Lactobacillus mucosus and application thereof in intensified fermentation of Nongxiang liquor

Abstract

The present application belongs to the technical field of microorganism, and particularly relates to a fermented Lactobacillus muci-depum and application thereof in intensified fermentation of soy sauce type liquor. Two kinds of fermented Lactobacillus muci-depum Q7 and Y8 are screened out through ethanol and acid tolerance, and the lactic acid production capacity is relatively optimal. Exogenous addition of the strain Q7 and Y8 is more significant in reducing the pH value of fermentation mash, and the pH value is basically stable within 3-15 days of fermentation, and the total acid content is significantly increased. The strain Y8 is used for intensified fermentation of soy sauce type liquor, and the content of ethyl lactate is increased by 675%, and the content of ethyl acetate is increased by 694% without affecting liquor output. Process optimization is carried out on the intensified fermentation of soy sauce type liquor by the strain Y8, and isobutyl acetate is a unique flavor substance after intensified fermentation, the content of ethyl acetate is increased by 3.4 times, the content of ethyl lactate is increased by 3.3 times, the content of characteristic higher alcohols is significantly reduced, the content of acetaldehyde and ketones is reduced respectively, and the quality of soy sauce type liquor is improved.

Description

Technical Field

[0001] This invention belongs to the technical field of microorganisms, specifically relating to a fermenting Lactobacillus mucilaginosus and its application in the enhanced fermentation of soy sauce-flavored baijiu. Background Technology

[0002] Soy sauce aroma type baijiu is mainly produced in the Pearl River Delta region of my country. It is named for its distinctive soy sauce aroma and has a unique southern flavor. The production process of soy sauce aroma type baijiu is unique, using rice as the raw material. It uses rice, soybeans, rice cake paste, fermented rice cake leaves, and rice cake balls as the saccharification and fermentation agent. Under fully liquid fermentation conditions, saccharification and fermentation occur simultaneously. After distillation, aging with meat, and blending, the resulting liquor is clear and pure, with a unique soy sauce aroma, mellow and smooth taste, and a clean finish. It is a typical representative of Chinese rice wine. The flavor compounds in Chinese baijiu are mainly composed of acids, esters, aldehydes, and alcohols. Among them, esters, mainly ethyl lactate and ethyl acetate, are the largest category of flavor compounds. The content and proportion of esters affect the style and quality of baijiu. Soy sauce aroma type baijiu uses a semi-solid-state fermentation process, which is easily affected by seasonal factors. The ester production in the fermentation mash is unstable, resulting in an overall low ester content. Due to seasonal factors and fermentation environment, the content of ethyl lactate in the liquor is unstable, which leads to problems such as insufficient aroma and insufficient body, resulting in fluctuations in product quality and making it difficult to meet consumers' higher requirements for liquor quality.

[0003] In the prior art, patent CN2024102766748 discloses an acid-resistant Lactobacillus acetotolerans La50 and its applications. This strain exhibits strong acid, salt, and high-temperature resistance. Its fermentation produces primarily lactic acid, followed by acetic acid and malic acid. It can increase the content of ethyl acetate but has a relatively small impact on the content of ethyl lactate. Therefore, this application aims to study the synthesis law of ethyl lactate during the post-fermentation period of soy sauce-flavored baijiu and the technical means to increase the ethyl lactate content during this period. This will increase the total ester content of rice wine, especially ethyl lactate, thereby enhancing the complex aroma of the rice wine without affecting its yield, improving the quality of the wine, increasing the rate of premium wine, and ultimately improving the product's market competitiveness. This research has significant implications. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a fermenting Lactobacillus myxobolus and its application in the enhanced fermentation of soy sauce-flavored Baijiu.

[0005] The technical content of this invention is as follows:

[0006] This invention provides a fermentation of *Lactobacillus mucinus*, including Q7 or Y8;

[0007] The fermenting Lactobacillus Q7, taxonomically named Limosilactobacillus fermentum, was deposited on September 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNO.65149. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1 of the sequence listing.

[0008] The fermenting Lactobacillus Y8, taxonomically named Limosilactobacillus fermentum, was deposited on September 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNO.65150. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.2 of the sequence listing.

[0009] This invention also provides the application of the above-mentioned fermented Lactobacillus mucilaginosus in the production of fermented foods or beverages;

[0010] The present invention also provides the application of the above-mentioned fermented Lactobacillus mucilaginosus in brewing soy sauce-flavored liquor. The method for brewing soy sauce-flavored liquor is as follows: take rice, add water and steam until cooked, cool to 32°C, add yeast and water and other auxiliary materials, then add brewing yeast and fermented Lactobacillus mucilaginosus liquid for fermentation. The fermentation temperature is 24-36°C and the fermentation time is 10-20 days.

[0011] The amount of brewing yeast added is 0.5-5‰ (w / w) of the rice content, preferably 0.5-2‰ (w / w);

[0012] The fermented *Lactobacillus mucinus* broth was obtained by activating and culturing *Lactobacillus mucinus*, and its concentration was 4 × 10⁻⁶. 9 CFU / mL;

[0013] The specific preparation method is as follows: Lactic acid bacteria preserved previously were streaked on agar plates and incubated statically at 37°C for 24 hours; single colonies were picked and incubated in liquid MRS medium at 37°C for 24 hours until the growth stabilized, resulting in primary seed culture; the primary seed culture was inoculated into 100 mL of MRS medium at an inoculation rate of 5% (V / V) and cultured at 37°C until mid-logarithmic growth, resulting in secondary seed culture; the activated bacterial culture was then centrifuged at 4°C and 8000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed with sterile physiological saline. This washing and centrifugation were repeated twice. The lactic acid bacteria were resuspended in sterile physiological saline, and the OD was adjusted. 600 The final concentration of the fermentation seed liquid was 4.00 × 10⁻⁶. 9 CFU / mL;

[0014] The amount of fermented Lactobacillus mucilaginosus liquid added is 1-10% (V / W, based on the volume of fermented Lactobacillus mucilaginosus liquid added per 200g of rice), preferably 1-5%, and more preferably 5%.

[0015] Preferably, the fermentation temperature is 24–27°C; more preferably, the fermentation temperature is 26°C.

[0016] Preferably, the fermentation time is 15 to 20 days.

[0017] The application aims to enhance the quality and flavor of soy sauce-flavored baijiu, which is reflected in the following aspects:

[0018] (1) Increase in the content of characteristic esters in baijiu;

[0019] (2) The decrease in the content of characteristic higher alcohols, aldehydes and ketones in baijiu;

[0020] (3) Increase in the content of characteristic acids in baijiu;

[0021] (4) Improvement of the diversity of flavor metabolism and quality of baijiu;

[0022] The characteristic esters include ethyl lactate, ethyl acetate, etc.

[0023] The characteristic acids include lactic acid, acetic acid, etc.

[0024] The beneficial effects of this invention are as follows:

[0025] The present invention utilizes two strains of *Lactobacillus fermentans*, Q7 and Y8, selected through ethanol and acid tolerance screening. Q7 and Y8 exhibit superior lactic acid production capabilities compared to other reported strains of the same genus. Exogenous addition of strains Q7 and Y8 significantly reduces the pH of the fermentation mash, which stabilizes within 3-15 days of fermentation. This significantly enhances the total acid content, increases lactic acid and acetic acid content, and raises the total ester content, particularly characteristic esters such as ethyl lactate and ethyl acetate. Furthermore, within the aroma threshold range, it significantly reduces the content of characteristic higher alcohols and acids in soy sauce-flavored baijiu. Strain Y8, when used to enhance the fermentation of soy sauce-flavored baijiu, increases ethyl lactate content by 675% and ethyl acetate content by 694% without affecting the yield. The fermentation process of soy sauce-flavored baijiu was optimized using strain Y8. The optimal conditions were: yeast addition of 0.8‰ (w / w), lactic acid bacteria inoculum of 6.8% (v / w), and fermentation temperature of 26℃. Under these conditions, the alcohol content of the soy sauce-flavored baijiu was 27.70±0.14% vol, and the ethyl lactate content reached 0.54±0.04 g / L. Thirty-nine characteristic flavor compounds were detected in the fermentation mash. Isobutyl acetate was a unique flavor compound after enhanced fermentation. The content of ethyl acetate increased by 3.4 times, and the content of ethyl lactate increased by 3.3 times. The content of characteristic higher alcohols was significantly reduced (isobutanol, isoamyl alcohol, and phenylethanol decreased by 34%, 43%, and 36%, respectively), and the contents of acetaldehyde and ketones decreased by 39% and 58%, respectively, thus improving the quality of the soy sauce-flavored baijiu. Analysis using metagenomics combined with metatranscriptomics revealed that the succession relationship between *Lactobacillus fermentatus*, *Saccharomyces cerevisiae*, and *Lactobacillus brevis* has a significant impact on the formation of flavor compounds in baijiu. Attached Figure Description

[0026] Figure 1 This represents the typical colony morphology of lactic acid bacteria in the sample.

[0027] Figure 2 The cell morphology of some strains under a microscope;

[0028] Figure 3 Agarose gel electrophoresis image of the PCR products of the strain;

[0029] Figure 4 A phylogenetic tree constructed using 16S rDNA;

[0030] Figure 5 To assess the strain's tolerance to different pH and ethanol concentrations;

[0031] Figure 6 The growth curves and acid production curves of the strain in MRS medium are shown (a: growth curve; b: acid production curve).

[0032] Figure 7The relationship between OD values ​​of bacterial strains and corresponding colony forming units (a: Q7; b: Y8; c: HPC6)

[0033] Figure 8 A flowchart for the enhanced fermentation process of soy sauce-flavored baijiu;

[0034] Figure 9 Changes in physicochemical indicators during the enhanced fermentation process of soy sauce-flavored baijiu;

[0035] Figure 10 The characteristic ester changes during the intensification fermentation process of soy sauce-flavored baijiu;

[0036] Figure 11 The content of higher alcohols, a characteristic of the fermentation endpoint of soy sauce-flavored baijiu, was determined (different letters in the same color indicate significant differences (P<0.05), and the same letter indicates no significant differences (P>0.05)).

[0037] Figure 12 To enhance the characteristic acid changes during the fermentation process of soy sauce-flavored baijiu;

[0038] Figure 13 The effect of the amount of brewing yeast added on the ethyl lactate content and alcohol content of fermented soy sauce-flavored baijiu;

[0039] Figure 14 The effect of lactic acid bacteria inoculum amount on the ethyl lactate content and alcohol content of fermented soy sauce-flavored baijiu;

[0040] Figure 15 The effect of fermentation temperature on the ethyl lactate content and alcohol content of fermented soy sauce-flavored baijiu;

[0041] Figure 16 Response surface curves and contour plots showing the interaction of various factors;

[0042] Figure 17 Changes in physicochemical indicators during the enhanced fermentation process of soy sauce-flavored baijiu;

[0043] Figure 18 The changes in the content of characteristic esters during the brewing process of soy sauce aroma-type baijiu;

[0044] Figure 19 The changes in the content of characteristic higher alcohols during the brewing process of soy sauce aroma-type baijiu;

[0045] Figure 20 Analysis of α-diversity of microbial communities in mash at different fermentation stages;

[0046] Figure 21 β-diversity analysis of microbial communities in mash at different fermentation stages;

[0047] Figure 22Succession of the microbial community in the fermentation mash (a: metagenomic genus-level species; b: metagenomic species-level species; c: metatranscriptome genus-level species; d: metatranscriptome species-level species);

[0048] Figure 23 Functional annotation analysis of KEGG (lever 3) in fermented liquor of soy sauce aroma type (Baijiu) using LEfSe;

[0049] Figure 24 To illustrate the relationship between environmental factors and the core microbial community (Top 8) (RDA analysis, blue arrows represent core microbial species; red arrows represent different environmental factors; dots represent samples from the experimental group and control group at different fermentation time points, a: metagenomic level samples; b: metatranscriptional level samples);

[0050] Figure 25 The correlation network between microorganisms and flavor compounds is shown (the line color is proportional to the Spearman value (P<0.05, |r|>0.7); red indicates a positive correlation, and blue indicates a negative correlation). Detailed Implementation

[0051] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0052] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0053] Microsoft Excel 2021 was used for data preparation; IBM SPSS Statistics 27.0.1 was used for one-way ANOVA, and Duncan's method was used for significance analysis, with P < 0.05 considered significant; MEGA software was used to construct phylogenetic trees; Design-Expert 13 was used for response surface methodology analysis and graphing; Origin Pro 2021 was used for data processing and graphing; Canoco5 was used for redundancy analysis; and Gephi was used for network visualization analysis. All experiments were performed in triplicate, and the data are expressed as mean ± standard error.

[0054] Example 1

[0055] 1. Screening and physiological and biochemical analysis of lactic acid bacteria

[0056] 1.1 Isolation and Preliminary Identification of Strains

[0057] 5g of raw materials (fermentation cake samples, fermented cake pellet samples, fermented mash samples, fermentation tank wall residue samples, coil bottom residue samples, and pickled vegetable samples) were suspended in sterile physiological saline and shaken at 37℃ and 120r / min for 3h, followed by serial dilution. 100μL of each dilution was evenly spread on solid MRS medium containing CaCO3 and incubated at 37℃ for 24–48h. After colony formation on the plates, the morphology of lactic acid bacteria colonies was observed to be predominantly characterized by a raised center, neat edges, and a smooth yellow or white surface. (See attached image for colony morphology.) Figure 1 Colonies that can produce calcium-dissolving zones are selected and repeatedly purified until they are pure cultures as seen under a microscope.

[0058] 1) Gram-positive strains were selected by observing the morphology of the strains under a microscope and transferred to MRS solid medium. They were then cultured at 37°C and repeatedly streaked until a pure culture was obtained.

[0059] 2) 141 Gram-positive strains with rod-shaped cell morphology under a microscope were initially screened and preliminarily identified as lactic acid bacteria. Colony morphology under a microscope is shown in [Figure showing colony morphology]. Figure 2 A total of 141 Gram-positive acid-producing bacterial strains were obtained. These were: strains B-1–B-20 and Q1–Q20 (40 strains from fermentation cake); strains W-1–W-20 (20 strains from fermentation cake pellets); strains Y1–Y20 (20 strains from fermentation mash); strains T-1–T-10 and T2-1–T2-10 (20 strains from fermentation tank wall residue); strains C-1–C-10 and T2-1–T2-6 (16 strains from bottom of fermentation coils); strains ZPC1–ZPC13 (13 strains from traditional kimchi); and strains HPC1–HPC12 (12 strains from Korean kimchi). Single colonies of each strain were obtained using the streak plating method, transferred to MRS liquid medium, and cultured at 37°C for 24 hours. The cultures were then mixed with an equal volume of 50% glycerol and stored at -80°C.

[0060] 3) A total of 141 strains from different sample sources were initially sampled in 8% ethanol-containing solid MRS medium with CaCO3. The growth of the strains and the size of the calcium dissolution zone were compared to preliminarily screen for acid-producing bacteria resistant to stress. The results are shown in Table 1. Regarding growth, 21 strains showed good growth with neat and smooth edges; 53 strains showed moderate growth with small or scattered colonies; the remaining strains did not grow. Among the 21 best-growing strains, the calcium dissolution zone was compared. Thirteen strains with large, clearly defined calcium dissolution zones, forming a sharp contrast with the surrounding medium, were selected and preliminarily identified as acid-producing bacteria resistant to 8% ethanol.

[0061] Table 1. Growth of lactic acid bacteria from different sample sources in 8% ethanol culture medium.

[0062]

[0063]

[0064] Growth status: - No growth; + Average growth; ++ Good growth. Calcium dissolution ring status: - No calcium dissolution ring; + Slightly transparent calcium dissolution ring; ++ Noticeably transparent calcium dissolution ring.

[0065] 1.2 Strain Identification

[0066] Based on the calcium dissolution zone and preliminary tolerance of the strains, 13 strains with better growth were selected for 16S rDNA sequence analysis, and the selected strains were identified for molecular biology identification.

[0067] 1) The isolated strains were cultured in MRS solid medium for 24 h, and single colonies were picked for PCR amplification. The PCR amplification reaction system is shown in Table 2, and the amplification reaction conditions are shown in Table 3. Universal bacterial primers were used to perform PCR amplification on 13 lactic acid bacteria strains. The PCR amplification products were subjected to agarose gel electrophoresis, and the results are shown below. Figure 3 As shown, the molecular weights of the PCR amplification products of lactic acid bacteria are all around 1500 bp, indicating successful amplification and suitability for sequencing.

[0068] Table 2 PCR reaction system

[0069]

[0070] The sequence of primer 27F is (SEQ ID NO.3): 5′-AGAGTTTGATCCTGGCTTAG-3′;

[0071] The sequence of primer 1492R is (SEQ ID NO.4): 5′-ACGGCTACCTTGTTACGACTT-3′;

[0072] Table 3 PCR reaction procedure

[0073]

[0074] 2) The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the sequences in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cg) to determine sequence homology. A phylogenetic tree was constructed using MEGAX 64, and the results are as follows: Figure 4As shown, the 13 selected lactic acid bacteria strains are 3 *Pediococcus acidilactici* strains, 2 *Lactobacillus fermentum* strains, 2 *Pediococcus pentosacchari* strains, and 6 *Lactobacillus plantarum* strains. Among them, W2, B8, and Q6 correspond to *Pediococcus acidilactici* with accession numbers MT538932.1, OP720973.1, and KT895267.1, respectively, with a similarity of 99%. Q7 and Y8 have a similarity of 99% to *Lactobacillus fermentum* with accession numbers MT464033.1 and MT604713.1, and are deposited at the Guangdong Provincial Microbial Culture Collection Center with accession numbers GDMCCNO: 65149 and GDMCCNO: 65150, respectively. T2 and T7 have a similarity of 99% to *Pediococcus pentosacchari* with accession numbers OR084828.1 and MT515895.1. The similarity to *Lactobacillus plantarum* (pendula) reached 99%; HPC6, HPC9, ZPC4, ZPC6, ZPC8, and ZPC10 showed 99% similarity to accession numbers MT510470.1, MT538454.1, and MT538454.1, respectively. The nucleotide sequences of the 16S rDNA of strains Q7 and Y8 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0075] 1.3 Comparison of strains' stress resistance

[0076] The growth environment of microorganisms has a significant impact on the growth of bacterial strains. In the baijiu brewing environment, the ethanol accumulated in the early stages of fermentation and the acidic environment created by acid-producing bacteria can inhibit the growth of bacterial strains. As fermentation progresses, only strains with good tolerance to ethanol and acidity can grow normally. Thirteen lactic acid bacteria strains identified through molecular biology were compared for their stress resistance, and strains with good growth were selected for use in the fortified brewing of soy sauce-flavored baijiu.

[0077] 1) Acid tolerance test: Thirteen strains of lactic acid bacteria seed culture were inoculated at a 5% (V / V) inoculation rate into modified MRS liquid medium with pH values ​​of 3.0, 3.5, 4.0, and 4.5, respectively. The cultures were incubated statically at 37℃ for 48 h, and the OD values ​​were measured. 600 value.

[0078] 2) Ethanol tolerance test: Thirteen strains of lactic acid bacteria seed culture were inoculated into modified MRS liquid medium with ethanol contents of 8%, 10%, 12%, and 14% at an inoculation rate of 5% (V / V). The cultures were incubated statically at 37°C for 48 h, and the OD values ​​were measured. 600 value.

[0079] The growth of the 13 lactic acid bacteria strains obtained from the initial screening under different pH values ​​and different ethanol concentrations is shown in the figure. Figure 5 .Depend on Figure 5 From a to d in the data, it can be seen that when pH = 4.5, except for strains T2 and T7, the OD of the other strains is... 600 The pH ranged from 2.79 to 5.82; as the pH decreased, strains HPC9, HPC6, Y8, Q7, and B8 showed better tolerance; when the pH was 3, strains Q7, Y8, HPC6, and HPC9 showed better tolerance, with OD... 600 The value ranges from 0.73 to 1.03. (From...) Figure 5 From e to h, we can see that when the ethanol concentration is 8%, the OD values ​​of strains Q7, B8, Q6, HPC6, and Y8 are... 600 A value greater than 2 indicates good tolerance; when the ethanol concentration is 10%, strain Q7OD... 600 The value was 2.35, for strain B8OD. 600 The value is 1.68, for strain HPC6OD. 600 The value was 1.47, for strain Y8OD. 600 The OD value of these four lactic acid bacteria was 1.06. As the ethanol concentration increased, the OD values ​​of these four lactic acid bacteria... 600 It still holds a dominant position among the 13 lactic acid bacteria strains. The regulation of stress resistance in lactic acid bacteria involves multiple mechanisms, including central metabolic pathways, proton pumps, changes in cell membrane composition and cell density, DNA and protein damage repair, and neutralization processes. The tolerance mechanisms of lactic acid bacteria under acid and ethanol stress show some similarities; they can enhance stability by adjusting the structure and composition of the cell membrane, and adapt to the environment by altering the activity of key enzymes and adjusting the production of metabolites. Taking all factors into consideration, three lactic acid bacteria strains—Q7, Y8, and HPC6—were selected for subsequent fortification experiments in soy sauce-flavored baijiu.

[0080] 2. Study on the fermentation characteristics of stress-resistant lactic acid bacteria

[0081] 2.1 Determination of growth curve

[0082] Selected stress-resistant lactic acid bacteria were inoculated into activation medium and incubated statically at 37°C for 24 hours. The activated bacterial solution was then inoculated into seed medium at a rate of 5% (V / V) and incubated statically at 37°C for 24 hours to obtain the seed culture. The seed culture was then inoculated into modified MRS liquid medium at a rate of 5% (V / V) and incubated statically at 37°C. Samples were taken every 2 hours to determine the OD of the samples. 600 Values, with cultivation time (x) as the x-axis, OD 600 The growth curve is plotted with the value (y) as the ordinate.

[0083] 2.2 Determination of lactic acid production capacity

[0084] Inoculate the lactic acid bacteria seed culture into 50 ml LMR broth medium at an inoculation rate of 5% (V / V), and incubate at 37°C for 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. Centrifuge the culture medium at 10000 r / min for 10 min, and take the supernatant to determine the lactic acid content.

[0085] The growth curves and acid production curves of three Lactobacillus strains fermented in MRS liquid medium at 37°C are shown below. Figure 6 As shown. By Figure 6 As shown in 'a', strains Q7 and Y8 grow slowly from 0 to 2 hours, enter the mid-logarithmic phase from 2 to 6 hours, and their cell concentration increases rapidly in an exponential manner. As the lactic acid bacteria grow, nutrients are gradually consumed, and metabolic products begin to accumulate, causing the growth rate to decrease to match the mortality rate. After 6 hours of culture, they enter the stationary phase, with OD... 600 The pH was maintained at 5.25. During growth, lactic acid bacteria produce large amounts of lactic acid and other organic acids, leading to a decrease in the pH of the culture medium. As the pH decreases, the cell growth rate may slow down, thus affecting the rate of increase in OD value. However, the strain remains active, and the CFU value continues to rise. Strain HPC6 showed a slow increase in bacterial concentration from 0 to 2 hours, a logarithmic mid-phase from 2 to 16 hours, with the fastest increase occurring from 2 to 8 hours, during which the strain's metabolism is vigorous. After 16 hours, it enters a stationary phase, and the OD value... 600 It remained between 8.50 and 9.30. (By...) Figure 6 As shown in b, during the 0–12 h culture period, the lactic acid production of strains Q7 and Y8 increased rapidly. Strain Q7 reached a lactic acid production of 15.92 g / L after 12 h of culture, while strain Y8 reached 16.92 g / L. With increasing culture time, the lactic acid production remained relatively stable. Strain HPC6 produced acid rapidly during the 0–24 h fermentation period, reaching a lactic acid production of 32.10 g / L at 24 h, indicating good growth and acid production capacity. During the 24–72 h culture period, the lactic acid content gradually decreased, possibly due to the accumulation of lactic acid and the gradual increase in environmental acidity, which inhibited the growth and metabolism of lactic acid bacteria, leading to a decrease in lactic acid production. An OD was established. 600 The relationship with CFU showed a linear correlation, indicating a good linear correlation. Growth curves and OD... 600 The corresponding colony-forming unit relationship is as follows: Figure 7 As shown. Figure 7 a and Figure 7 In b, after 4 hours of fermentation, strain Q7 OD 600 The CFU level reached 3.35, and the CFU level reached 4.87 × 10⁻⁶. 9 CFU / mL, strain Y8 OD 600 The CFU level reached 3.36, and the CFU level reached 13.44 × 10⁻⁶.9 CFU / mL; such as Figure 7 c in the text refers to strain HPC6 OD at 6 hours of fermentation. 600 The CFU level reached 5.17, and the CFU level reached 15.39 × 10⁻⁶. 9 CFU / mL. Based on the logarithmic growth phase of the strain's growth curve, subsequent experiments selected 4 hours as the seed culture time for Q7 and Y8, and 6 hours for HPC6.

[0086] 3. Lactic acid bacteria-enhanced fermented soy sauce-flavored baijiu

[0087] 3.1 Preparation of Fermentation Seed Liquid

[0088] Lactic acid bacteria from previous preservation were streaked on agar plates and incubated statically at 37°C for 24 hours. Single colonies were picked and incubated in liquid MRS medium at 37°C for 24 hours until the growth stabilized, which was the primary seed culture. The primary seed culture was inoculated into 100 ml MRS medium at an inoculum rate of 5% (V / V) and cultured at 37°C until the logarithmic midline, which was the secondary seed culture. The activated bacterial culture was centrifuged at 4°C and 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed with sterile physiological saline. The washing and centrifugation were repeated twice. The lactic acid bacteria were resuspended in sterile physiological saline and the OD was adjusted. 600 The final concentration of the fermentation seed liquid was 4 × 10⁻⁶. 9 CFU / mL.

[0089] 3.2 Fermentation of Soy Sauce Aroma Baijiu

[0090] The intensified fermentation process of soy sauce aroma type baijiu, such as Figure 8 As shown, the process can be roughly divided into three stages: steaming, fermentation, and distillation. 1 kg of clean rice is steamed with water and cooled to 32°C. It is then mixed evenly with prepared common yeast, water, and other auxiliary materials and put into a fermentation tank for fermentation. 1‰ (W / W) of brewing yeast is added, and the fermentation seed liquid is inoculated at 12% (V / W) of the feed amount. Fermentation is carried out at 30°C.

[0091] Samples were taken on days 3, 5, 7, 12, and 15 of fermentation. For each sample, 450–500 mL of mash was collected. 220 mL of the mash was weighed into a 1000 mL Erlenmeyer flask, 80 mL of water was added, and a reflux condenser was connected. The flask was slowly heated and distilled, and 100 mL of the distillate was collected and stored at 4 °C. 200 mL of the mash was centrifuged at 8000 rpm / min for 5 min at 4 °C, and the supernatant was collected and stored at -20 °C. The remaining mash was stored at -80 °C for later use.

[0092] 3.3 Determination of Physicochemical Indicators

[0093] 3.3.1 Quantitative analysis of acetic acid and lactic acid by high performance liquid chromatography

[0094] 1) Sample preparation: Take 500 μL of fermentation mash supernatant sample, filter it through a 0.22 μm inorganic aqueous filter membrane using a disposable sterile syringe, and place it in a sample bottle.

[0095] 2) Chromatographic conditions: Column: Ultimate AQ-C18 (250mm×4.6mm, 5μm); Mobile phase: 0.01mol / L diammonium hydrogen phosphate solution; Flow rate: 0.8mL / min; Wavelength: 210nm; Column temperature: 30℃; Injection volume: 20μL.

[0096] 3.3.2 Determination of ethyl acetate and ethyl lactate by gas chromatography

[0097] 1) Sample preparation: Take the distillate sample from each sample point, filter it through a 0.22μm inorganic aqueous filter membrane using a disposable sterile syringe, and inject 1mL into an automatic gas chromatograph vial for later use.

[0098] 2) Gas chromatography determination conditions

[0099] Chromatographic column: ZB-WAXPLUS (30m×0.25mm×0.25μm); Carrier gas: Nitrogen (purity ≥99.999%); Split ratio: 1:45; Column flow rate: 1mL / min; Injector temperature: 230℃; FID detector temperature: 270℃; Temperature program: Initial temperature 40℃, hold for 3 min, increase to 200℃ at 10℃ / min, and hold for another 8 min.

[0100] 3.3.3 Determination of Characteristic Components of Soy Sauce Aroma Baijiu by Gas Chromatography

[0101] 1) Sample preparation: Take the distillate sample from each sample point, filter it through a 0.22μm inorganic aqueous filter membrane using a disposable sterile syringe, and inject 1mL into an automatic gas chromatograph vial for later use.

[0102] 2) For the detection of characteristic alcohols, aldehydes, acids, esters, and ketones in soy sauce-flavored baijiu, modifications were made based on GB / T 10345-2022. Chromatographic column: ZB-WAXPLUS (30m×0.25mm×0.25μm); FID detector temperature: 250℃; injection port temperature: 250℃; constant flow mode: 1mL / min; injection volume: 1μL; split ratio: 40:1. Temperature program: initial temperature 35℃, hold for 1 min, increase to 70℃ at 3℃ / min, increase to 180℃ at 3.5℃ / min, then increase to 210℃ at 15℃ / min, hold for 6 min.

[0103] 3.3.4 Determination of Environmental Factors

[0104] The determination of alcohol content, residual reducing sugar content in fermentation mash, pH value of fermentation mash, total acid and total ester content shall be conducted in accordance with the national standard GB / T 16289-2018.

[0105] by Figure 8 The process with added lactic acid bacteria was used as the experimental group, and the process without added lactic acid bacteria was used as the blank control group. The results are as follows: Figure 9 As shown. By Figure 9 As shown in section 'a', the pH value of the control group's mash was 3.85–4.18, which is within the pH range of fermentation for soy sauce-flavored baijiu. The pH value of the experimental group was 3.48–3.76, significantly lower than the control group. The rapid growth and reproduction of exogenously added lactic acid bacteria accelerated the utilization and metabolism of the substrate, producing a large amount of acid and creating a more acidic environment. The exogenous addition of strains Q7 and Y8 had a more significant effect on reducing the pH value of the fermentation mash than the addition of HPC6. Within 3–15 days of fermentation, the pH value generally stabilized. In the early stage of fermentation (0–5 days), the yeast activity was high, reproduction was rapid, and metabolism was active, quickly utilizing the sugars in the raw materials for alcoholic fermentation. At this time, the alcohol content gradually increased, and the rate of increase was relatively fast. Figure 9 As shown in b, the alcohol content in the control group reached its maximum of 29.0% Vol on day 5 of fermentation, and then slowly decreased. At this time, the metabolic activity of yeast was inhibited by acid and other products, the substrate concentration decreased, and the alcohol production gradually slowed down. In the later stage of fermentation, ethanol, as a substrate, was utilized by other microorganisms and used as a precursor to generate other flavor substances such as ethyl lactate. Therefore, the alcohol content usually shows a trend of first rising and then falling throughout the brewing process. In the 15-day fermentation of the experimental group, the trend of alcohol content change was similar to that of the control group. In the early stage of fermentation, the alcohol content of the experimental group was significantly lower than that of the control group. On day 3 of fermentation, the alcohol content only reached 20.5-22.7% Vol, which may be due to the influence of the addition of lactic acid bacteria on the metabolism of yeast, slowing down the rate of alcohol production. On day 15 of fermentation, the alcohol content of the experimental group with exogenous addition of strain Q7 was only 24.37% Vol, while the exogenous addition of strains Y8 and HPC6 did not significantly affect the alcohol content, with the final alcohol contents being 26.40% Vol and 27.70% Vol, respectively. In the early stages of fermentation, substrates in raw materials such as rice and yeast are converted into reducing sugars by saccharifying enzymes, which are then available for microbial use. Figure 9 As shown in 'c', the residual reducing sugar content showed a continuous decreasing trend. At the fermentation endpoint, the residual amount in the control group mash was 1.2 g / L, and in the HPC6 group it was 2.2 g / L, indicating similar utilization. However, the residual reducing sugar contents in the Q7 and Y8 groups were 7.67 g / L and 13.73 g / L, respectively, indicating that the exogenous addition of strains Q7 and Y8 reduced the utilization of reducing sugars to some extent. Total acid content is one of the important indicators of baijiu quality. Suitable acidity is beneficial for starch gelatinization and saccharification. At the same time, abundant acid is also a structural component of baijiu flavor, an important component that imparts aroma to the liquor. Figure 9 As shown in d, in the control group, the total acid content at the fermentation endpoint was only 0.21 g / L, while the total acid content in the Q7 and Y8 groups was as high as 1.64 g / L. The exogenous addition of strains Q7 and Y8 significantly enhanced the total acid content, which is consistent with the result of the low pH value in the fermentation mash.

[0106] Characteristics of ester changes during the brewing process of soy sauce aroma-type baijiu: Esters not only affect the aroma of baijiu but also its taste, resulting in a richer and smoother body. The characteristic esters in soy sauce aroma-type baijiu mainly include ethyl lactate and ethyl acetate, which together contribute to the baijiu's delicate and subtle aroma. The changes in characteristic esters in soy sauce aroma-type baijiu during the intensified fermentation process are as follows: Figure 10 As shown. Figure 10 In the figure, 'a' represents the trend of total ester content. Total ester content is one of the important indicators for evaluating the quality of soy sauce-flavored baijiu. The total ester content of groups Q7 and Y8 was significantly higher than that of the experimental group, with the total ester content of group Q7 reaching 1.3 g / L, which was significantly higher than that of group Y8 (1.1 g / L). Figure 10 In the figure, 'b' represents the trend of ethyl lactate levels. Appropriate amounts of ethyl lactate can make the wine smoother, sweeter, and with a longer finish. Ethyl lactate is mainly produced in the later stages of fermentation, and studies have found that its content is positively correlated with the abundance of lactic acid bacteria. For example... Figure 10 In step b, at the fermentation endpoint, the ethyl lactate content in the experimental groups with exogenously added lactic acid bacteria was significantly higher than that in the control group. Among them, the ethyl lactate content in experimental groups Q7 and Y8 was significantly higher than that in the control group from the beginning of fermentation. The ethyl lactate content in group Q7 reached 0.33 g / L, which was 640% higher than that in the control group; the ethyl lactate content in group Y8 reached 0.34 g / L, which was 675% higher than that in the control group. Figure 10 In the figure, 'c' represents the trend of ethyl acetate. Ethyl acetate imparts a unique floral and fruity aroma to baijiu (Chinese liquor). When the ratio of ethyl lactate to ethyl acetate is appropriate, the two can harmonize and complement each other. Figure 10 As shown in c, the ethyl acetate content in the control group did not change much, reaching only 0.12 g / L at the end of fermentation; the synthesis of ethyl acetate in groups Q7 and Y8 increased rapidly from 5 to 12 days, and gradually stabilized after 12 days; the ethyl acetate content in group Q7 reached 1.05 g / L, an increase of 764% compared with the control group; the ethyl acetate content in group Y8 reached 0.96 g / L, an increase of 694% compared with the control group.

[0107] The effect of lactic acid bacteria-enhanced fermentation on characteristic higher alcohols in soy sauce-flavored baijiu: Proteins in the raw materials decompose into amino acids. Under the action of yeast, these amino acids undergo deamination and decarboxylation, releasing CO2 and ammonia, and generating higher alcohols. When the content of higher alcohols in baijiu is high, drinking it can cause congestion in the human nervous system, resulting in symptoms such as headache, nausea, and vomiting. The exogenous addition of lactic acid bacteria affects the content of characteristic higher alcohols in soy sauce-flavored baijiu as follows: Figure 11As shown. The ethanol content was 91.00 mg / L. Compared with the control group, the content of major higher alcohols in groups Q7 and Y8 was significantly reduced, while the change in group HPC6 was not significant. The content of isobutanol in group Q7 was 0.23 g / L, isoamyl alcohol was 0.20 g / L, and β-phenylethanol was 50.33 mg / L; the content of isobutanol in group Y8 was 0.24 g / L, isoamyl alcohol was 0.20 g / L, and β-phenylethanol was 52.33 mg / L. The flavor thresholds for isobutanol, isoamyl alcohol, and β-phenylethanol were 50, 30, and 14 mg / L, respectively. To ensure the flavor of the liquor, the content of higher alcohols should not be lower than their flavor thresholds. The exogenous addition of fermenting Lactobacillus mucilaginosus Q7 and Y8 can significantly reduce the characteristic higher alcohols in soy sauce-flavored baijiu within the aroma threshold range.

[0108] The Effect of Lactic Acid Bacteria-Enhanced Fermentation on Characteristic Acids in Soy Sauce Aroma Baijiu: The main acids in soy sauce aroma baijiu are lactic acid and acetic acid, along with butyric acid and hexanoic acid, accounting for over 90% of the total acid content. These are not only important aroma compounds in baijiu but also precursors to many other aroma compounds. The changes in characteristic acids during the enhanced fermentation of soy sauce aroma baijiu are shown below. Figure 12 As shown. By Figure 12 As shown in 'a', the lactic acid content in the fermented liquor of soy sauce-flavored baijiu was higher than that in the control group after the exogenous addition of lactic acid bacteria. Specifically, the lactic acid content in group HPC6 reached 5.37 g / L and remained relatively stable; in group Q7, the lactic acid content peaked at 10.75 g / L on the fifth day of fermentation and then slowly decreased; in group Y8, the lactic acid content reached 7.58 g / L on the third day of fermentation, significantly higher than other experimental groups, and continued to rise slowly, reaching its highest point on the 12th day of fermentation. Figure 12 As shown in b, in the early stage of fermentation, the acetic acid content in the fermentation mash of group HPC6 was not significantly different from that of the control group. At the end of fermentation, it was lower than that of the control group, only 1.19 g / L. The exogenously added *Lactobacillus plantarum* may have competed with acetic acid-producing microorganisms (such as yeast), thus inhibiting the production of acetic acid. The acetic acid content in groups Q7 and Y8 was significantly higher than that of the control group, reaching peak values ​​of 3.02 g / L and 2.86 g / L, respectively, after 12 days of fermentation. The exogenously added *Lactobacillus fermentatus* may have affected the microbial community structure and promoted the metabolism of acetic acid-producing microorganisms. After 12 days of fermentation, it slowly decreased, presumably because it was consumed as a precursor for the synthesis of flavor substances such as ethyl acetate.

[0109] 4. Single-factor experiment

[0110] Based on the results of previous experiments, Lactobacillus fermentans Y8 was selected to optimize the fermentation process of soy sauce-flavored baijiu. The brewing process of soy sauce-flavored baijiu was carried out according to the 1L system: 200g of rice was weighed, steamed with water and cooled to 32℃, 1‰ (W / W) of brewing yeast was added, and Lactobacillus fermentans was introduced at the feeding stage. The fermentation temperature was 24~36℃ and the fermentation time was 15 days.

[0111] The effects of brewer's yeast addition, lactobacillus inoculation amount, and fermentation temperature on the ethyl lactate content and alcohol content of soy sauce-flavored baijiu were investigated to select the most suitable fermentation process conditions. The single-factor experimental conditions are as follows:

[0112] 1) Effect of brewer's yeast addition amount on ethyl lactate yield and alcohol content

[0113] Five samples of soy sauce-flavored baijiu were brewed, and the inoculum was inoculated into the fermentation system. The addition rates of brewing yeast were set at 0.5‰, 1‰, 2‰, 3‰, and 4‰ (w / w), the lactic acid bacteria inoculation time was 0 h, the lactic acid bacteria inoculation amount was 10%, the fermentation temperature was 30℃, and the contents of other auxiliary materials were kept consistent. After fermentation, the distillate was collected, and the ethyl lactate content and alcohol content of the samples were determined.

[0114] Depend on Figure 13 It can be seen that with the increase of the amount of brewing yeast, the alcohol content of the fermented mash of soy sauce-flavored baijiu first increased and then remained basically unchanged, while the ethyl lactate concentration showed a trend of first increasing and then decreasing. When the amount of brewing yeast added was 1‰ (W / W), the ethyl lactate content of soy sauce-flavored baijiu was the highest, at 0.45±0.01 g / L, and the alcohol content was 25.05±0.35% vol. When the amount of brewing yeast added was 2‰ (W / W), the alcohol content was the highest, at 25.90±0.28% vol, but the ethyl lactate content was only 0.39±0.02 g / L. This may be because when the amount of brewing yeast added was low, the carbon source in the fermentation liquid was not fully utilized, resulting in a lower alcohol yield. When the amount of yeast added was high, the carbon source in the fermentation system was insufficient, and the addition of too much brewing yeast was not conducive to the growth and reproduction of lactic acid bacteria, resulting in incomplete fermentation of exogenously added lactic acid bacteria, reducing the amount of ethyl lactate synthesized in soy sauce-flavored baijiu, thus lowering the content. Taking all factors into consideration, the optimal amount of brewing yeast added for the enhanced fermentation process of soy sauce-flavored baijiu was determined to be 1‰ (W / W).

[0115] 2) Effect of fermentation *Lactobacillus mucilaginosus* Y8 inoculum size on ethyl lactate yield and alcohol content

[0116] Five samples of soy sauce-flavored baijiu were brewed, and the inoculum seed solution was inoculated into the fermentation system. The lactic acid bacteria inoculation amounts were set at 1%, 5%, 10%, 15%, and 20% of the rice feed weight (V / W, calculated based on the volume of seed solution added per 200g of rice feed). The lactic acid bacteria inoculation time was 0 hours, the brewing yeast addition amount was 1‰ (W / W), the fermentation temperature was 30℃, and the contents of other auxiliary materials were consistent. After fermentation, the distillate was collected, and the ethyl lactate content and alcohol content of the samples were determined.

[0117] Depend on Figure 14It can be seen that as the inoculum size increases within the range of 1%–5% (V / W), the alcohol content and ethyl lactate content of the fermented soy sauce-flavored baijiu show an upward trend. When the inoculum size is 5% (V / W), the alcohol content and ethyl lactate content reach their maximum values, at 27.00±0.22% vol and 0.51±0.02 g / L, respectively. When the inoculum size exceeds 5% (V / W), the alcohol content and ethyl lactate content show a downward trend. This may be because more lactic acid bacteria metabolize and produce lactic acid, consuming some of the substrate originally used for alcoholic fermentation, thus indirectly leading to a decrease in alcohol content. Excessive exogenous addition of lactic acid bacteria affects the stability of the fermentation community, impacting the metabolism of alcohol-producing microorganisms and thus affecting the alcohol yield. The concentration of ethanol, a precursor to ethyl lactate synthesis, decreases, causing a decrease in ethyl lactate content. When the inoculum size is 15% (V / W), the alcohol content slightly rebounds, while the ethyl lactate content decreases. Taking all factors into consideration, the optimal lactic acid bacteria inoculation amount was determined to be 5% (V / W) without significantly affecting the alcohol yield.

[0118] 3) Effect of fermentation temperature on ethyl lactate yield and alcohol content

[0119] Five samples of soy sauce-flavored baijiu were brewed, and the inoculum was inoculated into the fermentation system. Fermentation temperatures were set at 24℃, 27℃, 30℃, 33℃, and 36℃. The lactic acid bacteria inoculation time was 0 hours, the inoculation amount was 10%, and the amount of brewing yeast was 1‰ (w / w). The contents of other auxiliary materials remained consistent. After fermentation, the distillate was collected, and the ethyl lactate content and alcohol content of the samples were determined.

[0120] Depend on Figure 15 It can be seen that within the range of 24℃ to 30℃, the alcohol content of soy sauce-flavored baijiu tends to decrease with increasing fermentation temperature. This indicates that high temperatures are not conducive to increasing the alcohol yield, which is related to the alcohol-producing microorganisms in the baijiu brewing environment. Yeast is an important alcohol-producing microorganism, and its optimal growth temperature range is usually 20℃ to 30℃. When the temperature reaches 36℃, it seriously affects the alcohol yield. When the fermentation temperature is 27℃, the alcohol content reaches 25.35±0.07% vol, and the ethyl lactate content reaches its maximum value of 0.55±0.03 g / L, second only to fermentation at 24℃. Slightly higher temperatures result in slightly higher esterification enzyme activity, which promotes the esterification reaction. When the fermentation temperature is above 27℃, the ethyl lactate content decreases significantly. This may be because the increased fermentation temperature leads to the rapid growth of acid-producing microorganisms in the mash in the early stage, especially lactic acid-producing microorganisms, resulting in a higher acidity in the mash environment. This reduces the yield of ethanol, a precursor to ethyl lactate synthesis, thus affecting the proportion of esters. Taking all factors into consideration, the optimal fermentation temperature was determined to be 27℃.

[0121] 5. Response Surface Design

[0122] 1) Response surface methodology results

[0123] Based on the results of the single-factor experiments, the ethyl lactate content (M) and alcohol content (N) of soy sauce-flavored baijiu were used as response values. The amount of brewing yeast added (A), the amount of lactic acid bacteria inoculated (B), and the fermentation temperature (C) were selected as independent variables. According to the principle of central composite experimental design, a three-factor, three-level response surface experiment was used to optimize the fermentation process conditions. The experimental factors and levels are shown in Table 4.

[0124] Table 4. Response surface methodology and levels for optimizing the enhanced fermentation process of soy sauce-flavored Baijiu.

[0125]

[0126] The response surface methodology was designed using Design-Expert13 software. The results are shown in Table 5, and the analysis of variance is shown in Table 6.

[0127] Table 5. Response surface methodology and results for optimizing the enhanced fermentation conditions of soy sauce-flavored Baijiu.

[0128]

[0129] The results were fitted using Design-Expret 13 with multiple regression to obtain the regression equations for each factor on the alcohol content and ethyl lactate content of soy sauce-flavored baijiu. The results are as follows:

[0130] M=28.43+1.09*A-1.19*B-1.34*C+0.5756*AB+0.0169*AC-1.29*BC-0.7303*A 2 -0.002*B 2 -1.56*C 2 ;

[0131] N=0.4955-0.1058*A+0.0926*B+0.0383*C+0.0479*AB+0.0577*AC-0.0928*BC-0.0578*A 2 -0.1060*B 2 -0.0954*C 2 ;

[0132] Table 6 shows that the response surface regression model is significant (P < 0.01), and the lack-of-fit term is not significant (P values ​​are all greater than 0.05), indicating that the model can reflect the relationship between the response value and various factors. In the regression model for alcohol content, the coefficient of determination R0 is... 2 = 0.9727, adjusted coefficient of determination R 2 adj =0.9377, indicating that the model is reasonable and effective; according to the p-value analysis, the linear terms A, B, C, BC and the quadratic term C 2The effect on alcohol content was highly significant (P < 0.01), while other factors were not significant (P > 0.05). The F-values ​​indicate that the order of influence of each factor on alcohol content is: fermentation temperature (C) > lactic acid bacteria inoculum size (B) > yeast addition amount (A). Regression model analysis of ethyl lactate content showed a coefficient of determination R0. 2 =0.9641, adjusted coefficient of determination R 2 adj =0.9179, the regression equation of the model is reasonable and effective; according to the p-value analysis, the linear terms A, B, BC and the quadratic term A 2 B 2 C 2 The effect of yeast addition on ethyl lactate content was extremely significant (P < 0.01), and the effects of C and AC on ethyl lactate content were significant (P < 0.05), while other factors were not significant (P > 0.05). Based on the F-values, the order of influence of each factor on ethyl lactate content was: yeast addition amount (A) > lactic acid bacteria inoculum amount (B) > fermentation temperature (C).

[0133] Table 6. Analysis of variance of the regression model with ethyl lactate content and alcohol content as response values.

[0134]

[0135] 2) Response Surface Analysis

[0136] Based on the response surface methodology and the shape of the contour lines, the effects of yeast addition, lactic acid bacteria inoculum size, and fermentation temperature on the ethyl lactate content in fortified fermentation of soy sauce-flavored baijiu were analyzed. The steepness of the response surface methodology and the shape of the contour lines reflect the interactions between the factors. A steeper response surface indicates a more significant influence of the experimental factors on the response value. Figure 16 As can be seen from the response surface graph and contour plot of the interaction of various factors, the alcohol content and ethyl lactate content show a trend of first increasing and then decreasing with the increase of each factor. The steepness of the response curve indicates that the interaction between the factors has a significant impact on the ethyl lactate content. The contour plots of each factor are all elliptical, indicating that the interaction between the factors is significant, which is consistent with the results of the analysis of variance.

[0137] 3) Verification test

[0138] Analysis using Design-Expert 13 software, with ethyl lactate content as the target maximum value for the dependent variable, yielded the optimal fermentation conditions for enhanced soy sauce-flavored baijiu fermentation using exogenous lactic acid bacteria: yeast addition 0.82‰ (W / W), inoculum size 6.838% (V / W), and fermentation temperature 26.35℃. Under these optimized conditions, the alcohol content was 27.41% vol, and the ethyl lactate content reached 0.55 g / L. Considering practical feasibility, the optimal fermentation conditions were revised to: yeast addition 0.8‰ (W / W), lactic acid bacteria inoculum size 6.8% (V / W), and fermentation temperature 26℃. Under these conditions, the alcohol content and ethyl lactate content in the enhanced fermentation distillate of soy sauce-flavored baijiu reached 27.70±0.14% vol and 0.54±0.04 g / L, respectively. The error range between the actual values ​​and the model predictions was within 5%, indicating that the model can effectively analyze the optimal fermentation process and has real reliability. Meanwhile, the total acid content of the distillate from the enhanced fermentation of soy sauce-flavored baijiu under these conditions was determined by acid-base titration to be 0.96±0.08 g / L.

[0139] 6. Detection of flavor components in soy sauce-flavored baijiu under optimal brewing conditions

[0140] Based on the results of the response surface methodology, the brewing process of soy sauce-flavored baijiu was optimized according to the 1L system basic formula: 200g of rice was steamed with water and cooled to 32℃, mixed with koji and 0.80‰ (w / w) yeast was added, and the inoculum amount of lactic acid bacteria was 6.80% (v / w). The fermentation temperature was 26℃, and the fermentation time was 15 days. The sampling method was as described in 2.2.3.2. Simultaneously, centrifuged precipitates were collected at the corresponding time points. The precipitate samples were stored at -80℃ for high-throughput DNA sequencing analysis.

[0141] 6.1 Qualitative and quantitative analysis of the flavor of soy sauce aroma type Baijiu under optimal brewing conditions

[0142] 1) Analysis of volatile flavor compounds in lactic acid bacteria-enhanced fermentation of soy sauce-flavored baijiu

[0143] According to the optimal process formula, the fermented soy sauce-flavored baijiu with lactic acid bacteria Y8 was used as the experimental group, and the baijiu brewed with the same process and the addition of an equal amount of inactivated lactic acid bacteria Y8 was set as the control group. The volatile flavor substances in the soy sauce-flavored baijiu were detected by gas chromatography. The results are shown in Table 7.

[0144] Table 7. Effects of intensified fermentation on volatile flavor compounds in soy sauce-flavored Baijiu samples.

[0145]

[0146]

[0147] Table 7 shows that 39 substances were detected in the fermented baijiu (Chinese liquor) before and after intensified fermentation, including 14 alcohols, 15 esters, and 4 aldehydes. Isobutyl acetate was only detected after intensified fermentation. Isobutyl acetate mainly presents fruity, floral, and pineapple-like aromas in baijiu, giving it a unique fruity and floral fragrance and making its taste smoother and sweeter. Ethyl stearate was only detected in the control group, possessing a fat- or wax-like aroma, which can add a unique and mellow base flavor to the baijiu. The remaining components were present both before and after intensified fermentation. Higher alcohols also play an important role in the flavor composition of soy sauce-flavored baijiu. In the experimental group, the contents of n-propanol, n-butanol, isobutanol, isoamyl alcohol, 2,3-butanediol A, and β-phenylethanol were significantly reduced, decreasing by 44.66%, 59.34%, 34.36%, 41.30%, 50.51%, and 32.28%, respectively, compared to the control group. Ethyl acetate and ethyl lactate levels were significantly increased, with ethyl lactate showing a particularly significant increase. Ethyl acetate content increased 4.4 times compared to the control group, and ethyl lactate content increased 3.4 times, reaching 604.25±33.80 mg / L. For soy sauce-flavored baijiu, aldehyde and ketone content should not be too high to avoid losing its original flavor characteristics. After intensified fermentation, acetaldehyde content decreased by 39.11%, to only 39.93±0.92 mg / L. 3-hydroxy-2-butanone was detected in the liquor after intensified fermentation at 0.94±0.31 mg / L, a decrease of 58.41%. Isobutyric acid decreased by 56.40%, while n-decanoic acid increased by 65.47%, with little change in the content of other volatile aroma components. This indicates that intensified lactic acid bacteria fermentation has a significant impact on the characteristic flavor compounds of soy sauce-flavored baijiu, mainly manifested in the decrease of characteristic higher alcohols and the increase of characteristic acids and esters. Lactic acid bacteria-enhanced fermentation of soy sauce-flavored baijiu can promote the formation of characteristic ester components and reduce aldehyde components, thereby improving the flavor of soy sauce-flavored baijiu.

[0148] 2) Determination of physicochemical parameters during fermentation

[0149] During the intensified fermentation process of soy sauce-flavored baijiu, samples were taken at 3, 5, 7, 12, and 15 days of fermentation, and their physicochemical indicators were measured. The results are as follows: Figure 17 As shown. By Figure 17 As shown in 'a', enhanced fermentation can significantly reduce the pH value of the fermentation mash. On the third day of fermentation, the pH value of group Y8 was 3.34, then slowly rose to 3.47, and finally stabilized at around 3.58; while in the control group, the pH value was 3.54 on the third day of fermentation, and the pH value increased with the increase of fermentation time, reaching a maximum of 3.87. Figure 17As shown in b, on the third day of fermentation, the reducing sugar content in the experimental group and the control group were 1.00 g / L and 1.19 g / L, respectively. Because soy sauce-flavored baijiu uses a double-fermentation process, in the initial stage, molds and yeasts utilize macromolecules such as starch in the grain raw materials to hydrolyze them, producing reducing sugars such as glucose and fructose. In the first three days of fermentation, the utilization rate of reducing sugars by microorganisms is relatively fast, therefore the content of residual reducing sugars in the mash environment is not high. On the 15th day of fermentation, the content of residual reducing sugars in the mash remained at 0.85 g / L, and the utilization of reducing sugars was similar in both the experimental and control groups. Figure 17 As shown in 'c', the alcohol content of the baijiu gradually increases with the number of fermentation days, with the fastest increase occurring between days 3 and 5. Correspondingly, the content of residual reducing sugars in the mash also shows a steady downward trend. This indicates that in this fermentation system, the utilization of reducing sugars and the production of alcohol by microorganisms are continuously and dynamically occurring, unlike the traditional production system where alcohol fermentation is basically completed within the first 3 days. Figure 17 As shown in d, the total acid content in the mash after enhanced fermentation was significantly higher than that in the experimental group, which explains the decrease in pH value in the mash. The total acid content in the control group remained in the range of 0.25–0.29 g / L, while the total acid content in the experimental group was 0.84–0.99 g / L. The exogenous addition of lactic acid bacteria may have promoted the growth of other acid-producing microorganisms, and the increase in total acidity is also conducive to the positive synthesis of esters. Figure 17 As shown in equation e, the exogenous addition of *Lactobacillus fermentatus* did not significantly differ from the control group in the initial fermentation stage, with a content of 2.84 g / L. However, during fermentation days 5-7, the content rapidly increased to 7.19 g / L, reaching its peak, representing a 28.39% increase compared to the control group. Figure 17 As shown in f, acetic acid is the main contributor to total acidity in the fermentation mash. In the experimental group, the total acid content reached 1.14 g / L, which was 470% higher than that in the control group. This indicates that the exogenous addition of lactic acid bacteria not only increased the lactic acid content but also caused changes in the acetic acid content.

[0150] 3) Characteristics of changes in higher alcohols and esters

[0151] During the enhanced fermentation process, the characteristic ester changes in soy sauce aroma-type baijiu are as follows: Figure 18As shown in the figure, the total ester content, ethyl acetate content, and ethyl lactate content in the experimental group were all significantly higher than those in the control group. Figure a shows that the total ester content in the experimental group exhibited a significant upward trend, increasing from 0.37±0.030 g / L on day 3 of fermentation to 1.24±0.014 g / L at the end of fermentation, representing a 335.04% increase compared to the control group. Figure b shows the trend of ethyl lactate content changes during fermentation; the ethyl lactate content changed most rapidly on day 5, then showed a slow upward trend, reaching 0.60±0.03 g / L at the end of fermentation, a 331.26% increase compared to the control group. Figure c shows the change in ethyl acetate content; in the experimental group, the ethyl acetate content increased rapidly on days 5 and 12 of fermentation, reaching 0.72±0.02 g / L on day 12, then slowly decreasing; at the end of fermentation, the content reached 0.66±0.07 g / L, a 334.97% increase compared to the control group. In the control group, the content of characteristic esters increased from day 5 of fermentation, reaching its peak at day 12, and then decreased. Research indicates that ester formation during baijiu brewing mainly occurs in the middle and late stages of fermentation, followed by hydrolysis which slightly reduces ester content, consistent with the control group. In the experimental group, however, ester content consistently increased, with ethyl lactate and total esters showing a high growth rate between days 12 and 15 of fermentation. This suggests that the exogenous addition of *Lactobacillus mucilaginosus* provided abundant lactic acid as a precursor in the fermentation system. The increase in ester content was related to the decrease in acid content, while lactic acid content showed a decreasing trend between days 7 and 15 of fermentation. Figure 17 The reason for e) is that lactic acid is consumed as a precursor to generate ethyl lactate, which promotes the forward reaction of ethyl lactate synthesis.

[0152] The effect of exogenous lactic acid bacteria addition on the content of higher alcohols in fermented soy sauce-flavored baijiu: Figure 19 As shown. Isobutanol, isoamyl alcohol, and β-phenylethanol are the main higher alcohols in soy sauce-flavored baijiu. Figure 19 The results showed that the yields of the three characteristic higher alcohols in the experimental group were significantly lower than those in the control group: isobutanol (0.25±0.03 g / L), isoamyl alcohol (0.28±0.04 g / L), and β-phenylethanol (48.4±0.95 mg / L). This indicates that the exogenous addition of lactic acid bacteria can reduce the content of higher alcohols within the aroma threshold range. The study found that *Saccharomyces cerevisiae* secretes various metabolites, especially amino acids, in a nitrogen-rich environment, thus supporting the growth of lactic acid bacteria. The consumption and utilization of amino acids reduces the amino acids leading to the Ehrlich pathway, thereby reducing the production of higher alcohols.

[0153] Previous research indicates that during the first 0-5 days of fermentation, the alcohol content in the fermentation mash of soy sauce-flavored baijiu increases rapidly, while the reducing sugar content decreases rapidly. During this stage, various physicochemical indicators show significant changes. From 5-12 days of fermentation, the precursors ethanol and lactic acid have accumulated sufficiently, and the synthesis rate of ethyl lactate is fastest during this stage. Therefore, metagenomic sequencing selected samples from five fermentation time points (3, 5, 7, 12, and 15 days), while metagenomic transcriptome sequencing selected samples from three fermentation time points (5, 7, and 12 days).

[0154] 7. Metagenomics sequencing and analysis

[0155] 1) Sample DNA extraction: Total genomic DNA was extracted and its concentration and purity were determined; DNA integrity was detected by 1% agarose gel electrophoresis. DNA fragmentation (Covaris M220 DNA) was performed and fragments of approximately 350 bp were selected to construct a PE library.

[0156] 2) Library construction and sequencing: Library construction was performed using NEXTFLEX Rapid DNA-Seq, and sequencing was performed using Illumina NovaSeq. TM Metagenomic sequencing was performed using the XPlus sequencing platform.

[0157] 3) Data quality control: Cut the adapter sequences of the 3' and 5' of reads (https: / / github.com / OpenGene / fastp, version 0.20.0), remove reads with a length of less than 50bp and an average base quality value of less than 20, retain high-quality sequences, compare reads with the host DNA sequence, and remove contaminating reads with high alignment similarity.

[0158] 4) Assembly and gene prediction: After assembling the optimized sequence, contigs with a length ≥300bp were selected (https: / / github.com / voutcn / megahit, version 1.1.2); ORFs were predicted for the selected contigs (https: / / github.com / hyattpd / Prodigal, version 2.6.3), and genes with a nucleic acid length ≥100bp were selected and translated into amino acid sequences.

[0159] 5) Classification and Functional Annotation: The amino acid sequences of non-redundant gene sets are compared with those in major databases (https: / / github.com / bbuchfink / diamond, version 2.0.13). Species and functional annotations are obtained through the taxonomic information databases corresponding to the libraries. The abundance of the corresponding species is calculated by summing the gene abundance of the species.

[0160] Library construction and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd. The raw data has been submitted to NCBI, sequence number: PRJNA1197119.

[0161] 8. Metatranscriptomics Measurement and Analysis

[0162] 1) RNA extraction: Total RNA was extracted and its concentration and purity were detected. RNA integrity was detected by agarose gel electrophoresis and the RQN value was determined. Samples with a total RNA amount of 1 μg, a concentration ≥30 ng / μL, RQN>6.5, and OD260 / 280 between 1.8 and 2.2 were selected.

[0163] 2) Library construction and sequencing: mRNA was isolated from total RNA using Oligo(dT) magnetic beads and polyA for AT base pairing. Fragmentation buffer was added to randomly break the mRNA into fragments of approximately 300 bp. cDNA was synthesized using the mRNA as a template via reverse chromatography. End Repair Mix was added to create blunt ends, and an A base was added to the 3' end. After adapter ligation, purification and fragment sorting were performed, followed by PCR amplification. The final purified library was then sequenced.

[0164] 3) Differential Expression Analysis and Functional Enrichment: Expression levels of each transcript were calculated using the transcript per million reads (TPM) method. RSEM was used to quantify gene abundance. Differential expression analysis was performed using DESeq2 or DEGseq. DEGs with |log2FC|≧1 and FDR<0.05 (DESeq2) or FDR<0.001 (DEGseq) were considered differentially expressed genes. Functional enrichment was performed using Goatools and Python scipy software; DEGs were significantly enriched in metabolic pathways at P<0.05.

[0165] Library construction and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd. The raw data has been submitted to NCBI, sequence number: PRJNA1199900.

[0166] The metagenomic raw data was filtered to analyze the composition and quality distribution of bases. The basic information of the gene sequences is shown in Table 8.

[0167] Table 8. Basic Information on Metagenomic and Metatranscriptome Sequencing Data Statistics

[0168]

[0169] Table 8 shows that metagenomic sequencing produced a maximum of 54,148,282 raw reads, of which the maximum number of clean reads was 536,654,184, and the maximum number of clean bases was 808,512,899 bp, indicating a low average sequencing error rate for clean reads. Q20 and Q30 represent the proportion of bases with Phred quality values ​​greater than 20 and 30, respectively, both exceeding 95%, with a GC content close to 50%, indicating that the metagenomic sequencing data is valid and usable for subsequent analysis. Transcriptome analysis of 12 samples yielded 98.00 Gb of raw data, detecting 22,859 unique genes. After quality control, 92.00 GClean data were obtained, yielding a maximum of 66,648,940 clean reads. Q20 and Q30 were greater than 93%, and the GC content close to 50%, indicating that the metagenomic transcriptome sequencing data is valid and usable for subsequent analysis. As shown in Table 9, the average sequence length of non-redundant genes is 877 bp. The non-redundant gene sequence set was annotated in various functional databases, as shown in Table 10. A large number of genes were successfully annotated in the KEGG and NR databases.

[0170] Table 9. Statistics of metagenomic sequencing data

[0171]

[0172] Table 10. Statistics of metatranscriptome gene annotation results

[0173]

[0174] 9. Diversity analysis of microbial communities

[0175] 9.1 Analysis of α-diversity of microorganisms

[0176] The changes in the Shannon index at the species level of microorganisms in the fermentation system are as follows: Figure 20As shown in a, the microbial diversity in the control group initially decreased and then increased, possibly due to favorable conditions in the early stages of fermentation. On day 12, the accumulation of alcohol and acid levels inhibited the growth of some intolerant microorganisms in the mash. However, by the end of fermentation, microbial diversity rebounded, possibly because some microorganisms gradually adapted to the mash environment and began to grow and reproduce. The microbial diversity in the experimental group showed the opposite trend to the control group, initially increasing and then decreasing. In the early stages of fermentation, the exogenous addition of lactic acid bacteria affected the microbial community of the yeast itself; the slightly acidic environment inhibited the growth of some microorganisms, resulting in significantly lower microbial diversity in the early stages of fermentation compared to the control group. As fermentation progressed, microbial diversity gradually increased, peaking on day 12 and then decreasing. The consumption of nutrients also prevented some microorganisms from surviving. The changes in the species-level Chao index of microorganisms in the fermentation system are shown in the figure below. Figure 20 As shown in b, its changes are the same as those of the Shannon index, indicating that the exogenous addition of lactic acid bacteria to enhance the fermentation of soy sauce-flavored baijiu alters the changes in microbial diversity and richness, thereby affecting the succession of microorganisms in the brewing environment.

[0177] 9.2 Microbial β-diversity analysis

[0178] PCA analysis was performed on the abundance of the microbial community, and the results are shown in [the table below]. Figure 21 CK and Y8 were separated to varying degrees, indicating that the microbial communities in the environment before and after enhanced fermentation were different, especially on days 5, 7, and 12 of fermentation. The variance contribution rates of principal component 1 and principal component 2 were 78.99% and 14.87%, respectively, suggesting that selecting PC1 and PC2 for sample analysis has good reliability. Figure 21 In the diagram, 'b' represents PCA clustering at the macrotranscriptional level, with principal component 1 contributing 68.83% of the variance. In this direction, the CK and Y8 samples are completely separated, similar to the clustering results at the metagenomic level. This indicates that the exogenous addition of lactic acid bacteria to enhance the fermentation of soy sauce-flavored baijiu affects the microbial community. This difference is mainly observed on days 5, 7, and 12 of fermentation, which are also the peak periods for the synthesis of flavor compounds in baijiu.

[0179] 9.3 Microbial Composition Analysis of Fermentation Broth

[0180] Based on metagenomics and metatranscriptomics, the abundance changes of microorganisms in the mash during the brewing process of lactic acid bacteria-enhanced soy sauce-flavored baijiu were annotated. The results are as follows: Figure 22 As shown.

[0181] As shown in Figure a, the top 5 most abundant species annotated in metagenomic sequencing are *Lactiplantibacillus*, *Limosilactobacillus*, *Pediococcus*, *Levilactobacillus*, and *Lactobacillus*, indicating that lactic acid bacteria have a significant advantage in the baijiu fermentation system. Figure b shows that at the species level, *Lactobacillus plantarum* is the most dominant in CK, while *Limosilactobacillus fermentum* is the most dominant in Y8. As fermentation progresses, the abundance of *Limosilactobacillus fermentum* decreases in Y8, while the abundance of *Pediococcus pentosaceus* gradually increases. As shown in Figure c, in metatranscriptome sequencing, the most actively expressed microorganisms were *Lactiplantibacillus*, while *Saccharomyces*, *Levilactobacillus*, and *Limosilactobacillus* were mainly expressed in the experimental group. As shown in Figure d, at the species level, *Lactiplantibacillus* sp. was the most dominant in the CK group, with its expression abundance showing a trend of first increasing and then decreasing. In the Y8 group, the abundance of *Saccharomyces cerevisiae*, *Rhizopus microsporus*, and *Bacillus* sp. gradually increased, while the abundance of *Pediococcus pentosaceus* and *Lactobacillus brevis* decreased. It is speculated that the abundance of *Lactobacillus plantarum* significantly influences the production of flavor compounds during the brewing process of soy sauce-flavored baijiu. *Lactobacillus brevis* may compete with *Saccharomyces cerevisiae*, and the exogenous addition of *Lactobacillus fermentatus* creates competition with *Lactobacillus brevis*, slowing the growth of *Lactobacillus brevis* and thus reducing its inhibitory effect on *Saccharomyces cerevisiae*. Metagenomic and metatranscriptomic data show that *Lactobacillus fermentatus* was stably expressed in the experimental group. Although *Saccharomyces cerevisiae* had a relatively low abundance at the gene level, it and *Lactobacillus plantarum* were the two most active species in the fermentation system. The differences in microbial community structure based on DNA and RNA sequencing suggest that some highly abundant microorganisms may not play a crucial role. Therefore, metagenomic and metatranscriptomic analysis helps to discover active microorganisms and their novel functions. In this study, the exogenous addition of *Lactobacillus fermentatus* mainly affected the succession of microorganisms in the brewing environment, especially the abundance of *Saccharomyces cerevisiae* and *Lactobacillus brevis*.

[0182] 9.4 Microbial Functional Analysis of Fermentation Broth

[0183] KEGG level 3 is the most specific level of pathway classification, encompassing more detailed metabolic pathways and functional modules. Annotations of metabolic pathways (level 3) in the two mash samples based on the KEGG database are as follows: Figure 23 As shown, the top 10 functional metabolic pathways are displayed. In the experimental group (Y8), the pathways of microbial metabolism in diverse environments, biosynthesis of secondary metabolites, biosynthesis of amino acids, glycolysis / gluconeogenesis, and carbon metabolism were the most active. In the control group (CK), the ribosome pathway was the most active. Microbial metabolic activities in different environments produce various flavor compounds, and their enhancement promotes the formation of flavor compounds in baijiu brewing and improves fermentation efficiency. The enhancement of secondary metabolic biosynthesis pathways means that microorganisms can synthesize these flavor compounds more efficiently, thereby increasing the variety and content of flavor compounds in baijiu. This indicates that the exogenous addition of lactic acid bacteria alters the abundance of microorganisms, thereby changing their growth, reproduction, and metabolism; the altered metabolic pathways further improve the formation of flavor compounds.

[0184] 9.5 Correlation Analysis between Environmental Factors and Microorganisms

[0185] To investigate the relationship between the succession of fermentation microbial communities and changes in environmental factors (pH, alcohol content, reducing sugar, and total acid), redundancy analysis (RDA) was performed on the top 8 most abundant microbial species and bioactive species in the mash, along with their physicochemical properties. As shown in Table 11, at the metagenomic level, pH (P<0.01, explanatory power = 77.32%) and alcohol content (P<0.01, explanatory power = 72.97%) were key environmental factors affecting microorganisms; at the metatranscriptomical level, pH (P<0.01, explanatory power = 95.53%) and total acid (P<0.01, explanatory power = 98.45%) were also key environmental factors affecting microorganisms.

[0186] Table 11 Redundancy analysis at the gene and transcription levels: Importance of environmental factors to mash microorganisms

[0187]

[0188]

[0189] Based on RDA redundancy analysis, the correlation results between microorganisms and environmental factors are as follows: Figure 24 As shown, at the genomic and transcriptomic levels, the experimental and control group samples showed good clustering. The control group samples were significantly affected by pH and alcohol content, and showed stronger correlations with *Lactiplantibecillus* sp., *Saccharomyces cerevisiae*, *Rhizopus microsporus*, and *Bacillus* sp. The experimental group samples were more significantly affected by residual reducing sugar and total acid, and showed stronger correlations with *Levilactobacillus brevs*, *Lactobacillus* sp., and *Limosilactobacillus fermentum*, indicating that the exogenous addition of *Lactobacillus fermentum* had a significant impact on the experimental group samples.

[0190] like Figure 24 As shown in 'a', at the genus level, pH is positively correlated with Weissella, Levilactobacillus, Lactiplantibecillus, and Pediococcus; alcohol content is positively correlated with Saccharomyces and Rhizopus; while Lactobacillus and Limosilactobacillus are positively correlated with residual reducing sugar and total acid. Figure 24 As shown in b, at the transcriptome species level, *Lactiplantibecillus* sp. showed a positive correlation with pH, ​​while *Pediococcus pentosaceus* and *Levilactobacillus brevs* showed a negative correlation. *Saccharomyces cerevisiae*, *Rhizopus microsporus*, and *Bacillus* sp. showed a positive correlation with alcohol content, while *Lactobacillus* sp. and *Limosilactobacillus fermentum* showed a positive correlation with residual reducing sugar and total acid. This indicates that the exogenous addition of fermenting *Lactobacillus* increases the total acid in the fermentation system, affecting the utilization of reducing sugar by other microorganisms, thereby affecting the content of residual reducing sugar.

[0191] 9.6 Correlation analysis between microorganisms and flavor compounds

[0192] The correlation between microorganisms and flavor compounds plays a crucial role in analyzing the flavor formation mechanism and optimizing the fermentation process of baijiu. Using the Pearson correlation coefficient, a visual correlation network diagram of microorganisms and typical flavor compounds during the fermentation of soy sauce-flavored baijiu was constructed (|r|>0.70), as shown below. Figure 25 As shown, the top 8 most abundant microorganisms and 40 flavor compounds were analyzed, including alcohols, acids, esters, aldehydes, and ketones. *Saccharomyces cerevisiae* (brewing yeast) showed a positive correlation with most flavor compounds, including higher alcohols (excluding hexanol and isoamyl alcohol), long-chain fatty acid esters such as ethyl octanoate, ethyl linoleate, ethyl decanoate, and ethyl stearate, hexanoic acid and isobutyric acid, and acetaldehyde, propionaldehyde, acetal, hexanol, and 3-hydroxy-2-butanone. Therefore, the succession of *Saccharomyces cerevisiae* may be an important factor influencing flavor compounds. Lactic acid bacteria dominated the mash in the later stages of fermentation. Figure 25 It was found that *Limosilactobacillus fermention* was positively correlated with the contents of ethyl lactate and acetic acid, and negatively correlated with the contents of β-phenylethanol, isoamyl alcohol, and propionaldehyde; *Limosilactobacillus sp.* was positively correlated with acetic acid, and negatively correlated with the contents of propionaldehyde and isoamyl alcohol; *Lactiplanbacillus plantarum* was positively correlated with the contents of isobutanol, β-phenylethanol, and ethyl acetate, and negatively correlated with the contents of acetic acid; *Lactiplanbacillus sp.* was negatively correlated with the contents of acetic acid, and positively correlated with the contents of isoamyl alcohol; *Levilactobacillus brevis* was negatively correlated with the contents of acetic acid, ethyl acetate, and ethyl lactate. *Wessella confusa* was positively correlated with β-phenylethanol, isobutanol, and isoamyl alcohol, and negatively correlated with the contents of ethyl lactate, ethyl acetate, and lactic acid, indicating that *Weissella confusa* has a relatively small effect on the contents of esters. Exogenous addition of lactic acid bacteria Y8 can increase the abundance of *Saccharomyces cerevisiae* in the later stages of fermentation, while decreasing the abundance of short-lived *Lactobacillus*. Based on the correlation between these two factors and flavor compounds, it can be inferred that lactic acid bacteria may indirectly promote the growth of microorganisms that are conducive to ester synthesis by affecting the proportion of microorganisms in the yeast, thereby increasing the synthesis efficiency of ethyl lactate and reducing the content of higher alcohols.

Claims

1. A fermenting Lactobacillus mucinus Y8, characterized in that, Its taxonomic name is Limosilactobacillus fermentum The sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.65150. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.2 of the sequence listing.

2. The application of the fermenting Lactobacillus Y8 of claim 1 in the production of fermented foods.

3. The application of the fermenting Lactobacillus Y8 of claim 1 in the production of fermented beverages.

4. The application of the fermenting Lactobacillus mucilaginosus Y8 as described in claim 1 in the brewing of soy sauce-flavored baijiu.

5. The application according to claim 4, characterized in that, The method for brewing soy sauce-flavored baijiu is as follows: Take rice, add water and steam until cooked, spread it out to cool, add yeast and water, then add brewing yeast and fermenting lactobacillus liquid for fermentation. The fermentation temperature is 24~36℃, and the fermentation time is 10~20 days. The amount of brewing yeast added is 0.5-5‰ (w / w) of the rice content.

6. The application according to claim 5, characterized in that, The fermented *Lactobacillus mucinus* broth was obtained by activating and culturing *Lactobacillus mucinus* Y8, and its concentration was 4.00 × 10⁻⁶. 9 CFU / mL.

7. The application according to claim 6, characterized in that, The amount of fermented Lactobacillus mucin solution added is 1~10% (V / W).

8. The application according to claim 7, characterized in that, The amount of fermented Lactobacillus mucin solution added is 1~5% (V / W).

9. The application according to claim 5, characterized in that, The fermentation temperature is 24~27℃.

10. The application according to claim 4 or 5, characterized in that, The application aims to enhance the quality and flavor of soy sauce-flavored baijiu, which is reflected in the following aspects: (1) Increase in the content of characteristic esters in baijiu; (2) The decrease in the content of characteristic higher alcohols, aldehydes and ketones in baijiu; (3) Increase in the content of characteristic acids in baijiu; The characteristic esters include ethyl lactate and ethyl acetate; The characteristic acids include lactic acid, acetic acid, and n-decanoic acid; The characteristic higher alcohols include n-propanol, n-butanol, isobutanol, isoamyl alcohol, and 2,3-butanediol A. β -Phenylacetyl alcohol; The aforementioned higher aldehydes include acetaldehyde; The characteristic higher ketones include 3-hydroxy-2-butanone.

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