Bacillus subtilis JQBS002 and application thereof

Abstract

The present application relates to the field of microbial technology, and provides a bacillus subtilis JQBS002 Bacillus subtilis , which is preserved in the China General Microbiological Culture Collection Center on May 7, 2026, and the address is No. 1, Xili Beichen, Chaoyang District, Beijing, and the preservation number is CGMCC No. 38572. The strain can maintain good growth and metabolic activity in the black-like extract, promote the formation or accumulation of black-like related browning products, promote the formation of 2-methylpyrazine, 2,5-dimethylpyrazine and other Maillard reaction related roasted wheat flavor substances, and reduce the relative abundance of part of the fat oxidation taste and the relative abundance of the irritating alcohol taste related substances. This is also important for realizing the directional regulation of high-temperature Daqu Maillard reaction products and characteristic flavors.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus subtilis JQBS002 and its applications. Background Technology

[0002] High-temperature Daqu (a type of starter culture) is an important saccharification and fermentation agent and a source of flavor precursors in the brewing of Maotai-flavor Baijiu. During the Daqu production process, the starter culture undergoes a high-temperature, high-humidity, and open solid-state fermentation environment. Microbial metabolism, enzymatic degradation reactions, and non-enzymatic browning reactions all participate in the formation of the color, aroma, and flavor precursors of the Daqu. Among these, the Maillard reaction is one of the important reactions that contributes to the browning color and characteristic flavors such as roasted, nutty, Maotai, and roasted malt aromas in high-temperature Daqu.

[0003] The Maillard reaction involves the non-enzymatic browning of reducing sugars and amino acids under high-temperature conditions, producing a series of aroma compounds such as pyrazines, furans, and pyranones, characterized by roasted wheat, nutty, and caramel aromas. Melanoids are important brown polymers formed in the later stages of the Maillard reaction, reflecting the degree of browning and the accumulation level of Maillard reaction products. Pyrazines are important nitrogen-containing heterocyclic flavor compounds in high-temperature fermented foods and Daqu (a type of starter culture) systems, typically associated with background aromas of roasted wheat, nuts, roasting, and soy sauce.

[0004] However, the successful conduct of the Maillard reaction highly depends on two key prerequisites: first, the long-term maintenance of a high-temperature environment, allowing reducing sugars and amino acids to fully condense and degrade layer by layer; second, a sufficient supply of free amino acids and reducing sugars in the system. The former is the most important nitrogenous substrate in this reaction, and its type and concentration directly affect the quality of characteristic flavor compounds. Daqu fermentation requires a natural accumulation and heating process lasting several months. Sustained and stable high temperatures cannot always be guaranteed. If temperature fluctuations are too large or the required time is insufficient, the Maillard reaction cannot proceed fully. More importantly, although the Daqu system harbors a rich microbial community, the enzyme production and metabolic efficiency of these microorganisms vary, and their ability to degrade proteins is limited, resulting in a long-term deficiency of free amino acids available for the Maillard reaction in the fermentation system. This supply-demand imbalance directly restricts the generation of characteristic aroma compounds in Daqu, making it difficult for Daqu to fully exert its aroma-enhancing function. Therefore, screening functional strains with clearly defined origins and functions that can promote the formation of Maillard reaction products in Daqu is of great significance for improving the stability of Daqu flavor regulation and enhancing its quality.

[0005] Bacillus, as the dominant microorganism in high-temperature Daqu (a type of starter culture), possesses strong environmental adaptability and metabolic activity. It can participate in the degradation of substrates such as starch and proteins, providing precursors such as reducing sugars, amino acids, and small peptides for the Maillard reaction, thus accelerating the occurrence and progress of the Maillard reaction under suitable temperature conditions. However, there are many species of Bacillus, and different strains exhibit significant differences in enzyme secretion capacity and metabolite profiles. Not all Bacillus strains can effectively promote the formation of Maillard reaction products in Daqu. Therefore, screening functional Bacillus strains that promote the Maillard reaction from the Daqu ecological environment has clear application value. Summary of the Invention

[0006] The purpose of this invention is to provide a Bacillus subtilis strain JQBS002, which can effectively promote the formation or accumulation of melanoidin-related browning products, and at the same time promote the formation of Maillard reaction-related roasted wheat flavor substances such as 2-methylpyrazine and 2,5-dimethylpyrazine.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Bacillus subtilis JQBS002, classified as Bacillus subtilis. Bacillus subtilis It was deposited on May 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38572.

[0008] This invention provides the application of Bacillus subtilis JQBS002 in promoting the formation of macromellad reaction products.

[0009] Preferably, the Maillard reaction products of the large-grain liquor include melanoidin-related browning products and Maillard reaction-related volatile flavor compounds.

[0010] The present invention also provides the application of the Bacillus subtilis JQBS002 in promoting the formation of roasted wheat flavor compounds.

[0011] Preferably, the roasted wheat flavoring substances include pyrazine compounds.

[0012] The present invention also provides the application of the Bacillus subtilis JQBS002 in the preparation of fermentation agents.

[0013] This invention also provides a fermentation method for a melanin-like extract, comprising the following steps: (1) Mix Daqu with ethanol and extract, then centrifuge and collect the supernatant by rotary evaporation, and make up to volume to obtain melanoidin extract; (2) The bacterial culture of Bacillus subtilis JQBS002 is mixed with melanoidin extract and fermented by shaking to obtain a fermentation liquid with increased content of melanoidin-related browning products and increased roasted wheat flavor substances.

[0014] Preferably, the ratio of Daqu (a type of starter culture) to ethanol in step (1) is 2g:(30-40)mL.

[0015] Preferably, the extraction temperature in step (1) is 65-75℃ and the extraction time is 2-4h.

[0016] Preferably, the centrifugation speed in step (1) is 3000-4000 rpm and the centrifugation time is 10-20 min.

[0017] Preferably, the volume ratio of the Bacillus subtilis JQBS002 bacterial solution to the melanoidin extract in step (2) is (0.01-0.03):1.

[0018] Preferably, the OD of the Bacillus subtilis JQBS002 bacterial suspension in step (2) is... 600 It ranges from 0.8 to 1.2.

[0019] Preferably, the frequency of the oscillation fermentation in step (2) is 170-190 r / min, the temperature of the oscillation fermentation is 35-40℃, and the time of the oscillation fermentation is 45-50 h.

[0020] The present invention also provides a fermentation broth obtained by the fermentation method having increased content of melanin-related browning products and increased roasted wheat flavor compounds.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects: The Bacillus subtilis JQBS002 strain provided by this invention was derived from high-temperature Daqu (a type of starter culture). It was identified as Bacillus subtilis through colony morphology, Gram staining, and 16S rRNA gene sequence analysis. The strain has been preserved, and its origin, classification, and reproducibility are clear. The taxonomic name of the Bacillus subtilis JQBS002 is Bacillus subtilis. Bacillus subtilis It was deposited on May 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38572.

[0022] The Bacillus subtilis JQBS002 provided by this invention can survive and proliferate in melanoidin extract, thereby increasing the OD of the melanoidin extract. 420 Value. Compared with the unvaccinated control group, the OD value of melanoidin extract after inoculation with JQBS002 was higher.420 The significantly elevated value indicates that this strain can promote the formation or accumulation of melanoidin-related browning products.

[0023] The Bacillus subtilis JQBS002 provided by this invention can promote the formation of roasted wheat aroma-related flavor compounds such as 2-methylpyrazine and 2,5-dimethylpyrazine. These pyrazine compounds belong to the Maillard reaction-related nitrogen-containing heterocyclic flavor compounds and are associated with backgrounds of roasted wheat aroma, nutty aroma, baked aroma, and soy sauce aroma.

[0024] The Bacillus subtilis JQBS002 provided by this invention can regulate the volatile flavor composition of melanoidin extract, increasing the relative abundance of substances related to nutty, caramel, sweet, and fermented aromas, such as benzaldehyde, 2-methoxyphenol, 2-heptanone, and 2-nonanone, while decreasing the relative abundance of substances related to oxidized fats or pungent alcohols, such as nonanal, isoamyl alcohol, and some long-chain alcohols, thereby improving the flavor harmony of the melanoidin system.

[0025] Biological Preservation Instructions

[0026] This invention relates to Bacillus subtilis JQBS002, classified as Bacillus subtilis. Bacillus subtilis It was deposited on May 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38572.

[0027] This invention relates to Bacillus subtilis JQBS001, classified as Bacillus subtilis. Bacillus subtilis It was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35239. Attached Figure Description

[0028] Figure 1 This is a colony morphology diagram of Bacillus subtilis JQBS002. Figure 2 Gram staining morphology of Bacillus subtilis JQBS002 under an optical microscope; Figure 3 The growth curve of Bacillus subtilis JQBS002; Figure 4 OD values ​​of melanoidin extracts from each treatment group 420 Value change; Figure 5Total ion chromatogram of volatile products in melanoidin extract after fermentation of Bacillus subtilis JQBS002 for 48 h; Figure 6 Mass spectrometry identification chromatogram of 2-methylpyrazine ( Figure 6 The top image in the figure is the mass spectrum of Example 4-48h, and the bottom image is the NIST23 standard mass spectrum library matching diagram. Figure 7 The mass spectrometry identification chromatogram of 2,5-dimethylpyrazine ( Figure 7 The top image in the image shows the mass spectrum of Example 4-48h, and the bottom image shows the NIST 23 standard mass spectrum library matching diagram. Figure 8 Cluster heatmaps of volatile metabolites from melanoidin extracts at 0 h and 48 h in Comparative Example 1 and Example 4; Figure 9 The results of plate colony counts of Bacillus subtilis JQBS002 before and after fermentation of melanoidin extract ( Figure 9 In the table, 'a' represents the plate results of the sample treated for 0 h in Comparative Example 1; 'b' represents the plate results of the sample treated for 48 h in Comparative Example 1; and 'c' represents the plate results of the sample diluted 10 μL in Example 4 after 0 h of fermentation. -3 Plate results; d represents the sample from Example 4, fermented for 48 h, diluted 10⁻⁶. -3 Plate results; e represents the sample from Example 4, fermented for 0 hours, diluted 10... -4 Plate results; f is the sample from Example 4 fermented for 48 h and diluted 10. -4 (Tablet results). Detailed Implementation

[0029] This invention provides a Bacillus subtilis JQBS002, classified as Bacillus subtilis. Bacillus subtilis It was deposited on May 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO:38572.

[0030]

[0031] This invention provides the application of Bacillus subtilis JQBS002 in promoting the formation of macromellad reaction products.

[0032] In this invention, the Maillard reaction products of the large-grain liquor include melanoidin-related browning products and Maillard reaction-related volatile flavor compounds.

[0033] The present invention also provides the application of the Bacillus subtilis JQBS002 in promoting the formation of roasted wheat flavor compounds.

[0034] In this invention, the roasted wheat flavoring substance includes pyrazine compounds, more preferably including 2-methylpyrazine and 2,5-dimethylpyrazine.

[0035] The present invention also provides the application of the Bacillus subtilis JQBS002 in the preparation of fermentation agents.

[0036] This invention also provides a fermentation method for a melanin-like extract, comprising the following steps: (1) Mix Daqu with ethanol and extract, then centrifuge and collect the supernatant by rotary evaporation, and make up to volume to obtain melanoidin extract; (2) The bacterial culture of Bacillus subtilis JQBS002 is mixed with melanoidin extract and fermented by shaking to obtain a fermentation liquid with increased content of melanoidin-related browning products and increased roasted wheat flavor substances.

[0037] In this invention, the Daqu (a type of starter culture) is preferably high-temperature Daqu.

[0038] In this invention, Daqu (a type of starter culture) is mixed with ethanol for extraction. The preferred ratio of Daqu to ethanol is 2g:(30-40)mL, more preferably 2g:(32-38)mL, and even more preferably 2g:35mL. The volume concentration of ethanol in this invention is preferably 30-50%, more preferably 35-45%, and even more preferably 40%.

[0039] In this invention, water bath extraction is preferably used, and the extraction temperature is preferably 65-75℃, more preferably 68-72℃, and even more preferably 70℃; the extraction time is preferably 2-4h, more preferably 2.5-3.5h, and even more preferably 3h.

[0040] In this invention, after extraction, centrifugation is performed and the supernatant is collected. The centrifugation speed is preferably 3000-4000 rpm, more preferably 3200-3800 rpm, and even more preferably 3500 rpm. The centrifugation time is preferably 10-20 min, more preferably 12-18 min, and even more preferably 15 min.

[0041] In this invention, the supernatant is placed in a rotary evaporator to remove ethanol by rotary evaporation, obtaining a concentrated residue; then, the concentrated residue is dissolved in distilled water and brought to a final volume to obtain a melanoidin extract. The volume for final volume adjustment in this invention is preferably 40-60 mL, more preferably 45-55 mL, and even more preferably 50 mL.

[0042] In this invention, the process of obtaining the bacterial culture of Bacillus subtilis JQBS002 is as follows: Bacillus subtilis JQBS002 is inoculated into LB broth medium and cultured with shaking at 37°C for 24 h to obtain the seed culture; the concentration of the seed culture is adjusted to OD. 600 The value is 0.8-1.2, which gives the result.

[0043] In this invention, the OD of the bacterial culture of Bacillus subtilis JQBS002 is... 600 Preferably, it is 0.8-1.2, further preferably 0.9-1.1, and even more preferably 1.

[0044] In this invention, the preferred volume ratio of the bacterial culture of Bacillus subtilis JQBS002 to the melanoidin extract is (0.01-0.03):1, more preferably (0.015-0.025):1, and even more preferably 0.02:1.

[0045] In this invention, the frequency of the oscillation fermentation is preferably 170-190 r / min, more preferably 175-185 r / min, and even more preferably 180 r / min; the temperature of the oscillation fermentation is preferably 35-40℃, more preferably 36-39℃, and even more preferably 37℃; the time of the oscillation fermentation is preferably 45-50 h, more preferably 46-49 h, and even more preferably 48 h.

[0046] The present invention also provides a fermentation broth obtained by the fermentation method having increased content of melanin-related browning products and increased roasted wheat flavor compounds.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The LB nutrient agar of this invention is prepared by dissolving 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride and 12 g of agar in 1000 mL of distilled water, and then sterilizing it with steam at 121 °C and 0.105 MPa for 15 min.

[0049] The LB broth culture medium of this invention is prepared by dissolving 8 g of tryptone, 2 g of yeast powder and 10 g of sodium chloride in 1000 mL of distilled water, and then sterilizing by steaming at 121 °C and 0.105 MPa for 15 min.

[0050] Example 1: Isolation and Identification of Bacillus subtilis JQBS002 (one)

[0052] Take 1.0 g of high-temperature Daqu sample and place it in a sterile test tube containing 9 mL of sterile physiological saline. Shake thoroughly to mix, and obtain 10 -1 Gradient dilutions were prepared sequentially using a 10-fold serial dilution method. -2 10 -3 10 -4 10 -5 and 10 -6 Serial dilutions. Take 100 μL of each appropriate dilution and spread it evenly on the surface of LB nutrient agar plates. Incubate upside down at 37°C for 24 ± 2 h.

[0053] After incubation, observe the growth and morphological characteristics of the colonies on the plates. Select single colonies with typical Bacillus colony morphology and isolate and purify them by streaking continuously on LB nutrient agar medium using the streak plate method until a pure culture with consistent morphology, clear edges, and no contamination is obtained.

[0054] like Figure 1 As shown, the strain grew well on LB nutrient agar medium, with colonies ranging from grayish-white to milky-white, growing continuously along the streaked area. The colony surface was relatively rough with irregular edges, exhibiting an overall expanding growth characteristic, consistent with the typical colony morphology of Bacillus strains. (two)

[0056] Place a drop of sterile saline solution in the center of a clean glass slide. Pick a small amount of the isolated and purified single colony and spread it evenly in the sterile saline solution to form a thin and uniform bacterial film. After air drying, heat-fix the slide by quickly passing it several times over an alcohol lamp flame. Then, stain with crystal violet solution for 1 min, wash with water and blot away excess water around the slide; mordant with iodine solution for 1 min, wash with water and blot away excess water around the slide; after destaining, counterstain with safranin for about 40 s, wash with water and blot away excess water around the slide, and observe the bacterial morphology under an optical microscope.

[0057] like Figure 2 As shown, after Gram staining, the single colony appears as purple rod-shaped cells under an optical microscope. The bacterial morphology is relatively regular, and the cells are mainly distributed in single, paired, or short chain patterns, indicating that the strain is a Gram-positive bacillus. (three)

[0059]

[0060] The obtained 16S rRNA gene sequence was submitted to the NCBI database for BLAST homology comparison analysis. Table 1 shows that the 16S rRNA gene sequence of strain JQBS002 has high homology with related sequences of Bacillus subtilis in the NCBI database. Specifically, the sequence identity of JQBS002 with the partial 16S rRNA gene sequence of Bacillus subtilis strain LKM-BL and the complete genome sequence of Bacillus subtilis strain ATCC 13952 is 100%, with an E value of 0 and an alignment score of 2656. Based on colony morphology, Gram staining results, 16S rRNA gene homology comparison results, and strain preservation identification results, strain JQBS002 was confirmed as Bacillus subtilis.

[0061] It should be noted that the LKM-BL strain and ATCC 13952 strain in Table 1 are database reference strains, not the strain JQBS002 of this application. Eileen et al. (Eileen Rizlan Ross E, Hamzah A, SyaidatulAqma W. Endophytic bacteria from Theobroma cacao L. with antifungalactivities against Phytophthora palmivora[J]. Asian Journal of Agriculture and Biology, 2019, 7(3): 404-411) reported that LKM-BL is an endophytic bacterium isolated from healthy cacao tree (Theobroma cacao L.) tissue, derived from the Malaysia Cocoa Board Culture Collection, and its main purpose is to inhibit the pathogen of cacao black pod disease. Phytophthora palmivora The strain was identified as Bacillus subtilis by 16S rDNA sequence analysis and named... Bacillus subtilis LKM-BL, its GenBank accession number is KR560044.

[0062] ATCC strain 13952 is the reference strain of Bacillus subtilis preserved in ATCC. Its strain number is ATCC 13952, and its alternative name is 1346. ATCC publicly available information indicates that it is a sequenced strain and not a type strain. Tominaga M et al. (Tominaga M, Shimazaki K, Matsuno K, Yamashita M. Method of converting heterocyclic bases into the corresponding nucleosides by bacterial action. US Patent: 3535207. 1970) used... Bacillus subtilis ATCC 13952 is described as an inosine-producing strain.

[0063] In contrast, strain JQBS002, as described in this application, was isolated and purified by the applicant from a high-temperature Daqu (a type of starter culture) sample and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38572. Its purpose is to promote the formation of Maillard reaction products and roasted wheat flavor compounds in Daqu. Therefore, JQBS002 differs from LKM-BL and ATCC13952 in terms of isolation source, strain number, accession information, and application. The 16S rRNA gene sequence alignment results are only used to support the classification and identification of strain JQBS002 and cannot be used to conclude that JQBS002 is the same strain as the reference strain in the database.

[0064] Table 1. BLAST representation comparison results of the 16S rRNA gene sequence of strain JQBS002

[0065] Note: The results listed in the table represent the high sequence similarity, E-value of 0, and alignment score of the 16S rRNA gene sequence of strain JQBS002 after BLAST alignment with related sequences of Bacillus subtilis. Sequence similarity indicates the proportion of identical bases between the query sequence and the database reference sequence; the E-value indicates the significance of the alignment, and the closer the E-value is to 0, the lower the probability of a random match; the alignment score indicates the degree of sequence alignment matching. The LKM-BL strain and ATCC 13952 strain in the table are database reference strains, not the strain JQBS002 of this application; the 16S rRNA gene sequence alignment results are used to support the classification and identification of strain JQBS002.

[0066] The inventor deposited the Bacillus subtilis JQBS002 at the China General Microbiological Culture Collection Center on May 7, 2026. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC NO:38572.

[0067] Example 2: Analysis of the growth curve of Bacillus subtilis JQBS002

[0068] Activated Bacillus subtilis strain JQBS002 was inoculated into LB broth medium and cultured with shaking at 37°C for 24 h. During the culture process, samples were taken every 2 h to measure the absorbance (OD) of the culture medium at 600 nm. 600 Values. Each experiment was conducted in triplicate, and the average value was taken. The values ​​were then calculated based on the OD values ​​corresponding to different incubation times. 600 The growth curve of strain JQBS002 was plotted, and the results are as follows: Figure 3 As shown.

[0069] The results showed that Bacillus subtilis JQBS002 grew well under LB broth culture conditions at 37℃. The OD of the strain was [value missing] during the 0-6 h culture period. 600 The value changes little, indicating a lag phase; during 6-16 h of incubation, OD... 600 The value increased rapidly, indicating that the strain had entered the logarithmic growth phase; after 16 h of culture, the OD value... 600 The OD value tends to stabilize, and the strain enters the stationary phase. During the 18-24 h of culture, OD... 600 The value remained basically between 1.45 and 1.48, indicating that the strain has good proliferation ability and culture stability.

[0070] Example 3: Preparation of melanin extract

[0071] Weigh 2.0 g of high-temperature Daqu powder sample obtained from Gujinggong liquor, add 35 mL of 40% ethanol solution, mix thoroughly, and extract in a 70℃ water bath for 3 h. After extraction, centrifuge the resulting mixture at 3500 rpm for 15 min and collect the supernatant. Evaporate the ethanol from the supernatant in a rotary evaporator to obtain a concentrated residue. Then dissolve and dilute to 50 mL with distilled water to obtain a melanoidin extract.

[0072] Example 4: Fermentation application of Bacillus subtilis JQBS002 (one)

[0074] The melanoidin-like extract prepared in Example 3 was mixed with Bacillus subtilis JQBS002 bacterial culture at a volume ratio of 1:0.02 and fermented at 37°C with shaking at 180 r / min for 48 h. Samples were taken at 0 h, 12 h, 24 h, 36 h, and 48 h of fermentation to obtain fermented melanoidin-like extracts at different fermentation times, which were used as Example 4. The Bacillus subtilis JQBS002 bacterial culture was preferably cultured in LB broth medium at 37°C with shaking at 180 r / min until OD. 600 =1.00 of seed liquid; the inoculation amount is 2% of the volume of melanoidin extract.

[0075] The melanoidin extract prepared in Example 3 was taken and treated with shaking at 180 r / min for 48 h at 37 °C without inoculation with Bacillus subtilis JQBS002 bacterial culture. Samples were taken at 0 h, 12 h, 24 h, 36 h and 48 h to obtain unfermented melanoidin extracts at different treatment times, which served as Comparative Example 1.

[0076] Take the melanoidin-like extract prepared in Example 3 and the Bacillus subtilis JQBS001 bacterial culture (OD) 600 =1.00) were mixed evenly at a volume ratio of 1:0.02 and fermented at 37℃ with shaking at 180 r / min for 48 h. Samples were taken at 0 h, 12 h, 24 h, 36 h and 48 h of fermentation to obtain fermented melanin extracts at different fermentation times, which served as two comparative groups.

[0077] The Bacillus subtilis JQBS001 was deposited by the inventors on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35239.

[0078] (II) Determination of melanoidin content

[0079] Samples from Comparative Example 1, Comparative Example 2, and Example 4 were diluted 10 times with distilled water, and the absorbance of the samples at a wavelength of 420 nm was measured using a UV-Vis spectrophotometer. The absorbance was then expressed as OD. 420 The value represents the relative content of melanoidins in the sample. Each group of samples was set up with 3 parallel replicates. The results are expressed as average values, and a curve of melanoidin content variation was plotted.

[0080] Table 2. OD during fermentation of melanocytic extract in each group 420 value

[0081] Note: Data are expressed as mean ± standard deviation; different letters indicate significant differences. P <0.05.

[0082] From Table 2 and Figure 4 It can be seen that during the 48-hour oscillation treatment at 37℃ and 180 r / min, the OD of each treatment group... 420 The values ​​all showed an increasing trend over time, indicating that a certain degree of browning product accumulation occurred in the melanoidin extracts during the treatment process. Compared with control example 1 (uninoculated), the OD values ​​of the melanoidin extracts increased significantly after inoculation with different Bacillus strains. 420 The values ​​showed varying degrees of change, indicating that different strains had different effects on the formation or accumulation of melanoidin-related browning products. Comparative Example 1 showed OD values ​​within 48 hours of treatment. 420 The value increased from 1.3870 to 1.8800, an increase of 35.5%; in Comparative Example 2, it increased from 1.4697 to 2.1210, an increase of 44.3%; and in Example 4, it increased from 1.4560 to 2.3670, an increase of 62.6%. All of these values ​​were greater than those in Comparative Examples 1 and 2, and the OD value 48 h after inoculation with JQBS002 was significantly higher. 420 The value was 25.9% higher than that of the unvaccinated group (from 1.8800 to 2.3670).

[0083] Further comparison of OD values ​​of different treatment groups under the same fermentation time 420 The values ​​show that, after 24 hours of fermentation, the OD value of Example 4 was... 420 The value was 1.9593 ± 0.0232, which was 16.4% and 14.2% higher than Comparative Example 1 and Comparative Example 2, respectively, and significantly higher than each comparative example group. After 36 h of fermentation, the OD value of Example 4 was... 420 The value was 2.3075 ± 0.1435, which was 28.5% and 24.8% higher than Comparative Example 1 and Comparative Example 2, respectively, and significantly higher than each comparative example group. After 48 h of fermentation, the OD value of Example 4 was... 420 The value reached 2.3670±0.0285, which was 25.9% higher than the unvaccinated control group 1 and 11.6% higher than the control group 2, and was the highest among all treatment groups.

[0084] The above results indicate that, under the same bacterial concentration, inoculation ratio, and fermentation conditions, Bacillus subtilis JQBS002 is more effective than uninoculated treatment and treatment with Bacillus from other sources in promoting the OD of melanoidin extract. 420 An increase in the value indicates that this strain has a strain advantage in promoting the formation or accumulation of melanoidin-related browning products.

[0085] Example 5: Detection of volatile products in melanin extract

[0086] Sample preparation: Take 5 mL of melanoidin extract samples from Comparative Example 1 and Example 4 at 0 h and 48 h respectively, place them in 20 mL headspace vials, add 1.2 g NaCl and 5 μL of internal standard solution (2-octanol, concentration 1 mg / mL), seal the headspace vials and equilibrate at 50 °C for 10 min, then insert a solid-phase microextraction fiber for headspace adsorption for 30 min. After adsorption, immediately insert the extraction fiber into the gas chromatograph inlet and thermally desorb at 250 °C for 5 min for gas chromatography-mass spectrometry analysis. GC conditions: Volatile metabolites in the samples were analyzed using an Agilent 8890-5977B gas chromatograph-mass spectrometer. A DB-FFAP column (60m × 0.25mm, 0.25μm) was used with high-purity helium as the carrier gas at a flow rate of 1mL / min. The column temperature program was as follows: initial temperature 40℃, held for 5 min; then increased to 120℃ at 3℃ / min; then increased to 180℃ at 5℃ / min; finally increased to 230℃ at 10℃ / min and held for 5 min.

[0087] (3) MS conditions: Mass spectrometry detection uses an electron impact ionization source, and the ionization method is EI. + The electron energy was 70 eV, the ion source temperature was 230℃, the filament flow rate was 0.2 mA, and the detector voltage was 350 V. The full scan range of the mass spectrometer was 33-450 amu, and the scan frequency was 1 scan / s.

[0088] The obtained volatile metabolite mass spectrometry information was qualitatively compared with the standard mass spectrometry library NIST 23, and the relative content of each volatile metabolite was calculated using the internal standard method with 2-octanol as the internal standard. The detection results are as follows: Figures 5-8 As shown.

[0089] Depend on Figure 5 Multiple volatile metabolite signals were detected in the melanoidin extract after inoculation with Bacillus subtilis JQBS002 and fermentation for 48 h, indicating that JQBS002 can grow and metabolize in the melanoidin extract system and promote changes in the composition of volatile products in the system. Pyrazine compounds associated with roasted wheat aroma, nutty aroma, and roasted aroma were detected at retention times of approximately 30.8 min and 33.3 min in the chromatogram, respectively.

[0090] Depend on Figure 6 It can be seen that the mass spectrum fragment of the compound detected at a retention time of approximately 30.8 min matches the characteristic mass spectrum of 2-methylpyrazine in the NIST 23 standard mass spectrum library, indicating that this volatile compound can be identified as 2-methylpyrazine. Figure 7The mass spectrometric fragments of the compound detected at a retention time of approximately 33.3 min matched the characteristic mass spectrum of 2,5-dimethylpyrazine in the NIST 23 standard mass spectrometry library, indicating that the volatile compound could be identified as 2,5-dimethylpyrazine. Both 2-methylpyrazine and 2,5-dimethylpyrazine belong to typical nitrogen-containing heterocyclic flavor compounds, typically exhibiting roasted, nutty, and roasted wheat aromas. These results suggest that fermentation of the melanin extract by Bacillus subtilis JQBS002 can promote the formation of roasted wheat aroma-related pyrazine volatile flavor compounds.

[0091] Depend on Figure 8 It can be seen that the flavor profile of Example 4 at 48 h is significantly different from that of Comparative Example 1 and the initial state. Compared with the 48 h sample of Comparative Example 1 (Comparative Example 1-48 h), the color of the pyrazine region in the 48 h sample of Example 4 (Example 4-48 h) is significantly deeper than that of Comparative Example 1. The relative abundance of flavor compounds such as 2-methylpyrazine, 2,5-dimethylpyrazine, benzaldehyde, 2-methoxyphenol, 2-heptanone, and 2-nonanone is increased. These compounds are associated with typical Daqu flavor characteristics such as roasted wheat aroma, nutty aroma, roasted aroma, caramel aroma, sweet aroma, and fermented aroma. Among them, 2-methylpyrazine and 2,5-dimethylpyrazine are Maillard reaction-related nitrogen-containing heterocyclic flavor compounds. Their increase indicates that JQBS002 can promote the formation of Maillard reaction-related flavor compounds in the melanin-like system. At the same time, the relative abundance of compounds such as nonanal, isoamyl alcohol, and some long-chain alcohols associated with fatty oxidation flavor or pungent alcohol flavor is decreased in Example 4-48 h. The above results indicate that Bacillus subtilis JQBS002 can promote the formation of beneficial flavor compounds in the Daqu-type melanin system, while reducing the abundance of some undesirable flavor-related compounds, thereby improving the roasted aroma, roasted wheat aroma and soy sauce aroma-related flavor characteristics of the melanin-type system.

[0092] Example 6: Plate colony count of Bacillus subtilis JQBS002 before and after fermentation of melanin extract

[0093] Samples of melanoidin extract taken from Comparative Example 1 and Example 4 at 0 h and 48 h were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the lower precipitate was collected. 1 mL of sterile physiological saline was added to the precipitate and the mixture was thoroughly mixed to obtain the bacterial suspension to be tested.

[0094] Comparative Example 1 sample: its 0 h and 48 h precipitate resuspension was spread onto LB nutrient agar plates; Example 4 sample: its 0 h and 48 h bacterial suspension was serially diluted to 10⁻¹⁰. -3 and 10 -4The cultures were spread onto LB nutrient agar plates. Each plate was incubated upside down at 37°C for 24 hours. After incubation, colony growth was observed and the colony count was recorded. Plates with a total colony count exceeding 300 were considered inaccurately countable. Results are as follows: Figure 9 As shown in Table 3.

[0095] Table 3. Colony count results of Bacillus subtilis JQBS002 in melanoidin extract.

[0096] Note: "-" indicates that the total number of colonies on the plate exceeds 300 and cannot be accurately counted.

[0097] Depend on Figure 9 As shown in Table 3, Comparative Example 1 was not inoculated with JQBS002, and the plate results were used to exclude the interference of background colonies in the melanoidin extract itself on the counting results of Example 4. Example 4 consisted of melanoidin extract inoculated with Bacillus subtilis JQBS002. At 0 h of fermentation, 10... -3 The dilution plate count was 326 colonies, 10 -4 The dilution plate showed 51 colony-forming units, indicating that the fermentation starter system already contained a certain number of viable JQBS002 bacteria. After 48 hours of fermentation, the sample from Example 4 was tested at 10... -3 and 10 -4 The number of bacterial colonies on the plates at all dilutions exceeded 300, making accurate counting impossible, indicating that Bacillus subtilis JQBS002 can survive and further proliferate in the melanoidin extract system. These results demonstrate that Bacillus subtilis JQBS002 can adapt to the melanoidin extract environment and maintain strong growth and proliferation capabilities under fermentation conditions at 37℃.

[0098] In summary, the Bacillus subtilis JQBS002 isolated from high-temperature Daqu (a type of starter culture) of this invention can survive and proliferate in melanoidin-like extract, promoting the formation or accumulation of melanoidin-related browning products and increasing the OD (oxidative stress) of the fermentation system. 420 It enhances the composition of Maillard reaction-related pyrazine flavor compounds such as 2-methylpyrazine and 2,5-dimethylpyrazine, while reducing the relative abundance of some fat oxidation flavor and pungent alcohol flavor compounds. It has the application value of improving the composition of Maillard reaction products of Daqu and enhancing the flavor characteristics of roasted wheat.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis JQBS002, characterized in that, The species, classified as Bacillus subtilis, was deposited on May 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38572.

2. The application of Bacillus subtilis JQBS002 as described in claim 1 in promoting the formation of products of the macromolecular Maillard reaction, characterized in that, The Maillard reaction products of the Daqu include melanoidin-related browning products and Maillard reaction-related volatile flavor compounds.

3. The application of Bacillus subtilis JQBS002 as described in claim 1 in promoting the formation of roasted wheat flavor compounds, characterized in that, The roasted wheat flavoring substances include pyrazine compounds.

4. The application of Bacillus subtilis JQBS002 as described in claim 1 in the preparation of fermentation inoculants.

5. A fermentation method for a melanin-like extract, characterized in that, Includes the following steps: (1) Mix Daqu with ethanol and extract, then centrifuge and collect the supernatant by rotary evaporation, and make up to volume to obtain melanoidin extract; (2) The bacterial culture of Bacillus subtilis JQBS002 as described in claim 1 is mixed with melanoidin extract and fermented by shaking to obtain a fermentation liquid with increased content of melanoidin-related browning products and increased roasted wheat flavor substances.

6. The fermentation method according to claim 5, characterized in that, The ratio of Daqu (a type of starter culture) to ethanol in step (1) is 2g:(30-40)mL; The extraction temperature is 65-75℃, and the extraction time is 2-4 hours.

7. The fermentation method according to claim 5, characterized in that, In step (1), the centrifugation speed is 3000-4000 rpm and the centrifugation time is 10-20 min.

8. The fermentation method according to claim 5, characterized in that, In step (2), the volume ratio of the Bacillus subtilis JQBS002 bacterial solution to the melanoidin extract is (0.01-0.03):

1. The OD of the bacterial culture of Bacillus subtilis JQBS002 600 It ranges from 0.8 to 1.

2.

9. The fermentation method according to claim 5, characterized in that, The frequency of the shaking fermentation in step (2) is 170-190 r / min, the temperature of the shaking fermentation is 35-40℃, and the time of the shaking fermentation is 45-50 h.

10. A fermentation broth obtained by the fermentation method according to any one of claims 5-9, which has increased content of melanoidin-related browning products and increased roasted wheat flavor compounds.

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