activity of key hydrolytic enzymes such as protease, saccharifying enzyme, and amylase. It efficiently degrades the raw materials into nutrient substrates for the selenium-enriched conversion and alcohol ester synthesis of yeast, optimizes the fermentation microenvironment, and lays the foundation for the selenium-enriched function and excellent flavor of the product from the source.

[0022] 4. The Aspergillus oryzae CGMCC 42103 used in the protein-fortified selenium-enriched bran koji of this invention is an indigenous strain with excellent high-yield acidic protease-glutaminase activity isolated and screened from Shanxi aged vinegar koji in our laboratory. It is rich in protease system, containing not only neutral protease but also abundant acidic protease, which can adapt to the acidic environment of the acetic acid fermentation stage of Shanxi aged vinegar. At the same time, it can enhance the umami flavor of the product through the complex action of glutaminase.

[0023] 5. This invention adopts a dual solid-state fermentation process of "solid-state alcoholic fermentation and solid-state acetic acid fermentation". The alcoholic fermentation stage is carried out in an underground tank. The stable micro-oxygen environment of the underground tank and the porous structure of the solid substrate form a synergistic advantage, which slows down the oxygen transfer rate during fermentation, provides suitable conditions for the metabolism of selenium-enriched yeast, and promotes the generation and accumulation of flavor substances such as esters. At the same time, the solid substrate can adsorb and enrich the selenium element in the raw materials, reduce the loss, and enhance the selenium-enrichment conversion activity of native yeast, so as to achieve simultaneous enhancement of flavor and selenium-enrichment function.

[0024] 6. The active nano-selenium-chitosan complex uses highly biocompatible nano-selenium as the core material and the natural polymer chitosan as the carrier. It is prepared through an in-situ reduction-physical coating coupling process to form a stable composite system. Compared to the shortcomings of traditional free nano-selenium, which is prone to agglomeration and oxidation, chitosan can block the interaction between nano-selenium particles. When added to fresh leached vinegar, it maintains structural stability in the acidic fermentation system, significantly increasing and maintaining a long-term stable total selenium content in the finished product.

[0025] 7. This invention uses selenium-enriched raw materials, combined with protein-fortified selenium-enriched bran koji, supplemented with sodium selenite, and inoculated with indigenous brewing yeast strains selected from Shanxi aged vinegar mash with strong selenium enrichment capacity and excellent alcohol and ester production characteristics. After solid-state alcoholic fermentation and solid-state acetic acid fermentation in earthen pits, the mash is smoked, vinegar is extracted, and active nano-selenium-chitosan complex is added. After aging, filtration, sterilization, and bottling, selenium-enriched aromatic aged vinegar is obtained, achieving full selenium enrichment throughout the process. Total selenium ≥ 0.23 mg / L, total acid ≥ 5.50 g / 100 mL, amino acid nitrogen ≥ 0.30 g / 100 mL, non-volatile acid ≥ 1.80 g / 100 mL, total ester ≥ 3.50 g / 100 mL, and rich in organic acids and volatile aromas.

[0026] The brewing yeast of this invention ( Saccharomyces cerevisiae JY2418 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 34906 and deposit date of June 16, 2025. Aspergillus oryzae ( Aspergillus oryzae QM81 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42103 and deposit date of June 16, 2025. Attached Figure Description

[0027] Figure 1 The images show the colony and cell morphology of selenium-enriched yeast strain JY2418 on PDA medium. (Left image: colony morphology; Right image: cell morphology). Figure 2 ITS sequencing phylogenetic tree of selenium-enriched yeast strain JY2418; Figure 3 Results of enzyme activity assays for different Aspergillus oryzae strains; Figure 4 This image shows the colony morphology of Aspergillus oryzae strain QM81 on a PDA plate. Figure 5 This is an ITS sequencing phylogenetic tree diagram of Aspergillus oryzae strain QM81. Detailed Implementation

[0028] The present invention is described below through specific embodiments. These embodiments should be understood as illustrative, not limiting, of the scope of the invention, which is defined solely by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in the embodiments, without departing from the spirit and scope of the invention, also fall within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0030] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The Shanxi aged vinegar mash used in the following examples was sourced from Yangquan Yushengyuan Agricultural Products Development Co., Ltd.; the saccharifying enzymes used in the following examples were purchased from Shandong Longket Enzyme Preparation Co., Ltd.; the commercial selenium-enriched yeast agent used in the following examples was purchased from Angel Yeast Co., Ltd.; and the fungal DNA extraction kit used in the following examples was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0032] Example 1: Screening and identification of superior selenium-enriched yeasts; 1. Screening of superior selenium-enriched yeast strains: Ten yeast strains derived from Shanxi aged vinegar mash and one commercially available selenium-enriched yeast strain were screened for superior overall performance by measuring their alcohol production, ester production, and selenium enrichment capabilities. The commercially available selenium-enriched yeast strain was purchased from Angel Yeast Co., Ltd.

[0033] (1) Determination of the selenium enrichment performance of the strain Eleven yeast strains were activated using PDA liquid medium. Simultaneously, a selenium solution was prepared with sterile water and added to the malt extract medium to achieve a total selenium concentration of 16 μg / mL, thus creating a selenium-enriched malt extract medium. The activated yeast was inoculated into this medium at a rate of 4%, and cultured at 30°C for 48 h, with growth observed. After culture, the cells were collected by centrifugation at 6000 r / min for 10 min, washed multiple times with distilled water, and then freeze-concentrated to prepare dry cells. The selenium content of the dry cells was then determined.

[0034] Total selenium content determination method: The total selenium content was determined by hydride atomic fluorescence spectrometry in GB 5009.93—2017 "National Food Safety Standard - Determination of Selenium in Food".

[0035] The PDA liquid culture medium formula is as follows: 20 g potato, 2 g glucose, 0.2 g peptone, 0.2 g KH2PO4, 0.1 g MgSO4, 100 mL distilled water, sterilized at 121℃ for 20 min.

[0036] The method for preparing the selenium-enriched malt extract culture medium is as follows: dry malt is pulverized to 20 mesh using a pulverizer, malt powder and water are mixed in a 4:1 ratio, dissolved in water at 65 ℃, saccharified for 3-4 h, saccharification is completed by iodine titration, 20 mL of high-temperature amylase is added to the saccharified solution and stirred continuously, and then filtered after boiling. The saccharified solution is filtered through 4-6 layers of gauze, the filtrate is diluted to 5-6 °Brix, pH about 6.4, and sterilized by autoclaving at 121 ℃ for 20 min for later use.

[0037] (2) Determination of alcohol and ester production performance of the strain After activation by inoculating the above 11 yeast strains into PDA liquid medium, they were inoculated into 200 mL sorghum juice medium at an inoculation rate of 4%. After aerobic culture at 30℃ for 24 h, the mixture was sealed with plastic sheeting for anaerobic fermentation for 48 h. The alcohol content of the fermentation broth was determined by distillation, and the total ester content of the fermentation broth was determined by saponification.

[0038] The method for preparing the sorghum juice culture medium is as follows: 200 g of sorghum is crushed into four to six pieces, 800 mL of water and 200 μL of high-temperature amylase are added, and the mixture is cooked at 100 ℃ for 60 min. When the mixture is cooled to 55 ℃, 1 mL of saccharifying enzyme is added. After keeping the mixture at 55 ℃ for 4 h, the mixture is filtered to obtain the filtrate, and then sterilized at 121 ℃ under high pressure for 20 min for later use.

[0039] As shown in Table 1, the selenium-enriched yeast JY2418 exhibits excellent comprehensive fermentation performance, with a total selenium content of 3000 μg / g, a total ester content of 5.72 g / 100 mL, and an alcohol content of 7.85% vol. Compared with commercial selenium-enriched yeast, JY2418 has a selenium enrichment capacity of 3.60 times, an ester production capacity of approximately 2.05 times, and an alcohol production capacity of 60.38 times, indicating that this strain has significant advantages and potential in the production of selenium-enriched foods.

[0040] Table 1. Screening of superior selenium-enriched yeast strains for aged vinegar. 2. Identification of superior selenium-enriched yeasts Morphological identification: A small amount of yeast JY2418 was picked up with an inoculation loop and streaked onto PDA agar plates to isolate single colonies. The colonies were photographed and recorded, and then their cell morphology was observed under a microscope using simple staining. See below for colony and cell morphology of yeast JY2418 on PDA agar. Figure 1 Colony morphology: round, 2.0-3.0 mm in diameter, milky white, with raised hemispherical colonies, neat edges, smooth and moist surface with a slight luster; cell morphology: oval, single and tightly packed.

[0041] ITS Sequencing: Yeast strain JY2418 cells were freeze-ground, and DNA was extracted using a kit for ITS sequencing and strain identification. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. BLAST was employed for sequence homology analysis, and an ITS sequencing phylogenetic tree was constructed. The ITS sequencing phylogenetic tree of yeast strain JY2418 is shown below. Figure 2 Strain JY2418 was identified as *Saccharomyces cerevisiae* (Saccharomyces Saccharomyces cerevisiae It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 34906 and deposit date of June 16, 2025.

[0042] Example 2: Screening and identification of superior Aspergillus oryzae strains 1. Screening of superior Aspergillus oryzae strains: Ten Aspergillus oryzae strains isolated from Shanxi aged vinegar starter were selected in the laboratory. The commercially available Hu Niang AS 3.042 strain was used as a control group. Enzyme activities, including acidic protease, neutral protease, glutaminase, amylase, saccharifying enzyme, aminopeptidase, cellulase, xylanase, and β-glucosidase, were measured. The specific methods are as follows: Screening of Aspergillus oryzae strains: Aspergillus oryzae strains were activated by slant agar inoculation, and spores were scraped off using an inoculation spatula and placed in sterile physiological saline to prepare 10... 8 CFU / mL Aspergillus oryzae spore suspension was transferred to a 4 mL Erlenmeyer flask containing barley-pea medium. After shaking, the flask was incubated at 30°C for 72 h, shaking once every 12 h. The above barley-pea medium was prepared as follows: barley and peas were crushed into granules and mixed evenly at a mass ratio of 7:3. Water was added to the mixture at a mass-volume ratio of 1:1 and mixed well. 30 g of the mixture was placed into a 250 mL Erlenmeyer flask and sterilized at 121°C for 20 min.

[0043] Crude enzyme extraction: Accurately weigh 2.50 g of the cultured Aspergillus oryzae strain solid culture in an Erlenmeyer flask and immerse it in 50 mL of the corresponding buffer solution. Incubate at 150 pm and 40℃ with constant temperature and shaking for 60 min. Pass the crude enzyme solution through a 200-mesh double-layer filter cloth and dilute to 250 mL in a volumetric flask with the corresponding buffer solution for later use.

[0044] Methods for determining enzyme activity: Protease activity assay: Protease activity was determined by ultraviolet spectrophotometry according to GB / T 23527.1—2023 "Quality requirements for enzyme preparations Part 1: Protease preparations". Neutral protease activity was determined using sodium phosphate buffer at pH 7.2, and acidic protease activity was determined using lactate-sodium lactate buffer at pH 3.0.

[0045] Glutaminase activity assay: A glutaminase activity assay kit was used. Glutaminase is a key functional enzyme for enhancing the flavor of vinegar. Its activity determines the conversion efficiency of glutamine into glutamic acid, a flavor substance, while also increasing the amino acid nitrogen content of vinegar, ensuring that the finished vinegar meets the standards for flavor and physicochemical indicators.

[0046] Amylase activity assay: Extraction, filtration, and volume adjustment were performed using phosphate-citrate buffer at pH 6.0. Under constant temperature water bath conditions of 60℃, amylase extract, soluble starch solution, and buffer were reacted accurately for 5 min. The reaction was then terminated by adding 0.5 mL of 0.1 mol / L HCl, followed by adding 2.5 mL of dilute iodine solution and vortexing. The absorbance was then measured at 660 nm.

[0047] Saccharifying enzyme activity assay: Extraction was performed using an acetate-sodium acetate buffer solution at pH 4.6, followed by filtration and volume adjustment. Under constant temperature water bath conditions of 40℃, the saccharifying enzyme extract was accurately reacted with a soluble starch solution for 30 min. NaOH was then added to terminate the reaction. The terminated solution was vortexed with DNS, and the reaction was terminated by boiling in a water bath for 5 min. The absorbance was measured at 540 nm.

[0048] Aminopeptidase activity assay: Extraction was performed using a Trimethylolamine buffer solution at pH 8.0, followed by filtration and volume adjustment. At a constant temperature of 40℃, 90 μL of the aminopeptidase extract was reacted with 10 μL of leucine-p-nitroaniline solution in a 96-well plate for 10 min. The reaction was then terminated with 100 μL of anhydrous ethanol, and the result was measured at 405 nm using a microplate reader. Aminopeptidase hydrolyzes peptides to generate free amino acids, enriching the variety of amino acids in vinegar and increasing the amino acid nitrogen content. This provides precursors for the formation of umami and flavor compounds, ensuring the umami flavor profile and physicochemical quality of the finished vinegar.

[0049] Cellulase and xylanase activity determination: Cellulase activity was determined according to GB / T 23881—2009 "Determination of Cellulase Activity in Feed - Filter Paper Method"; xylanase activity was determined according to GB / T 23874—2009 "Determination of Xylanase Activity in Feed Additives - Spectrophotometric Method". Cellulase and xylanase synergistically hydrolyze cellulose, xylan and other polysaccharides in vinegar brewing raw materials into fermentable sugars, thereby improving raw material utilization and fermentation efficiency.

[0050] β-glucosidase activity assay: Extraction was performed using an acetate-sodium acetate buffer solution at pH 4.6, followed by filtration and volume adjustment. Under constant temperature water bath conditions at 50℃, 30 μL of the glucosidase extract was accurately reacted with 270 μL of 0.15% p-Nitrophenol-Glucoside (pNPG) solution for 10 min. Then, 600 μL of Na2CO3 was added to terminate the reaction. The absorbance was measured at 410 nm using an enzyme-linked immunosorbent assay (ELISA) reader. β-glucosidase can hydrolyze bound glycosides and flavor precursors in vinegar brewing raw materials, releasing free aromatic substances, enriching the aroma layers of the finished vinegar. Simultaneously, it assists in the degradation of carbohydrates to generate fermentable sugars, improving the utilization rate of raw materials and fermentation efficiency.

[0051] The enzyme activity assay results for different Aspergillus oryzae strains were as follows: Figure 3 As shown in Table 2, Aspergillus oryzae strain QM81 exhibits the strongest enzyme production capacity, producing high levels of acidic protease and glutaminase. Its acidic protease activity and glutaminase activity reach 630.25 U / g and 7.03 U / g, respectively, which are 19.96% and 48.00% higher than those of commercially available Hu Niang AS 3.042. It also possesses a rich enzyme system including amylase, saccharifying enzyme, aminopeptidase, cellulase, β-glucosidase, pectinase, and xylanase.

[0052] Table 2. Results of enzyme activity assays for different Aspergillus oryzae strains 2. Identification of superior Aspergillus oryzae strains Morphological identification: Aspergillus oryzae QM81 spores were picked up with an inoculation loop and spot-inoculated onto PDA agar plates. The colonies growing on the plates were photographed and recorded. The colonies of Aspergillus oryzae QM81 on PDA agar plates are shown below. Figure 4 The colonies of Aspergillus oryzae QM81 on PDA medium are greenish-yellow in the middle, white at the edge, loose on the surface, and flocculent in texture.

[0053] ITS sequencing: After the strain was cultured on an slant, mycelia were scraped, frozen, and ground. DNA was extracted using a kit for ITS sequencing and strain identification. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. Homology comparison was performed using BLAST software, and an ITS sequencing phylogenetic tree was constructed. The ITS sequencing phylogenetic tree of Aspergillus oryzae strain QM81 is shown below. Figure 5 QM81 strain and Aspergillus oryzae ( Aspergillus oryzae CBD-OH-S1 is highly homologous to Aspergillus flavus (CBD-OH-S1). Aspergillus flavusGlinf027 also showed high homology, indicating that ITS sequence alignment alone cannot definitively distinguish strain QM81 as Aspergillus oryzae. This strain was isolated from brewing koji and its morphological characteristics matched those of Aspergillus oryzae; combining morphological observation and phylogenetic analysis, strain QM81 was identified as Aspergillus oryzae (…). Aspergillus oryzae It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42103 and deposit date of June 16, 2025.

[0054] Example 3: Preparation of active nano-selenium-chitosan complex A chitosan solution with a mass concentration of 10 mg / mL was taken, and a sodium selenite solution of 50 mmol / L was added to it. Then, an ascorbic acid solution with a concentration of 80 mmol / L was added dropwise to the mixture. The volume ratio of the chitosan solution, sodium selenite solution, and ascorbic acid solution was 1:1:1. The mixture was stirred continuously during the dropwise addition to obtain a primary active nano-selenium-chitosan complex. The primary complex was transferred to a dialysis bag and dialyzed in sufficient deionized water for 2-3 days. After dialysis, the volume was adjusted to 10 times the initial volume of the chitosan solution with deionized water to finally obtain a high-purity active nano-selenium-chitosan complex with a particle size of 50-100 nm.

[0055] The method for preparing the 10 mg / mL chitosan solution is as follows: accurately weigh 0.1 g of chitosan, place it in a beaker and dissolve it with deionized water. Stir for 20 min at room temperature (25 ℃) and magnetic stirring speed (200 r / min) until the chitosan is completely dissolved and the system is homogeneous and transparent. Then transfer it to a 100 mL volumetric flask, dilute to the mark with deionized water, shake well and set aside. The method for preparing the 50 mmol / L sodium selenite solution is as follows: accurately weigh 0.8648 g of sodium selenite (Na2SeO3) powder using an analytical balance, place it in a beaker, add an appropriate amount of deionized water and stir thoroughly to dissolve, transfer it to a 100 mL volumetric flask, dilute to the mark with deionized water, shake well and set aside. The method for preparing the 80 mmol / L ascorbic acid solution is as follows: 1.4090 g of ascorbic acid powder is weighed quickly using an analytical balance. Since ascorbic acid is easily oxidized, it is immediately transferred to a beaker after weighing. Deionized water is added to the beaker to completely dissolve the powder. The solution is then transferred to a 100 mL volumetric flask and diluted to the mark with deionized water. The solution is shaken well and used immediately to avoid oxidation and inactivation due to prolonged storage.

[0056] Example 4: During the alcoholic fermentation stage, 60% selenium-enriched wheat bran koji and 0.1% selenium-enriched brewing yeast CGMCC 34906 were added; an active nano-selenium-chitosan complex was added to the freshly leached vinegar. Selenium-enriched sorghum and wheat are ground to 50-60 mesh and then mixed with selenium-enriched wheat bran, rice husks, and rice husks in a mass ratio of 2.5:1:2:2:0.19. Water at 140% of the sorghum mass is added and the mixture is stirred until homogeneous. The mixture is then moistened for 24 hours. After moistening, the mixture is steamed for 90 minutes, cooled to 28-32°C, and then saccharifying enzyme at 0.1% of the sorghum mass is added, along with sodium selenite at 0.01% of the sorghum mass. Selenium-enriched wheat bran koji at 40% of the sorghum mass and selenium-enriched brewing yeast CGMCC 34906 fermentation agent are added, and water at 100% of the sorghum mass is added. The mixture is then placed in an alcoholic fermentation tank for solid-state fermentation for 8-20 days, achieving an alcohol content of 8-10%. %vol, to end alcoholic fermentation and obtain mash; the fermented mash is divided into acetic acid fermentation tanks, and 10% of fire mash is added, and fermentation is carried out for 11-15 days, with a total acid of 5-6 g / 100 mL, and then the acetic acid fermentation is ended; after smoking the mash and leaching the vinegar, new leached vinegar is obtained, and 1% of active nano-selenium-chitosan complex (particle size of 50-100 nm) is added to it. After sun-drying and aging for one year, after filtration, sterilization and bottling, selenium-enriched aromatic ester aged vinegar is obtained.

[0057] The method for preparing selenium-enriched wheat bran koji is as follows: Selenium-enriched soybeans and selenium-enriched wheat are mixed and crushed at a mass ratio of 1:1. Ten times the mass of soybeans' wheat bran and ten times the mass of soybeans' water are added and mixed evenly to form a mixture. Simultaneously, a nylon bag containing 25% of the soybeans' mass of dried wheat bran and the mixture are steamed together for 40 minutes. After steaming, the mixture is spread out and cooled to 40℃ to form koji material. The sterilized wheat bran in the nylon bag is mixed with 0.3% of the soybeans' mass of Aspergillus oryzae CGMCC 42103 koji seed, and then inoculated into the cooled koji material and mixed evenly. This mixture is then placed in a koji bed, and the room temperature and product temperature are controlled at 32–35℃ throughout the process. The mixture is turned over every 12 hours, for a total of two times. During the first turning, the state of the koji material shows no obvious change; during the second turning, white mycelium can be seen growing. After culturing for 36 hours, the mixture is divided into blocks, and ventilation continues for about two days until the koji material matures. It is then dried and stored for later use.

[0058] The preparation method of Aspergillus oryzae CGMCC 42103 spore suspension is as follows: Transfer 4 mL of Aspergillus oryzae CGMCC 42103 spore suspension (10 7 ~10 8(CFU / mL) was injected into a 250 mL Erlenmeyer flask containing 30 g of wheat bran and soybean flour culture medium. The flask was placed in a 30℃ incubator, and the flask was tapped every 12 hours. After 72 hours of incubation, the flask was dried in a 40℃ constant temperature drying oven for 3 hours to obtain Aspergillus oryzae inoculum. The preparation method of wheat bran and soybean flour culture medium was as follows: soybeans were crushed into flour-like powder, and wheat bran and soybean flour were mixed evenly at a mass ratio of 9:1. Water was added at 0.5 times the dry weight of the culture medium and mixed. The mixture was then sterilized in a high-pressure steam sterilizer at 0.1 MPa and 121℃ for 30 minutes. After sterilization, the raw materials were allowed to cool to about 40℃.

[0059] The Aspergillus oryzae CGMCC 42103 is an excellent indigenous strain isolated and screened from Shanxi aged vinegar koji. It has high acid protease-glutaminase activity. After being cultured in soybean bran flour medium for 3 days, its acid protease activity and glutaminase activity were 630.25 U / g and 7.03 U / g, respectively. It also has amylase, β-glucosidase, aminopeptidase, cellulase and xylanase enzyme systems. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

[0060] The preparation method of the selenium-enriched Saccharomyces cerevisiae CGMCC 34906 fermentation agent is as follows: Activated selenium-enriched Saccharomyces cerevisiae CGMCC 34906 is inoculated into PDA liquid culture medium at an inoculation rate of 3%, and then statically cultured at 30 ℃ for 24 h until the cell concentration reaches 10. 9 CFU / mL; collect bacterial cells by centrifugation at 8000 r / min for 10 min, wash the cells twice with sterile physiological saline, add 3 times the amount of cell mass protectant, and freeze-dry under vacuum to obtain the starter culture powder, which is the selenium-enriched brewer's yeast CGMCC 34906, with a viable count ≥10. 10 CFU / g; The protective agent formulation is as follows (w / v): 12% maltodextrin, 1.5% sodium caseinate, 6% whey powder, 2.5% lactose, 4% glucose, 0.3% sodium citrate, 0.2% ascorbic acid, 0.1% Tween 80, and the remainder is water.

[0061] In Comparative Example 1, 60% selenium-enriched wheat bran koji and 0.1% commercial selenium-enriched yeast starter were added during the alcohol fermentation stage; no active nano-selenium-chitosan complex was added to the fresh vinegar, and the rest of the process was the same as in Comparative Example 4.

[0062] In Comparative Example 2, 60% selenium-enriched wheat bran koji and 0.1% selenium-enriched yeast CGMCC34906 fermentation agent were added during the alcohol fermentation stage; no active nano-selenium-chitosan complex was added to the fresh vinegar, and the remaining process operations were the same as in Example 4.

[0063] Example 5: Determination of Physicochemical Indicators and Flavor Compounds in Selenium-Enriched Aged Vinegar (1) Determination methods for total selenium, total acid, non-volatile acid, total ester and amino acid nitrogen ① Determination method of total selenium: The determination method is the same as in Example 1.

[0064] ② Determination of total acid, amino acid nitrogen, non-volatile acid and total ester content: Take 20 mL of vinegar solution, add 180 mL of distilled water to dilute 10 times, stir evenly with a glass rod and set aside. Refer to the method of national standard GB / T 19777—2013 "Geographical Indication Product Shanxi Aged Vinegar" to determine the total acid, amino acid nitrogen, non-volatile acid and total ester content.

[0065] (2) Methods for determining the content of organic acids and volatile aroma components ① Determination of organic acids: Take 1000 μL of acetic acid solution, dilute to 10 mL with ultrapure water, filter through a 0.22 μm microporous membrane, and inject the sample to determine the content of eight organic acids (oxalic acid, tartaric acid, pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid). High performance liquid chromatography conditions: Agilent ZORBAX-C 18 A chromatographic column (4.6 mm × 150 mm × 5 μm) was used; 20 mmol / L NaH₂PO₄ was used as the mobile phase; the injection volume was 20 μL; the flow rate was 0.8 mL / min; the wavelength was 210 nm; and the column temperature was 30℃. The external standard method was used for quantitative analysis of organic acids in acetic acid solution. Qualitative analysis was based on the retention time of each organic acid, while quantitative analysis was performed using the external standard method with peak area, with standard solutions of different concentrations of organic acids injected separately.

[0066] ② Determination of volatile aroma components: The extraction head was aged at 270 ℃ at the injection port until no impurity peaks were observed. 100 μL of a mixed internal standard (tert-amyl alcohol, n-amyl acetate, and 2-ethylbutyric acid, with mass concentrations of 297.88 mg / L, 296.10 mg / L, and 295.87 mg / L, respectively) was added to the acetic acid solution. The mixture was equilibrated in a 45 ℃ incubator for 30 min, followed by headspace adsorption for 30 min. The extraction head was then inserted into the injection port, and desorption was performed at 270 ℃ for 5 min to extract the volatile aroma components from the sample. Chromatographic conditions: The column was a VF-5MS (30 m × 0.25 mm × 0.25 μm); carrier gas: helium, purity 99.999%, flow rate 1 mL / min, splitless. Temperature program: initial temperature 40 ℃, held for 3 min, then increased to 160 ℃ at a rate of 4 ℃, held for 1 min. The temperature was then increased to 270 °C at a rate of 10 °C / min and held for 5 min. Mass spectrometry conditions: interface temperature 280 °C, ion source temperature 280 °C, electron energy 70 eV, scan mass range 41–500 amu, and internal standard method was used for quantitative analysis.

[0067] As shown in Table 3, the total selenium content of Example 4 increased by 1150% and 78.57% compared to Comparative Example 1 and Comparative Example 2, respectively. This is mainly due to the double selenium enrichment formed by the addition of active nano-selenium-chitosan complex and the efficient biotransformation of selenium-enriched brewer's yeast CGMCC 34906. The total ester content of Example 4 increased by 36.65% compared to Comparative Example 1. The non-volatile acids increased by 30.34% compared to Comparative Example 1, fully demonstrating the synergistic optimization effect of protein-fortified selenium-enriched bran koji and temperature control process.

[0068] Table 3. Determination results of total selenium content and physicochemical indicators in aged vinegar with different selenium-enriched esters. As shown in Table 4, the total organic acid content in the selenium-enriched aged vinegar of Example 4 was 52.94 g / L, which was 10.96% higher than that of Comparative Example 1. This is because Aspergillus oryzae CGMCC 42103 in the protein-fortified selenium-enriched bran koji secreted abundant acidic protease and glutaminase, which efficiently degraded the raw material protein to generate amino acids and improved the degradation efficiency of the raw material to promote the generation of organic acids.

[0069] Table 4. Determination results of organic acids in selenium-enriched aged vinegar. As shown in Table 5, a total of 48 volatile aroma components were detected in Example 4, including 14 esters, 8 acids, 11 alcohols, 5 aldehydes, 2 ketones, 2 pyrazines, and 6 other types. The total content of esters was 3.433 mg / 100 mL, and the total content of acids was 2.558 mg / 100 mL, which were 78.90% and 89.48% higher than those in Comparative Example 1, respectively.

[0070] Table 5. Determination results of volatile aroma components in selenium-enriched aged vinegar. Note: "-" indicates that it was not detected.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing selenium-enriched aged vinegar using a combination of high-selenium-enriched indigenous yeast and active nano-selenium-chitosan complex, characterized in that: Using selenium-rich sorghum, wheat, wheat bran, and rice husks as raw materials, and using selenium-rich soybeans, wheat, and wheat bran as saccharification and fermentation agents, and using the indigenous brewing yeast CGMCC 34906 as a fermentation agent for solid-state alcoholic fermentation and solid-state acetic acid fermentation, after fermentation, the mash is smoked and leached to obtain new leached vinegar. Active nano-selenium-chitosan complex is added to the new leached vinegar, and after aging, filtration, sterilization, and bottling, selenium-rich aromatic ester aged vinegar is obtained.

2. The preparation method according to claim 1, characterized in that: The specific steps include: Selenium-enriched sorghum and wheat are crushed to 50-60 mesh and mixed with selenium-enriched bran, rice husks, and rice husks in a mass ratio of 2.5:1:2:2:0.

19. Water equal to 140% of the sorghum mass is added and mixed thoroughly. The mixture is allowed to soak for 24 hours. After soaking, the mixture is steamed for 90 minutes, then cooled to 28-32°C. Saccharifying enzyme (0.1% of sorghum mass) and sodium selenite (0.01%-0.02% of sorghum mass) are added, along with selenium-enriched bran koji (40% of sorghum mass) and brewing yeast CGMCC 34906 (0.1-0.5% of sorghum mass). Water equal to 100% of the sorghum mass is added, and the mixture is mixed thoroughly. The mixture is then placed in an alcohol fermentation tank for solid-state fermentation for 8-20 days, achieving an alcohol content of 8-10%. The alcoholic fermentation was stopped at %vol to obtain the mash. The fermented mash was then divided into acetic acid fermentation tanks, and then 10% of the mash was added to the fermented mash. The fermentation was carried out for 11 to 15 days until the total acid content was 5 to 6 g / 100 mL. The acetic acid fermentation was then stopped. After smoking the mash and leaching the vinegar, new leached vinegar was obtained. Then, active nano-selenium-chitosan complex with a particle size of 50 to 100 nm and a mass-volume ratio of 1% to 2% of the new leached vinegar were added. The mixture was then sun-dried and aged for one year. After filtration, sterilization and bottling, the selenium-enriched aged vinegar was obtained.

3. The preparation method according to claim 2, characterized in that: The method for preparing selenium-enriched wheat bran koji is as follows: Selenium-enriched soybeans and selenium-enriched wheat are mixed and crushed at a mass ratio of 1:

1. Ten times the mass of soybean bran and ten times the mass of soybean water are added and mixed evenly to form a mixture. Simultaneously, a nylon bag containing 25% of the soybean mass of dry wheat bran and the mixture are steamed together for 40 minutes. After steaming, the mixture is spread out and cooled to 40℃ to form koji material. The sterilized wheat bran in the nylon bag is mixed with 0.3% of the soybean mass of Aspergillus oryzae CGMCC 42103 koji seed, and then inoculated into the cooled koji material and mixed evenly. This mixture is then placed in a koji bed, and the room temperature and product temperature are controlled at 32–35℃ throughout the process. The mixture is turned over every 12 hours for a total of two times. During the first turning, the state of the koji material shows no significant change; during the second turning, white mycelium can be seen growing. After culturing for 36 hours, the mixture is divided into blocks, and ventilation continues for about two days until the koji material matures. It is then dried and stored for later use.

4. The preparation method according to claim 3, characterized in that: The preparation method of the Aspergillus oryzae CGMCC 42103 spawn is as follows: Transfer 4 mL of 10 7 ~10 8 CFU / mL Aspergillus oryzae CGMCC 42103 spore suspension was injected into a culture medium containing 30 g of wheat bran and soybean powder, placed in a 30℃ incubator, and the bottle was tapped every 12 h. After 72 h of culture, it was placed in a constant temperature drying oven at 40 ℃ for 3 h to obtain Aspergillus oryzae inoculum. The wheat bran and soybean powder culture medium was prepared as follows: soybeans were crushed into flour, and wheat bran and soybean powder were mixed evenly at a mass ratio of 9:

1. Water was added at 0.5 times the dry weight of the culture medium and mixed. The mixture was sterilized at 121℃ for 30 min at 0.1 MPa. After sterilization, the raw materials were allowed to cool to 40℃.

5. The preparation method according to claim 3, characterized in that: Aspergillus oryzae CGMCC 42103 is an excellent indigenous strain isolated and screened from Shanxi aged vinegar starter. It has high acid protease-glutaminase activity. After culturing in soybean bran flour medium for 3 days, its acid protease activity and glutaminase activity are 630.25 U / g and 7.03 U / g, respectively. It also has amylase, β-glucosidase, aminopeptidase, cellulase and xylanase enzyme systems. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

6. The preparation method according to claim 2, characterized in that: The preparation method of the selenium-enriched Saccharomyces cerevisiae CGMCC 34906 fermentation agent is as follows: Activated selenium-enriched Saccharomyces cerevisiae CGMCC 34906 is inoculated into PDA liquid culture medium at an inoculation rate of 3%, and then statically cultured at 30 ℃ for 24 h until the cell concentration reaches 10. 9 CFU / mL; collect bacterial cells by centrifugation at 8000 r / min for 10 min, wash the bacterial cells twice with sterile physiological saline, add 3 times the amount of cell mass protectant, and freeze-dry under vacuum to obtain bacterial powder, which is the selenium-enriched brewer's yeast starter, with a viable count ≥10. 10 CFU / g; The protective agent formulation is as follows (w / v): 12% maltodextrin, 1.5% sodium caseinate, 6% whey powder, 2.5% lactose, 4% glucose, 0.3% sodium citrate, 0.2% ascorbic acid, 0.1% Tween 80, and the remainder is water.

7. The preparation method according to claim 6, characterized in that: The selenium-enriched brewing yeast CGMCC 34906 is an excellent indigenous strain isolated and screened from Shanxi aged vinegar mash. It has a strong selenium enrichment capacity. In selenium-enriched malt extract medium, with an initial selenium concentration of 16 μg / mL and an inoculum amount of 4%, the selenium enrichment capacity reaches 3000 μg / g dry cells after 48 h of shake-flask fermentation at 30℃. It also has excellent fermentation characteristics with high alcohol and ester production. In sorghum juice medium, the alcohol production is 7.85% vol and the ester production is 5.72 g / 100 mL. It is deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

8. The preparation method according to claim 2, characterized in that: The preparation method of the active nano-selenium-chitosan complex is as follows: Take a chitosan solution with a mass concentration of 5-15 mg / mL, add 40-60 mmol / L sodium selenite solution to it, and then add an ascorbic acid solution with a concentration of 60-100 mmol / L dropwise to the mixture. The volume ratio of the chitosan solution, sodium selenite solution, and ascorbic acid solution is 1:1:

1. Stir continuously during the dropwise addition to obtain a primary active nano-selenium-chitosan complex. Transfer the primary complex into a dialysis bag and dialyze it in sufficient deionized water for 2-3 days. After dialysis, adjust the volume with deionized water to 10 times the initial volume of the chitosan solution. By adjusting the concentration, ensure the uniform dispersion of the composite particles and accurately control the particle size range of the product to finally obtain a high-purity active nano-selenium-chitosan complex with a particle size of 50-100 nm.

9. The preparation method according to claim 2, characterized in that: The prepared selenium-enriched aged vinegar is clear and bright, with a rich and harmonious ester aroma, possessing both the smoky and aged fragrance unique to aged vinegar; it has a mellow and long-lasting sour taste; the total selenium content is ≥0.23 mg / L, the total acid content is ≥5.50 g / 100 mL, the amino acid nitrogen content is ≥0.30 g / 100 mL, the non-volatile acid content is ≥1.80 g / 100 mL, the total ester content is ≥3.50 g / 100 mL, and it is rich in organic acids and volatile aromas.

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