Salt-tolerant tetragenococcus TL39-3 for regulating the fermentation community homeostasis and flavor quality of soybean paste and its identification method and application

Abstract

The application belongs to the technical field of fermented food microorganisms, and particularly relates to a salt-tolerant tetragenoccus sp. TL39-3 for regulating the fermentation community homeostasis and flavor quality of soybean paste and an identification method and application thereof. The application takes a synthetic microbial community which can reproduce the physicochemical and flavor characteristics of traditional naturally fermented soybean paste as a research object, establishes a one-way community disturbance system of full community control and bacterial single strain elimination, realizes accurate identification of key bacteria and causal confirmation of community level function through multidimensional evaluation of flavor metabolic characteristics and community structure of soybean paste at the end of fermentation, and confirms the key bacteria of the synthetic bacterial community of soybean paste from the causal level. T. halophilus TL39-3 is a key bacterium of the synthetic bacterial community of soybean paste: the strain can regulate community homeostasis, regulate lipid metabolism, phenylalanine metabolism, organic acid metabolism and peptide metabolism, improve the incense, fermentation, fruit, umami and strong taste of the product, and inhibit the undesirable flavor.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology for fermented foods, specifically relating to a halophilic tetracoccus TL39-3 that regulates the homeostasis and flavor quality of fermented soybean paste communities, as well as its identification method and application. Background Technology

[0002] Soybean paste is a typical representative of traditional fermented soybean products in my country and a core pillar product of the condiment industry, possessing multiple values ​​including nutrition, flavor, and cultural significance. Its fermentation process involves a series of biochemical reactions mediated by a complex microbial community. Bacteria, as the core functional group in the fermentation system, hydrolyze large soybean proteins through the secretion of extracellular enzymes such as proteases and peptidases, generating flavor-enhancing amino acids and active polypeptides, which form the core material basis for the product's umami and rich taste. Furthermore, they regulate the pH of the system by generating organic acids through metabolism, constructing a stable microecological environment, inhibiting the proliferation of putrefactive and pathogenic bacteria, and ensuring the product's biosafety. In addition, they can synergistically interact with yeasts, molds, and other groups to generate characteristic flavor substances such as esters and alcohols, forming the main flavor profile of the product. Therefore, the composition, interspecies interactions, and functional state of the bacterial community in the fermentation system directly determine the stability of soybean paste's quality, the controllability of its production, and its core industrial value.

[0003] In the complex bacterial community of fermented soybean paste, not all strains contribute equally. Key bacteria are the core group driving the fermentation process, determining product phenotype, and maintaining community structure and functional homeostasis. Their presence or absence directly determines whether the fermentation system can fully realize its functions. Current traditional natural fermentation suffers from core pain points such as complex and uncontrollable microbial composition, long fermentation cycles, and large batch-to-batch quality fluctuations, making it difficult to meet the demands of industrial standardization and upgrading. Numerous studies have shown that using synthetic microbial communities for inoculation fermentation is an important strategy to solve the dilemma of modernizing traditional fermented soybean paste. However, the prerequisite for the rational construction of synthetic microbial communities is to clarify the functions of microorganisms in traditionally naturally fermented soybean paste. Many scholars have conducted in-depth research on microorganisms involved in raw material degradation and flavor synthesis in traditional soybean paste, but the key microbial composition and related functions regulating community structure and functional homeostasis remain unclear.

[0004] Currently, the most common method for identifying key microorganisms in fermented foods in the field of microbial ecology is the topological index method. This method analyzes the dynamic changes of the microbial community during fermentation using techniques such as amplicon sequencing and metagenomics, and combines Pearson / Spearman correlation analysis and co-occurrence network analysis to screen for high-abundance or high-node microorganisms associated with fermentation phenotypes. However, this method can only establish a correlation between the microbial community and the phenotype, and cannot confirm the causal relationship of function. On the one hand, it cannot distinguish whether the function of the strain is a direct contribution or an indirect mediating effect, and it is easy to identify high-abundance concomitant bacteria as key bacteria, while it may miss low-abundance but core pivot bacteria that play a regulatory role in the community. Therefore, the results obtained are only theoretically relevant and cannot provide clear functional targets and causal evidence for the precise design of synthetic microbial communities, making it difficult to directly support the rational construction of industrially synthesized microbial communities.

[0005] The core bacterial groups in the soybean paste fermentation system, including *Bacillus*, *Tetracoccus*, and *Weissella*, are widely recognized as important participants in the fermentation process. *Tetracoccus halophilus*, as a typical salt-tolerant lactic acid bacterium in high-salt fermentation environments, has demonstrated its potential in organic acid production, tolerance to high-salt stress, and participation in amino acid metabolism in various high-salt fermented foods. However, due to the technical limitations of existing identification methods, whether *Tetracoccus halophilus* plays a key role in maintaining community homeostasis and driving flavor formation in the complex community of soybean paste has not yet been empirically proven at the causal level. Furthermore, existing research largely focuses on the enzyme activity and metabolic characteristics at the single-strain level, lacking functional confirmation in the context of community interactions. This results in a lack of precise theoretical basis for the application of *Tetracoccus halophilus* in the construction of the synthetic microbial community of soybean paste, limiting its potential for industrial application.

[0006] The elimination-by-one method is a classic approach for validating the function of microbial communities. By removing specific taxa one by one from a community and observing changes in phenotype and function, a causal relationship between microbial taxa and function can be directly established. However, this method struggles to achieve precise removal of single taxa in complex natural communities. Synthetic microbial communities, on the other hand, provide a controllable and reproducible research platform for the elimination-by-one method. Currently, there are no systematic reports combining the controllability of synthetic microbial communities with the causal validation capability of the elimination-by-one method for the precise identification of key bacteria in soybean paste. This has resulted in the screening of key functional bacteria in soybean paste fermentation remaining at the level of correlation inference for a long time, making it difficult to support the precise design and industrial application of standardized synthetic microbial communities.

[0007] Therefore, there is an urgent need to establish a key bacterial identification technology system that has been verified by cause and effect, and to screen out key strains that have the function of maintaining community homeostasis and regulation, so as to provide core technical support for the precise construction of microbial communities for soybean paste synthesis and the modernization and upgrading of the industry. Summary of the Invention

[0008] The purpose of this invention is to provide a halophilic tetracoccus TL39-3 that regulates the homeostasis and flavor quality of soybean paste fermentation communities.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a halophilic tetracoccus that regulates the homeostasis and flavor quality of soybean paste fermentation communities. Tetragenococcus halophilus The halophilic tetracoccus TL39-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50147 and deposit date of August 24, 2026.

[0010] This invention provides an application of the above-mentioned halophilic tetracoccus TL39-3 in soybean paste fermentation, the application including any one or more of the following: (a) Preparation of fermentation starter culture for soybean paste; (b) Regulating the homeostasis of the microbial community during the fermentation process of soybean paste; (c) Enhance the flavor quality of soybean paste, including increasing milky aroma, fermented aroma, fruity aroma, umami and richness, and / or suppressing the generation of undesirable flavors.

[0011] This invention provides a method for identifying a halophilic tetracoccus that regulates the homeostasis and flavor quality of soybean paste fermentation communities, comprising the following steps: (1) Construction of the synthetic microbial community system: A complete synthetic microbial community was obtained by compounding six microbial strains that can reproduce the physicochemical and flavor characteristics of traditional naturally fermented soybean paste. At the same time, a single-bacterial exclusion method was used to disturb the community by constructing individual bacterial strains for each strain. The six microbial strains include Bacillus belyssus ( B. velezensis BL13-3-1, Weissella Greekis ( W. hellenica ), Halophilic tetrad cocci ( T. halophilus TL39-3, Hansenula polymorpha ( D. hansenii ), short-stemmed broom mold ( S. brevicaulis ) and Penicillium ( Penicillium sp.); (2) Fermentation verification: The whole community of synthetic bacteria and the single bacterial deletion disturbance community were respectively inoculated into the soybean paste fermentation system for fermentation; (3) Multidimensional index determination: The volatile and non-volatile flavor metabolites and microbial community structure of fermented soybean paste were determined; (4) Identification of key bacteria: Through multivariate statistical analysis, the differences in volatile and non-volatile metabolites and the changes in microbial community structure between each single bacterial deletion group and the whole community control group were compared. The bacteria that have the greatest impact on community structure and flavor function are the key bacteria in soybean paste. The key bacteria mentioned in step (4) is the aforementioned halophilic tetracoccus ( T. halophilus TL39-3.

[0012] Preferably, the Bacillus belyssus BL13-3-1 in step (1) is deposited at the China General Microbiological Culture Collection Center, with the culture collection number CGMCC No. 34948 and the deposit date being June 19, 2025.

[0013] Preferably, the inoculum amounts of *Cladosporium brevis* and *Penicillium* in step (1) are 10... 6 CFU / g and 10 5 The inoculation ratio of CFU / g to CFU / g is 10:1.

[0014] Preferably, the inoculation amounts of Bacillus belye BL13-3-1, Weissella hellenica, Halophilic tetracoccus TL39-3, and Hansenula d'Barry yeast in step (1) are 10 8 10 7 10 7 and 10 6 CFU / g.

[0015] Preferably, the soybean paste fermentation system described in step (2) is prepared by the following method: soybeans are washed, soaked, and cooked until soft and tender. After cooling, they are inoculated with *Pseudomonas septum* and *Penicillium*, and then fermented and dried to obtain soybean koji. After adding salt and water to the soybean koji, the corresponding bacterial groups are inoculated and fermented under natural conditions.

[0016] Preferably, the volatile flavor metabolomic profile in step (3) is determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS); the non-volatile flavor metabolomic profile is determined by liquid chromatography-mass spectrometry (LC-MS); and the microbial community structure is determined by 16S rRNA gene amplicon sequencing and ITS gene amplicon sequencing, respectively, to determine the bacterial and fungal community structures.

[0017] Preferably, the multivariate statistical analysis in step (4) includes partial least squares discriminant analysis (PLS-DA) on volatile and non-volatile flavor components and principal coordinate analysis (PCoA) on the microbial community structure; the missing bacteria corresponding to the missing group that is furthest from the whole community control group sample in the PLS-DA score map and PCoA score map are identified as key bacteria.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through a combined identification system of synthetic microbial communities and the exclusion method, overcomes the limitations of traditional methods that can only perform correlation inferences, and confirms the causal relationship of *Halophilic tetracoccus* (HTH). Tetragenococcus halophilusTL39-3 is a core strain that regulates the community structure and flavor function of soybean paste fermentation, filling a technological gap in the functional positioning of this strain. This strain can effectively maintain the microecological homeostasis during fermentation, ensuring the stability of the fermentation process, while promoting the accumulation of flavor components and improving the flavor quality of the product, providing reliable strain support for the standardized and controllable fermentation of soybean paste. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is the phylogenetic tree of TL39-3.

[0021] Figure 2 PLS-DA model score plot (A) and substitution test plot (B) for volatile flavor components.

[0022] Figure 3 PLS-DA model score plot (A) and displacement test plot (B) for non-volatile flavor components.

[0023] Figure 4 Figures show the β-diversity and genus-level microbial composition of microorganisms in soybean paste. Specifically, Figures A and B show the β-diversity and genus-level species composition of bacteria in soybean paste fermented for 56 days, respectively; Figures C and D show the β-diversity and genus-level species composition of fungi in soybean paste fermented for 56 days, respectively; Figures E and F show the β-diversity and genus-level species composition of bacteria in the whole community group and the halophilic tetracoccus TL39-3 deletion group, respectively; and Figures G and H show the β-diversity and genus-level species composition of fungi in the whole community group and the halophilic tetracoccus TL39-3 deletion group, respectively.

[0024] Figure 5 Differences in volatile flavor metabolites of fermented soybean paste inoculated with two synthetic microbial communities were analyzed. Figures A and B represent the score plots and permutation test plots of OPLS-DA analysis for the whole community group and the halophilic tetracoccus TL39-3 deletion group, respectively. Figure C represents the important variable projection values ​​(VIP) based on the OPLS-DA model. Figure D represents a comparative heatmap of key differential volatile flavor components (VIP > 1) between the whole community group and the halophilic tetracoccus TL39-3 deletion group.

[0025] Figure 6Differences in non-volatile flavor components of fermented soybean paste inoculated with two synthetic microbial communities were analyzed. Figures A and B represent the score plots and permutation test plots of OPLS-DA analysis of non-volatile flavor components in soybean paste from the whole community group and the group lacking *Tetracoccus halophilus* TL39-3, respectively. Figure C represents the important variable projection values ​​(VIPs) based on the OPLS-DA model. Figure D represents a comparative heatmap of key difference flavor components (VIP > 1) between soybean paste from the whole community group and the group lacking *Tetracoccus halophilus* TL39-3.

[0026] Preservation information of strains Collection Name: TL39-3; Taxonomous Tetracoccus Tetragenococcus halophilus ; Preservation institution: China General Microbiological Culture Collection Center; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 50147; Date of preservation: August 24, 2026. Detailed Implementation

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0029] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0030] Example 1: Preparation of spore solution and bacterial suspension 1.1 The synthetic bacterial culture used in this embodiment was composed of 6 self-isolated bacterial strains, namely: Bacillus belyssus ( B. velezensis BL13-3-1, Weissella Greekis ( W. hellencia M036, Halophilic tetrad cocci ( T. halophilus TL39-3, Hansenula polymorpha ( D. hansenii L11-8Y, short-stalk broom enzyme (S. brevicaulis MicL1, Penicillium ( Penicillium sp.)ML8-10-1.

[0031] Among them, *Halophilic tetracoccus* TL39-3 and *Bacillus belye* BL13-3-1 have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 50147 and CGMCC No. 34948, respectively, and accession dates of August 24, 2026 and June 19, 2025, respectively. The remaining strains are common functional microorganisms in traditional fermented foods, and those skilled in the art can obtain similar strains from naturally fermented soybean paste using conventional isolation and screening techniques.

[0032] In addition, Bacillus belysium BL13-3-1 has been deposited and disclosed in patent CN121086951A.

[0033] 1.2 S. brevicaulis MicL1 and Penicillium sp. Preparation of ML8-10-1 spore solution Will S. brevicaulis MicL1 and Penicillium sp. After culturing ML8-10-1 in PDA medium at 28 ℃ for 7 days, spores were collected using sterile physiological saline containing 0.05% Tween 80. Sterile glass beads were added to the collected spore solution, and the mixture was shaken in a shaker at 200 r / min for 15 min. After filtration through 8 layers of sterile gauze to remove excess hyphae, the filtered spore solution was centrifuged (5 min, 5000 r / min), washed 3 times with sterile physiological saline, and resuspended in sterile physiological saline to obtain the spore solution.

[0034] 1.3 B. velezensis Preparation of BL13-3-1 bacterial suspension Will B. velezensis BL13-3-1 was inoculated into LB medium and cultured at 37 °C for 24 h on a shaker at 150 r / min. Then, 2% of the culture was inoculated into fresh LB medium and cultured at 150 r / min for 24 h to prepare a seed culture. The seed culture was centrifuged (5 min, 5000 r / min), washed three times with sterile physiological saline, and resuspended in sterile physiological saline. B. velezensis Bacterial suspension of BL13-3-1.

[0035] 1.4 W. hellenica Preparation of M036 bacterial suspension Will W. hellenicaM036 was inoculated into MRS medium and cultured at 37 ℃ for 18 h. Then, it was inoculated at a 2% inoculum into fresh MRS medium and cultured at 37 ℃ for 12 h to prepare a seed culture. The seed culture was centrifuged (5 min, 5000 r / min), washed three times with sterile physiological saline, and resuspended in sterile physiological saline. W. hellenica M036 bacterial suspension.

[0036] 1.5 T. halophilus Preparation of TL39-3 bacterial suspension Will T. halophilus TL39-3 was inoculated into 8% MRS medium and cultured at 30°C for 48 h. Then, 2% of the culture was inoculated into fresh 8% MRS medium and cultured at 30°C for another 48 h to prepare a seed culture. The seed culture was centrifuged (5 min, 5000 r / min), washed three times with sterile physiological saline, and resuspended in sterile physiological saline. T. halophilus A bacterial suspension of TL39-3. Among them, *Hydrophilic tetracoccus* TL39-3 was confirmed by whole-genome sequencing. Tetragenococcus halophilus ( Figure 1 It has been deposited at the China General Microbiological Culture Collection Center.

[0037] 1.6 D. hansenii Preparation of L11-8Y bacterial suspension Will D. hansenii L11-8Y culture medium was inoculated into PDB medium and cultured at 28 °C for 24 h using a shaker at 150 r / min. Then, a 2% inoculum was added to fresh PDB medium and cultured at 150 r / min for 24 h to prepare the seed culture. The seed culture was centrifuged (5 min, 5000 r / min), washed three times with sterile physiological saline, and resuspended in sterile physiological saline. D. hansenii L11-8Y bacterial suspension.

[0038] Example 2: Preparation and collection of fermented soybean paste from whole-community and bacterial-deficient microbial communities. 2.1 Composition Weigh out 2000 g of soybeans, wash them thoroughly with clean water, soak them overnight, then steam them in a jacketed kettle for 1.5 hours until they are soft. Remove the soybeans and let them cool. Simultaneously, inoculate with... S. brevicaulis MicL1 and Penicillium sp .ML8-10-1, S. brevicaulis MicL1 and Penicillium sp The inoculation dose for ML8-10-1 was 10. 6The inoculation ratio of CFU / g to soybeans was 10:1. After inoculation, the soybeans were placed in a well-ventilated plastic basket. A sterilized, breathable cloth was placed at the bottom of the basket, and then another cloth was placed on top of the soybeans. The basket was then left to ferment for 72 hours. After fermentation, the soybeans were sun-dried until semi-dry.

[0039] 2.2 Fermentation Sterilize the fermentation jars with alcohol, then place the sun-dried koji into the jars. Add 200g of coarse salt and 1500g of boiled water to each jar. S. brevicaulis MicL1 and Penicillium sp Microorganisms other than those in ML8-10-1 were inoculated into the fermented soybean paste according to the inoculation strategy in Table 1 and mixed thoroughly. B. velezensis BL13-3-1 W. hellenica M036 T. halophilus TL39-3, D. hansenii The vaccination doses for L11-8Y were 10 8 10 7 10 7 and 10 6 The inoculation ratios of the above-mentioned strains in groups A, AB, AW, and AT were 100:10:10:1, 0:10:10:1, 100:0:10:1, and 100:10:0:1, respectively. After sealing the top of the jar with a double layer of cloth, fermentation was carried out under natural conditions. The soybean paste was stirred daily with a sterilized spoon.

[0040] Table 1. Inoculation strategies for different synthetic microbial communities

[0041] 2.3 Sample Collection Soybean paste samples were collected at 0, 7, 14, 28, and 56 days. The collected samples were divided into three aliquots: one aliquot was stored at -80°C for 16S, ITS, and metabolomics testing; the other aliquot was stored at -20°C for physicochemical and volatile metabolite detection. Physicochemical, metabolomics, and microbiome analyses were performed within one week of all sample collection. Three independent fermentation experiments were conducted in each experimental group.

[0042] Example 3: Identification of Key Microorganisms 3.1 Effects of bacteria on the volatile flavor metabolite profile of soybean paste The volatile components of soybean paste were determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0043] Accurately weigh 2 g of sample into a 25 mL solid-phase microextraction (SPME) bottle, add 2 g NaCl and 6 mL of CO2-free distilled water, and add 5 μL of 25 mg / L 2-octanol as an internal standard. After placing the bottle in a magnetic rotor, seal the cap and place it in a constant temperature water bath at 55 ℃ with magnetic stirring. Use the SPME extraction head to adsorb volatile compounds for 45 min. After extraction, place the extraction head in the GC-MS inlet and desorb at 250 ℃ for 5 min.

[0044] For the separation of volatile components, an FFAP capillary column was used, and the instrument parameters were set as follows: the carrier gas was high-purity helium (>99.999%), the flow rate was 1 mL / min, and the splitless mode was used; the temperature program was as follows: the initial temperature was 40 ℃ and held for 3 min, then the temperature was increased to 180 ℃ at a rate of 5 ℃ / min and held for 5 min, and then increased to 220 ℃ at a rate of 5 ℃ / min and held for 8 min.

[0045] Mass spectrometry conditions: ion source temperature 230 °C, electron energy 70 eV, mass range 33–450 amu. Volatile compounds were qualitatively identified using the NIST 11.0 database, and the content of each volatile component was calculated based on the peak area ratio of the target compound to the internal standard 2-octanol.

[0046] A total of 47 volatile flavor components were detected in all soybean paste samples, and their contents are shown in Table 2. The results of PLS-DA analysis of these components are as follows: Figure 2 As shown, R²X, R²Y, and Q² are all greater than 0.85, and the intercept in the permutation test plot is negative, confirming that the PLS-DA model has good fit and predictive ability, and that the model is robust, reliable, and free from overfitting risk.

[0047] PLS-DA score graph T. halophilus The TL39-3 missing group soybean paste showed a greater European distance from the whole community group soybean paste, indicating that... T. halophilus TL39-3 is the most important bacterium affecting the volatile flavor components of soybean paste.

[0048] Table 2. Content of volatile components in soybean paste

[0049] Note: - indicates not detected. Volatility levels are expressed as mean ± standard deviation; different lowercase letters in the same line indicate significant differences. P<0.05, the odor descriptions in the table are based on the reference flavor databases (http: / / www.flavornet.html) and (https: / / www.femaflavor.org).

[0050] 3.2 Effects of bacteria on the nonvolatile metabolic profile of soybean paste Non-volatile components in soybean paste were determined using liquid chromatography-mass spectrometry (LC-MS). The specific procedure was as follows: An appropriate amount of sample was placed in a 2 mL centrifuge tube, and 600 µL of a methanol solution containing 4 ppm 2-chloro-L-phenylalanine was added. The mixture was vortexed for 30 s. A steel ball was added, and the mixture was then placed in a tissue homogenizer and homogenized at 55 Hz for 60 s. The homogenate was sonicated at room temperature for 15 min. Subsequently, the mixture was centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was filtered through a 0.22 µm filter membrane, and the filtrate was transferred to a detection vial for LC-MS analysis. Analysis was performed using a Vanquish ultra-high performance liquid chromatography (UHPLC) system. The chromatographic column used was an ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm), and the column temperature was 40 ℃. The mobile phase flow rate was 0.3 mL / min, and the injection volume was 2 μL. The mobile phases for LC-ESI(+)-MS analysis were (B2) 0.1% formic acid acetonitrile (v / v) and (A2) 0.1% formic acid water (v / v). The gradient elution program was: 0–1 min, 8% B2; 1–8 min, 8%–98% B2; 8–10 min, 98% B2; 10–10.1 min, 98%–8% B2; 10.1–12 min, 8% B2. For LC-ESI(-)-MS analysis, the mobile phases were (B3) acetonitrile and (A3) ammonium formate (5 mM), with the gradient elution program as follows: 0–1 min, 8% B3; 1–8 min, 8%–98% B3; 8–10 min, 98% B3; 10–10.1 min, 98%–8% B3; 10.1–12 min, 8% B3. Mass spectrometry conditions: An Orbitrap Exploris 120 spectrometer with an ESI ion source was used, and MS1 and MS / MS data were acquired simultaneously in full MS-ddMS2 mode. Parameter settings were as follows: sheath gas pressure, 40 arb; auxiliary gas flow rate, 10 arb; spray voltage, 3.50 kV for ESI(+) and -2.50 kV for ESI(-), respectively; capillary temperature, 325 ℃; MS1 range, 100–1000 m / z; MS1 resolution, 60000 FWHM; number of correlation scans per cycle, 4; MS / MS resolution, 15000 FWHM; normalized collision energy, 30%; dynamic exclusion time, automatic.

[0051] A total of 2462 non-volatile metabolites were detected under both positive and negative ion modes. Analysis of the flavor characteristics of these metabolites revealed 42 flavor-enhancing metabolites, as shown in Table 3. PLS-DA analysis was performed on the flavor metabolites of the whole community group and the bacterial-deficient community group of soybean paste; the results are shown in Table 3. Figure 3 R²X, R²Y, and Q² are all greater than 0.75, and the intercepts in the permutation test plots are negative, confirming that the PLS-DA model has good goodness of fit and predictive ability, is robust and reliable, and has no risk of overfitting. The flavor metabolites of soybean paste samples from the whole community group and the single-bacterial deletion group showed significant differences. The flavor metabolite profiles of the AB and AW groups were more similar, while the flavor metabolite profile of the AT group differed most from that of the whole community group. This indicates... T. halophilus The loss of TL39-3 had the most significant impact on the flavor metabolites of soybean paste.

[0052] Table 3 Peak areas of non-volatile flavor components

[0053] Note: Peak areas of flavor components are expressed as mean ± standard deviation, and different lowercase letters in the same row represent significant differences. P <0.05).

[0054] 3.3 The Influence of Bacteria on the Microbial Structure of Soybean Paste Genomic DNA was extracted from soybean paste using the EZNA soil DNA kit. The concentration and purity of the DNA were determined by 1% agarose gel electrophoresis.

[0055] The primer pairs for the V3-V4 region of the 16S rRNA gene were 341F 5'-CCTAYGGGRBGCASCAG-3' (SEQ ID NO.1) and 806R 5'-GGACTACNNGGGTATCTAAT-3' (SEQ ID NO.2). The universal primer pairs for the ITS region of the fungal gene were ITS-1F 5'-CTTGGTCATTTAGAGGAAGTAA-3' (SEQ ID NO.3) and ITS-1R 5'-GCTGCGTTCTTCATCGATGC-3' (SEQ ID NO.4). The amplification conditions were: 98 ℃ for 1 min, 98 ℃ for 10 s, 50 ℃ for 30 s, 72 ℃ for 30 s, for a final extension at 72 ℃ for 5 min.

[0056] PCR products were purified using the Qiagen gel extraction kit. Sequencing libraries were prepared using the Illumina TruSeq® DNA PCR-Free Sample Preparation Kit. Paired-end (2×50) sequencing was performed using the Illumina NovaSeq platform. The resulting amplicon sequence variants (ASVs) were then used as characteristic sequences, and abundance tables for species annotation were generated. The Silva database (https: / / www.arb-silva.de) and the UNITE database (https: / / unite.ut.ee) were used for bacterial and fungal taxonomic annotation, respectively.

[0057] The microbial communities of fermented soybean paste were analyzed in the whole community group and the bacterial-deficient community group. The results are shown in […]. Figure 4 β-diversity analysis showed ( Figure 4 A and 4C), T. halophilus The TL39-3 deletion group had the greatest European distance from the whole community soybean paste, indicating that... T. halophilus TL39-3 had the greatest impact on the bacterial and fungal community structure of soybean paste, and the community composition analysis results ( Figure 4 B and 4D) are consistent with the above conclusions.

[0058] Further comparison of the community succession dynamics throughout the fermentation process of the two groups of soybean paste ( Figure 4 E, 4G): At the bacterial community level, days 0-7 were critical windows for microbial colonization and primary community succession in both soybean paste groups; the whole bacterial community showed tight aggregation from day 14 onwards, indicating a stable community state. The synthetic microbial community lacked... T. halophilus At TL39-3, the bacterial community underwent continuous succession throughout the fermentation process, but a stable community structure did not form. At the fungal community level, the entire bacterial group underwent rapid succession from 0 to 7 days, and the structure tended to stabilize after day 7; the synthetic community lacked [specific details needed]. T. halophilus At TL39-3, the fungal community structure underwent significant changes between 14 and 28 days, indicating a lag in the community succession process. The dynamic structure of the community composition is shown in [reference needed]. Figure 4 F and 4H.

[0059] By day 7 of fermentation, the bacterial community structure of the two groups of soybean paste showed significant differentiation: in the group inoculated with the whole bacterial community, *Tetracoccus* proliferated rapidly due to its strong tolerance to high salt, reaching a relative abundance of 50% and becoming the dominant genus; while... T. halophilus In the TL39-3 deletion group, no single dominant bacterial genus was found in the soybean paste. Enterobacteriaceae, Weissella, and Proteus were all dominant. In the middle and late stages, the abundance of Weissella was significantly higher than that of the whole community group, becoming the core dominant bacteria.

[0060] In terms of fungi, *Debaliyces* is the dominant genus in the late stage of fermentation. T. halophilus The abundance of TL39-3 in the soybean paste was significantly reduced in the TL39-3 deletion group, indicating that...T. halophilus TL39-3 can promote the proliferation and colonization of the Debali yeast genus.

[0061] In conclusion, T. halophilus TL39-3 can regulate the dynamic succession of microbial communities and promote bacterial community stability. The absence of this strain will make it difficult for the bacterial community to reach a steady state, while the succession window of the fungal community will be significantly delayed.

[0062] 3.4 Identification of key microorganisms By comparing the volatile and non-volatile flavor components, microbial community structure, and composition of soybean paste in the whole community group and the single bacterial absence group, it was found that... T. halophilus TL39-3 had the most significant disturbance effect on soybean paste, and this bacterium was identified as a key microorganism affecting the microbial community structure and flavor homeostasis.

[0063] Example 4 T. halophilus The function of TL39-3 in the production of soybean paste flavor 4.1 T. halophilus Effect of TL39-3 on the volatile flavor of soybean paste Volatile components of soybean paste were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). 2 g of sample was accurately weighed into a 25 mL headspace solid-phase microextraction bottle, followed by the addition of 2 g NaCl, 6 mL of CO2-free distilled water, and 5 μL of 25 mg / L 2-octanol as an internal standard. The bottle was then placed in a magnetically stirred water bath at 55 ℃, and the SPME extraction head was used to adsorb volatile compounds for 45 min. After extraction, the extraction head was placed in the GC-MS inlet, and desorption was performed at 250 ℃ for 5 min. The volatile components were separated using an FFAP capillary column with the following instrument parameters: carrier gas was high-purity helium (>99.999%), flow rate was 1 mL / min, and splitless mode was used; the temperature program was as follows: initial temperature 40 ℃ held for 3 min, increased to 180 ℃ at a rate of 5 ℃ / min, held for 5 min, then increased to 220 ℃ at a rate of 5 ℃ / min, and held for 8 min. Mass spectrometry conditions were: ion source temperature 230 ℃, electron energy 70 eV, and mass range 33–450 amu. The volatile compounds were qualitatively identified using the NIST 11.0 database, and the content of each volatile component was calculated based on the peak area ratio of the target compound to the internal standard 2-octanol.

[0064] For the whole community group and T. halophilus Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the volatile flavor of fermented soybean paste containing the TL39-3 missing microbiota. The results are shown in [Figure number missing]. Figure 5 The OPLS-DA score chart shows that the whole-culture group of soybean paste and T.halophilus The soybean paste in the TL39-3 deletion group showed obvious separation along the first principal component, indicating that... T. halophilus TL39-3 can alter the overall volatile flavor composition of soybean paste. Based on the OPLS-DA model, 28 volatile components with a variable importance projection value (VIP) greater than 1 were selected, which are the key markers causing the differences in volatile flavor between the two groups of soybean paste. Among them, the content of 19 volatile flavor components was significantly upregulated, and 9 were significantly downregulated, indicating that... T. halophilus TL39-3 exhibits a strong regulatory effect on the formation of volatile flavor compounds. Compared to a complete synthetic microbial community system, the synthetic microbial community lacks... T. halophilus TL39-3 significantly reduced the content of fatty acids (such as 4-methylhexanoic acid and octanoic acid) that impart a sour taste and cheese aroma to soybean paste; at the same time, the content of aromatic metabolites such as benzaldehyde, phenylacetaldehyde, and phenylacetic acid, which impart floral and fruity aromas, decreased substantially, while esters such as ethyl phenylpropionate and ethyl decanoate accumulated in large quantities. In addition, the content of compounds related to greasy, musty, earthy, and phenolic off-flavors, such as palmitic acid, decanal, 3-octanol, acetophenone, and p-cresol, increased significantly.

[0065] 4.2 T. halophilus Effects of TL39-3 on non-volatile flavor components of soybean paste Non-volatile components in soybean paste were determined using liquid chromatography-mass spectrometry (LC-MS). The specific procedure was as follows: An appropriate amount of sample was placed in a 2 mL centrifuge tube, and 600 µL of a methanol solution containing 4 ppm 2-chloro-L-phenylalanine was added. The mixture was vortexed for 30 s. A steel ball was added, and the mixture was then placed in a tissue homogenizer and homogenized at 55 Hz for 60 s. The homogenate was sonicated at room temperature for 15 min. Subsequently, the mixture was centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was filtered through a 0.22 µm filter membrane, and the filtrate was transferred to a detection vial for LC-MS analysis. Analysis was performed using a Vanquish ultra-high performance liquid chromatography (UHPLC) system. The chromatographic column used was an ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm), and the column temperature was 40 ℃. The mobile phase flow rate was 0.3 mL / min, and the injection volume was 2 μL. The mobile phases for LC-ESI(+)-MS analysis were (B2) 0.1% formic acid acetonitrile (v / v) and (A2) 0.1% formic acid water (v / v). The gradient elution program was: 0–1 min, 8% B2; 1–8 min, 8%–98% B2; 8–10 min, 98% B2; 10–10.1 min, 98%–8% B2; 10.1–12 min, 8% B2. For LC-ESI(-)-MS analysis, the mobile phases were (B3) acetonitrile and (A3) ammonium formate (5 mM), with the gradient elution program as follows: 0–1 min, 8% B3; 1–8 min, 8%–98% B3; 8–10 min, 98% B3; 10–10.1 min, 98%–8% B3; 10.1–12 min, 8% B3. Mass spectrometry conditions: An Orbitrap Exploris 120 spectrometer with an ESI ion source was used, and MS1 and MS / MS data were acquired simultaneously in full MS-ddMS2 mode. Parameter settings were as follows: sheath gas pressure, 40 arb; auxiliary gas flow rate, 10 arb; spray voltage, 3.50 kV for ESI(+) and -2.50 kV for ESI(-), respectively; capillary temperature, 325 ℃; MS1 range, 100–1000 m / z; MS1 resolution, 60000 FWHM; number of correlation scans per cycle, 4; MS / MS resolution, 15000 FWHM; normalized collision energy, 30%; dynamic exclusion time, automatic.

[0066] For the whole community group and T. halophilus Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the non-volatile flavor components of fermented soybean paste from the TL39-3 deletion group. The results are shown in [Figure number missing]. Figure 6 The OPLS-DA score chart shows that the whole-culture group of soybean paste and T. halophilus The soybean paste in the TL39-3 deletion group showed obvious separation along the first principal component, indicating that... T. halophilusTL39-3 can alter the overall flavor composition of fermented soybean paste. Based on the OPLS-DA model, 25 volatile components with a variable importance projection value (VIP) greater than 1 were selected as key markers causing the flavor differences between the two groups of fermented soybean paste. Among them, the content of 13 volatile flavor components was significantly increased, and that of 12 was significantly decreased, indicating that... T. halophilus TL39-3 exhibits a strong regulatory effect on the formation of non-volatile flavor compounds. Compared to a complete synthetic microbial community, the absence of *Tetracoccus halophilus* TL39-3 significantly reduced the content of umami compounds (Asp and Asp-Glu), the strong-flavor compound Gly-Glu, and the sweet compound mannitol in fermented soybean paste. This indicates that this strain can directly or indirectly promote the formation of these substances, while the content of bitter amino acids (Leu, His, Phe, Trp, and Met) and acidic organic acids (malic acid and succinic acid) was significantly reduced. T. halophilus The significant increase in TL39-3 after deletion indicates... T. halophilus TL39-3 can consume and transform bitter substances during fermentation, and can convert highly irritating malic acid into other acids, thus balancing the sourness of the product and maintaining the harmony and palatability of the bean paste flavor.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A halophilic tetracoccus that regulates the homeostasis and flavor quality of soybean paste fermentation community ( Tetragenococcus halophilus TL39-3, characterized in that, The halophilic tetracoccus TL39-3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50147 and deposit date of August 24, 2026.

2. The application of the halophilic tetracoccus TL39-3 described in claim 1 in soybean paste fermentation, characterized in that, The application includes any one or more of the following: (a) Preparation of fermentation starter culture for soybean paste; (b) Regulating the homeostasis of the microbial community during the fermentation process of soybean paste; (c) Enhance the flavor quality of soybean paste, including increasing milky aroma, fermented aroma, fruity aroma, umami and richness, and / or suppressing the generation of undesirable flavors.

3. A method for identifying a halophilic tetracoccus TL39-3 that regulates the homeostasis and flavor quality of the fermented soybean paste community as described in claim 1, characterized in that, Includes the following steps: (1) Construction of the synthetic microbial community system: A complete synthetic microbial community was obtained by compounding six microbial strains that can reproduce the physicochemical and flavor characteristics of traditional naturally fermented soybean paste. At the same time, a single-bacterial exclusion method was used to disturb the community by constructing individual bacterial strains for each strain. The six microbial strains include Bacillus belyssus ( Bacillus velezensis BL13-3-1, Weissella Greekis ( Weissella hellenica ), Halophilic tetrad cocci ( T. halophilus TL39-3, Hansenula polymorpha ( Debaryomyces hansenii ), short-stemmed broom mold ( Scopulariopsis brevicaulis ) and Penicillium ( Penicillium sp . ); (2) Fermentation verification: The whole community of synthetic bacteria and the single bacterial deletion disturbance community were respectively inoculated into the soybean paste fermentation system for fermentation; (3) Multidimensional index determination: The volatile and non-volatile flavor metabolites and microbial community structure of fermented soybean paste were determined; (4) Identification of key bacteria: Through multivariate statistical analysis, the differences in volatile and non-volatile metabolites and the changes in microbial community structure between each single bacterial deletion group and the whole community control group were compared. The bacteria that have the greatest impact on community structure and flavor function are the key bacteria in soybean paste. The key bacteria mentioned in step (4) is the halophilic tetracoccus described in claim 1 ( T. halophilus TL39-3.

4. The method according to claim 3, characterized in that, The Bacillus belyssus BL13-3-1 described in step (1) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34948 and accession date of June 19, 2025.

5. The method according to claim 3, characterized in that, The inoculation amounts of *Cladosporium brevesense* and *Penicillium* in step (1) were 10... 6 CFU / g and 10 5 The inoculation ratio of CFU / g to CFU / g is 10:

1.

6. The method according to claim 3, characterized in that, In step (1), the inoculation amounts of Bacillus belye BL13-3-1, Weissella hellenica, Halophilic tetracoccus TL39-3, and Hansenula d'Barry yeast were 10. 8 10 7 10 7 and 10 6 CFU / g.

7. The method according to claim 3, characterized in that, The soybean paste fermentation system described in step (2) is prepared by the following method: Soybeans are washed, soaked, and cooked until soft and tender. After cooling, they are inoculated with *Pseudomonas septum* and *Penicillium*. The fermented soybean paste is then made into koji and dried. After adding salt and water to the koji, the corresponding microbial groups are inoculated and fermented under natural conditions.

8. The method according to claim 3, characterized in that, The volatile flavor metabolomic profile described in step (3) was determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS); the non-volatile flavor metabolomic profile was determined by liquid chromatography-mass spectrometry (LC-MS); and the microbial community structure was determined by 16S rRNA gene amplicon sequencing and ITS gene amplicon sequencing, respectively, to determine the bacterial and fungal community structures.

9. The method according to claim 3, characterized in that, The multivariate statistical analysis described in step (4) includes partial least squares discriminant analysis (PLS-DA) of volatile and non-volatile flavor components and principal coordinate analysis (PCoA) of microbial community structure; the missing bacteria corresponding to the missing group that is furthest from the whole community control group sample in the PLS-DA score map and PCoA score map are identified as key bacteria.

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