Bacillus amyloliquefaciens capable of producing ethyl lactate and application of bacillus amyloliquefaciens in improvement of fermented ethyl lactate of rice-flavor liquor

Abstract

The invention belongs to the technical field of microorganisms and baijiu brewing, and particularly relates to bacillus amyloliquefaciens capable of producing ethyl lactate and application of the bacillus amyloliquefaciens to improvement of fermented ethyl lactate of rice-flavor baijiu. The strain is separated from later-stage fermentation liquor of rice-flavor liquor, and has the characteristics of acid resistance, high-concentration ethanol resistance and high-yield ethyl lactate. According to the invention, the strain is added in the later period of fermentation of the rice-flavor liquor for enhanced fermentation, and then physical temperature control is carried out, so that the concentration of ethyl lactate can be synergistically increased, flavor substances such as total ester of the rice-flavor liquor can be remarkably increased, and meanwhile, the liquor yield is not influenced, so that the compound fragrance of rice wine is increased, and the liquor quality is improved. The method is good in stability among batches, can obtain the rice-flavor baijiu with high ethyl lactate content, and is suitable for popularization and application in baijiu brewing.

Description

Technical Field

[0001] This invention belongs to the field of microbial and liquor brewing technology, and specifically relates to a strain of Bacillus amyloliquefaciens that produces ethyl lactate and its application in enhancing the production of ethyl lactate in rice-flavored liquor fermentation. Background Technology

[0002] Rice-aroma baijiu is one of the important aroma types of traditional Chinese baijiu, including rice-aroma and soy-sauce-aroma baijiu. Its typical flavor characteristics are closely related to the content and proportion of esters such as ethyl lactate and ethyl acetate. Rice-aroma baijiu is "made from rice as the main raw material, using small-grain yeast as the saccharification and fermentation agent, and fermented, distilled, aged, and blended using traditional semi-solid-state methods, possessing a complex aroma primarily composed of ethyl lactate and β-phenylethanol." Soy-sauce-aroma baijiu is a derivative of rice-aroma baijiu. It uses carefully selected rice as raw material, which is steamed, cooled, mixed with large yeast cakes, fermented with water, matured, and then distilled in a pot. After clarification and maceration with aged fatty pork, it develops a unique soy-sauce aroma. It has a mellow taste, a unique soy-sauce aroma, a rich and sweet flavor, and a clean aftertaste.

[0003] Rice-aroma baijiu is a popular type of liquor in the Guangdong and Guangxi regions. It has a relatively low alcohol content, and ethyl lactate is its most abundant ester. Ethyl lactate is a miscible ester with both water and ethanol, acting as a solubilizer for other esters while also buffering the aftertaste and adding richness. For low-alcohol baijiu, ethyl lactate is crucial for overcoming a watery taste and enhancing the overall flavor. Therefore, ethyl lactate plays a vital role in the flavor of rice-aroma and soy-sauce-aroma baijiu. However, rice-aroma baijiu uses a semi-solid-state fermentation process, making it susceptible to seasonal influences. The ester production in the fermentation mash is unstable, resulting in an overall low ester content. Furthermore, the unstable ethyl lactate content due to seasonal and fermentation environmental factors leads to insufficient aroma and body, making it difficult to maintain consistent quality in rice-aroma baijiu.

[0004] Currently, the main methods to increase the ethyl lactate content in wine include: optimizing the raw material ratio, adjusting fermentation process parameters (such as temperature and acidity), and adding exogenous esterases or functional strains to enhance fermentation.

[0005] However, the existing technology has the following limitations: (1) Simply adjusting the process parameters has a limited effect on increasing ethyl lactate and may affect the balance of other flavor substances; (2) Directly adding commercial esterifying enzymes is costly, and the stability and efficiency of the enzymes in complex fermentation systems need to be improved; (3) When using functional microorganisms for enhancement, the adaptability of the strains, the specificity of their esterification ability, and their synergy with the original mash microbial community are often insufficient, resulting in unstable effects. For example, Chinese patent application CN2024102766748 discloses an acid-resistant Lactobacillus acetotolerans La50 and its application. Its fermentation produces mainly lactic acid, followed by acetic acid and malic acid. It can increase the content of ethyl acetate, but has little effect on the content of ethyl lactate. Moreover, in actual production, when lactic acid reaches a certain amount, the increase in ethyl lactate is still limited, that is, simply increasing lactic acid cannot solve the problem of insufficient ethyl lactate content; (4) A single microbial strategy or physical strategy is difficult to meet the ethyl lactate content required for production.

[0006] Therefore, there is an urgent need for a new technology that is efficient, stable, and cost-controllable in the targeted increase of ethyl lactate content in rice-flavored baijiu. Summary of the Invention

[0007] To address the aforementioned problems, the primary objective of this invention is to provide a strain of *Bacillus amyloliquefaciens* that produces ethyl lactate. This *Bacillus amyloliquefaciens* is resistant to acid and high ethanol concentrations, and produces high levels of ethyl lactate even in acidic and ethanol-containing environments.

[0008] Another object of the present invention is to provide the application of the above-mentioned ethyl lactate-producing Bacillus amyloliquefaciens.

[0009] Another objective of this invention is to provide a method for increasing the ethyl lactate content in rice-flavored baijiu through bio-enhanced fermentation and synergistic physical treatment.

[0010] Another object of the present invention is to provide an application of the above method and a rice-scented liquor.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A strain of Bacillus amyloliquefaciens that produces ethyl lactate, named Bacillus amyloliquefaciens FB13, with accession number GDMCC No: 67597, was deposited on January 5, 2026, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0013] The application of the aforementioned ethyl lactate-producing Bacillus amyloliquefaciens in Baijiu brewing.

[0014] The preferred type of baijiu is rice-aroma baijiu.

[0015] A method for increasing the ethyl lactate content of rice-flavored Baijiu through bio-enhanced fermentation and synergistic physical treatment includes the following steps:

[0016] (1) Inoculate the seed culture of Bacillus amyloliquefaciens that produces ethyl lactate into the enzyme-producing medium and culture it. Centrifuge the resulting culture solution A and take the precipitate.

[0017] (2) Add the precipitate obtained in step (1) to the rice-fragrant liquor post-fermentation liquid for enhanced fermentation;

[0018] (3) Temperature control treatment is carried out after fermentation is completed;

[0019] (4) After adding water to the mash obtained in step (3), distill to obtain the base liquor.

[0020] The ethyl lactate-producing Bacillus amyloliquefaciens mentioned in step (1) is the ethyl lactate-producing Bacillus amyloliquefaciens mentioned in claim 1.

[0021] The seed liquid described in step (1) is preferably obtained by the following steps: Bacillus amyloliquefaciens that produces ethyl lactate is inoculated into a seed culture medium and cultured with shaking at 35-40°C until the logarithmic growth phase to obtain the seed liquid.

[0022] The seed culture medium is a culture medium containing nitrogen and carbon sources; its preferred composition is as follows: 2.5-3.5 g / L beef extract, 8-12 g / L peptone, 4-6 g / L glucose, and 4-6 g / L sodium chloride; more preferably, it is as follows: 3 g / L beef extract, 10 g / L peptone, 5 g / L glucose, and 5 g / L sodium chloride.

[0023] The preferred rotation speed for the oscillation culture is 150–250 rpm; more preferably 150–200 rpm.

[0024] The preferred oscillation culture time is 16–30 h; more preferably 23–25 h.

[0025] The preferred inoculation amount of the seed liquid in step (1) is 0.5% to 5% by volume; more preferably, it is 1% by volume.

[0026] The enzyme-producing culture medium mentioned in step (1) is a culture medium that is conducive to the production of enzymes by Bacillus amyloliquefaciens, which produces ethyl lactate. It is a nitrogen-containing culture medium, and its composition is preferably as follows: 2.5-3.5 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride; more preferably as follows: 3 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride.

[0027] The preferred culture conditions in step (1) are shaking culture at 35-40℃ for 48-96 hours; more preferably shaking culture at 37℃ for 72 hours.

[0028] The preferred rotation speed for the oscillation culture is 150–250 rpm; more preferably 150–200 rpm.

[0029] The centrifugation conditions described in step (1) are preferably 10,000 to 15,000 rpm for 3 to 10 minutes; more preferably 12,000 rpm for 5 minutes.

[0030] The precipitate mentioned in step (2) is the precipitate obtained by centrifugation of culture medium A, which is calculated as 5-20% of the volume of the fermentation liquid in the later stage of rice-fragrant liquor; preferably, the precipitate is obtained by centrifugation of culture medium A, which is calculated as 10-15% of the volume of the fermentation liquid in the later stage of rice-fragrant liquor (15% is the best result).

[0031] In step (2), when adding the precipitate to the rice-fragrant liquor fermentation liquid, it is preferable to use the rice-fragrant liquor fermentation liquid or PBS to disperse the precipitate evenly before adding it to the rice-fragrant liquor fermentation liquid.

[0032] The amount of PBS used is 1-2% of the volume of the rice wine fermentation liquid in the later stage.

[0033] The rice-scented liquor fermentation liquid mentioned in step (2) is the fermentation liquid after 15 days of rice-scented liquor fermentation.

[0034] The preferred conditions for enhanced fermentation in step (2) are fermentation at 28–32°C for 6–9 days under sealed conditions; more preferably, fermentation at 30°C for 7–8 days under sealed conditions.

[0035] The preferred conditions for the temperature control treatment in step (3) are 80-100℃ for 4-8 hours; more preferably 90-100℃ for 6-8 hours; and most preferably 90℃ for 6 hours.

[0036] The amount of water used in step (4) is preferably 30 to 40% of the volume of the mash.

[0037] The application of the above methods in the brewing of rice-scented baijiu.

[0038] A type of rice-flavored liquor, obtained through the above method, is rich in ethyl lactate.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] This invention provides a synergistic strategy to significantly increase the ethyl lactate content of rice-flavored baijiu (Chinese white liquor). By specifically screening for ester-producing bacteria capable of tolerating 15° ethanol and an acidic environment of pH 3.65, and capable of synthesizing ethyl lactate, these bacteria are added to the later stages of fermentation for enhanced fermentation. Temperature control is then applied after fermentation to increase the total ester content, especially ethyl lactate, in the rice liquor. This enhances the complex aroma of the rice liquor without affecting the yield, improving the quality and increasing the percentage of premium liquor, thereby enhancing the product's market competitiveness. This invention has significant research value. The invention has the following characteristics:

[0041] Highly targeted: By screening specific esterase-producing strains through the later fermentation broth, the biosynthetic pathway of ethyl lactate is specifically enhanced, increasing the content of the target product. This invention is the first to discover that Bacillus amyloliquefaciens plays an important role in promoting the synthesis of ethyl lactate.

[0042] Synergistic effect: The two-step method of "microbial-enhanced late-stage fermentation" and "post-fermentation temperature control" produces a synergistic effect. Late-stage inoculation avoids fierce competition during the main fermentation period, allowing functional strains to play a more effective role; temperature control accelerates non-enzymatic esterification reactions and promotes the formation of various flavor compounds, thereby improving the flavor of baijiu.

[0043] Significant and stable results: This method can significantly increase the total ester content in fermentation broth and base liquor, especially the ethyl lactate content (verified by examples, the fermentation broth can be increased by more than 95%, and the base liquor by about 78%), and has good batch-to-batch stability without compromising the inherent style of rice-flavored baijiu.

[0044] Costs are controllable and easy to promote: The functional strains used can be cultivated independently and are adapted to the environment in the later stage of rice-flavored baijiu fermentation. The temperature control process is simple and highly feasible, making it suitable for application in baijiu brewing enterprises. Attached Figure Description

[0045] Figure 1 This is a graph showing the screening results of strains with high esterase activity; where A represents the screening results for bacteria, B represents the screening results for lactic acid bacteria, and C represents the screening results for fungi.

[0046] Figure 2 This is a graph showing the results of ethyl lactate and ethyl acetate production by strains undergoing further screening for high esterase activity. A represents ethyl lactate production by lactic acid bacteria, B represents ethyl lactate production by bacteria, C represents ethyl lactate production by fungi, D represents ethyl acetate production by lactic acid bacteria, E represents ethyl acetate production by bacteria, and F represents ethyl acetate production by fungi.

[0047] Figure 3 The images show the colony morphology of Bacillus amyloliquefaciens FB13 (A), an oil immersion (×100) photograph (B), and a growth curve (C).

[0048] Figure 4The figure shows the effect of concentrated Bacillus amyloliquefaciens FB13 inoculum amount on ethyl lactate content.

[0049] Figure 5 The figure shows the effect of the number of fermentation days after concentrated inoculation with Bacillus amyloliquefaciens FB13 on the content of ethyl lactate.

[0050] Figure 6 The graph shows the effect of different temperature treatments on the synthesis of ethyl lactate in the late fermentation broth of rice-flavored baijiu.

[0051] Figure 7 This is a graph showing the effect of bio-enhanced fermentation and synergistic temperature control on the relative abundance of various flavor compounds in the base liquor. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0053] Example 1: Isolation and Identification of Bacillus amyloliquefaciens FB13

[0054] (1) Enrichment of esterase-producing strains in late-stage fermentation broth with high ethanol concentration and high acidity

[0055] Enrichment media (all liquid media, solvent: deionized water): bacteria (beef extract 3 g / L, peptone 5 g / L); lactic acid bacteria (MRS broth); fungi (potato dextrose agar, PDB, with streptomycin sulfate added to a final concentration of 50 µg / mL before unplating after sterilization). Add 5% (50 mL) of rice-flavored baijiu late-stage fermentation broth to the enrichment media. 3 The large-volume baijiu fermentation liquid (provided by Guangdong Jiujiang Winery Co., Ltd., hereinafter the same) was enriched, in which bacteria and lactic acid bacteria were cultured at 37℃ and 180rpm for 48h, and fungi were cultured at 30℃ and 180rpm for 48h.

[0056] (2) Preliminary isolation and purification of esterase-producing strains

[0057] Preparation of tributyric acid ester plates: Weigh 3g of polyvinyl alcohol (type 1799) into 80mL of distilled water, heat and stir in a boiling water bath until completely dissolved, cool, and then dilute to 100mL with distilled water to obtain a 3.0% w / v polyvinyl alcohol solution. Use the 3.0% polyvinyl alcohol solution as a solvent to prepare a 100g / L tributyric acid ester emulsion. Mix the culture medium (bacterial medium composition: beef extract 3g / L, peptone 5g / L, agar 25g / L; lactic acid bacteria medium composition: MRS broth medium supplemented with 25g / L agar; fungal medium: PDA medium) with the above emulsion at a volume ratio of 4:1, and allow to settle at the natural pH. Sterilize at 121℃ for 20min, then pour and cool to prepare tributyric acid ester plates.

[0058] The bacterial culture obtained in step (1) was diluted with the appropriate enrichment medium by a dilution factor of 10. -2 10 -4 10 -6 and 10 -8 100 μL of each solution was diluted and plated onto tributylate plates, with three replicates for each dilution. The plates were incubated at 37°C for 2-3 days. Colonies exhibiting a clear zone on the tributylate plates were observed and selected. The plates were streaked twice for purification. The size of the clear zone was photographed and recorded, and the colonies were numbered. A total of 21 bacterial strains, 9 lactic acid bacteria, and 10 fungal strains with esterase-producing capabilities were obtained through screening.

[0059] (3) Screening of esterase-producing strains for esterase activity

[0060] The bacteria screened in step (2) were inoculated into seed culture medium (3 g / L beef extract, 10 g / L peptone, 5 g / L glucose, 5 g / L sodium chloride) and cultured at 37℃ and 180 r / min for 24 h. The seed culture was then inoculated into enzyme-producing culture medium (3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride) at an inoculation rate of 1% v / v and cultured at 37℃ and 180 r / min for 48 h. 1 mL of culture medium was taken and centrifuged at 4℃ and 8000 r / min for 10 min to precipitate the cells. The supernatant was collected and diluted 5 times to obtain the crude enzyme solution.

[0061] The lactic acid bacteria obtained in step (2) were inoculated into MRS broth medium and cultured at 37℃ and 180r / min for 48h. 1mL of the culture medium was taken and centrifuged at 4℃ and 8000r / min for 10min. The supernatant was diluted 5 times to obtain the crude enzyme solution.

[0062] The fungal strains screened in step (2) were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of PDB medium and cultured on a shaker at 30 °C and 180 r / min. Samples were taken at 2, 4, and 6 days. The sample solution was centrifuged at 8000 r / min and 4 °C for 10 min, and the supernatant was diluted 5 times to obtain the crude enzyme solution.

[0063] 50 μL of crude enzyme solution was added to 400 μL of 50 mM phosphate buffer (pH 7.5) and 50 μL of 10 mM p-nitrophenyl acetate substrate solution (ethanol solution), and then the solution was incubated at 30 °C for 10 min. The release of p-NP was quantified by measuring the absorbance at 410 nm. One unit of enzyme activity was defined as the amount of 1 μmol of p-NP produced per μL of fermentation broth per minute at pH 7.5 and 30 °C.

[0064] Esterase activity assay results are as follows Figure 1 As shown, bacterial strains FB9, 10, 11, 12, 13, 15, and 16 exhibited good esterase activity. Based on the results of the clear zone on the plates, these 7 strains, along with FB19, 20, and 21, were selected for further screening (see [link to image]). Figure 1 (A in the text); while four lactic acid bacteria strains, FL1, 2, 5 and 8 (see...) Figure 1 (B in the text) and five fungal strains FG1, 3, 4, 5 and 8 (see...) Figure 1 C) in the sample showed good esterase activity and was selected for the next screening step.

[0065] (4) Screening of lactate ethyl ester synthesis ability of strains with high esterase activity

[0066] The high esterase activity strains obtained in step (3) were cultured. The bacterial seed culture was cultured at 37℃ and 180r / min for 24h and then inoculated into the enzyme-producing medium and cultured under the same conditions for 48h. The lactic acid bacteria were inoculated into MRS medium and cultured at 37℃ and 180r / min for 48h. The fungi were inoculated into PDB medium and cultured at 30℃ and 180r / min for 5d.

[0067] Fermentation was carried out by inoculating 10% v / v culture medium of the above-mentioned strain into 5 mL of rice-fragrant liquor fermentation broth on day 5 at 30℃ for 5 days. Each group had three replicates. The control group consisted of rice-fragrant liquor fermentation broth on day 5 with the same volume of the corresponding liquid culture medium. The ethyl lactate and ethyl acetate content after fermentation was used for secondary screening to identify ester-producing bacteria with high ethyl lactate production. The secondary screening results are shown below. Figure 2 As shown, FL1 exhibited better ethyl lactate synthesis ability, increasing by approximately 13% compared to the control group (see...). Figure 2 (A in the control group), but its ethyl acetate content was lower than that of the control group (see A in the control group). Figure 2 The presence of D in the figure indicates that this strain and its produced enzymes and metabolites may inhibit ethyl acetate synthesis. FB13 showed the best effect in promoting ethyl lactate synthesis, followed by FB10, 9, and 20, which increased the synthesis rate by approximately 15%, 13%, 12%, and 11% respectively compared to the control group (see D). Figure 2 (see B in the text), and the ethyl acetate content of these strains was not significantly different from that of the control (see...). Figure 2 The presence of E in the figure indicates that it does not affect the synthesis of ethyl acetate during the promotion of ethyl lactate esterification. Among the FG strains, FG8 exhibits better ethyl lactate synthesis ability, increasing it by approximately 13% (see [reference needed]). Figure 2 (C in the text), and does not affect the content of ethyl acetate (see...) Figure 2 (F in the text). Therefore, strain FB13 is preferred for enhanced fermentation.

[0068] Gas chromatography determination of ethyl lactate and ethyl acetate content:

[0069] Sample pretreatment: The sample solution to be tested was centrifuged at 6200g for 2min, and the supernatant was diluted 3 times and mixed with an equal volume of acetonitrile containing about 800 mg / L butyl acetate. The mixture was stored in a -20℃ refrigerator, centrifuged at 12000r / min for 2min, and filtered through a 0.22μm organic filter membrane.

[0070] The injection port temperature was 250℃, and the FID detector temperature was 300℃; the chromatographic column was a GsBP Inowax column (30m×0.32mm×0.40μm); the injection volume was 1μL; the split ratio was 10:1; the carrier gas was high-purity nitrogen, and the flow rate was set to 2.52mL / min; the temperature program was as follows: the initial column temperature was set to 45℃, and the temperature was increased to 55℃ at a rate of 5℃ / min, and then increased to 120℃ at a rate of 35℃ / min and held for 4.5min.

[0071] Standard curve: Using 15% (v / v) ethanol aqueous solution as solvent, prepare 1000 mg / L ethyl lactate solution and 500 mg / L ethyl acetate solution respectively. Take 10, 25, 50, 100, 200 and 400 μL of the above solutions and add them to 400 μL of 800 mg / L butyl acetate solution (solvent is acetonitrile). Make up to 800 μL with 15% v / v ethanol aqueous solution. Store in a -20℃ refrigerator, centrifuge at 12000 r / min for 2 min, filter through a 0.22 μm organic filter membrane and perform GC analysis.

[0072] (5) Identification of high ethyl lactate-producing strain FB13

[0073] Colony morphology as Figure 3 As shown in Figure A, the colonies on LB medium are pale yellow and opaque, with a rough surface, raised areas, and irregular edges.

[0074] Gram staining: Microscopic examination was performed after Gram staining. The staining method was a three-step method (refer to Huang Yuantong, Cui Jie. Three-step method and quality control of Gram staining [J]. Acta Microbiologica Sinica, 1996, 36(1):3.). The results are as follows: Figure 3 As shown in B, it is a Gram-positive bacterium that appears rod-shaped under an oil immersion microscope, a typical bacillus.

[0075] Growth curve determination: The revived and activated strain was inoculated into LB medium in triplicate, with samples taken every 3 hours to measure its OD. 600 Plot it as a curve, such as Figure 3 As shown in C, it reaches a stable period 15 hours after inoculation.

[0076] Molecular biological identification (16S rRNA gene sequencing identification):

[0077] After inoculating the bacterial strain into LB medium for 18-20 h, 1.2 mL of the culture medium was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 min. The supernatant was removed, and 50 μL of biosynthetic buffer was added to the bacterial cells. The mixture was incubated at 80 °C for 5 min to obtain DNA extract. A fragment of approximately 1000 bp was amplified using primers 27F and 1492R. The amplification system consisted of 50 μL of 2×SanTaq PCR Mix (25 μL), 2 μL each of forward and reverse primers, 2 μL of DNA template, and sterile deionized water to a final volume of 50 μL. The amplification program was as follows: 95 °C for 6 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, 35 cycles; 72 °C for 10 min; 4 °C infinity. The PCR product containing the target fragment obtained in the above steps was sent to Genewiz Biotechnology Co., Ltd. for sequencing, and the sequence sequencing results were compared with the NCBI database for similarity.

[0078] The comparison results identified it as Bacillus amyloliquefaciens, with accession number GDMCC No: 67597, named Bacillus amyloliquefaciens FB13. This strain was deposited on January 5, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0079] Primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0080] Primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0081] The sequence of the 16S rRNA gene is as follows:

[0082] TAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAG

[0083] CGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTA

[0084] ACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTG

[0085] AACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGC

[0086] GCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGA

[0087] GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG

[0088] GGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTT

[0089] TCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCT

[0090] TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA

[0091] GGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTC

[0092] TGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGC

[0093] AGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACAC

[0094] CAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGC

[0095] GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGG

[0096] GTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCG

[0097] CAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA

[0098] ATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATA

[0099] GGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGT

[0100] GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAG

[0101] TTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAAT

[0102] CATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGC

[0103] GAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAA

[0104] CTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACG

[0105] TTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGG

[0106] TGAGGTAACCTTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT

[0107] AACAAGGTAACCA。

[0108] Example 2: Effect of fermentation parameters on the production of ethyl lactate by Bacillus amyloliquefaciens FB13

[0109] Enzyme production time: Bacillus amyloliquefaciens FB13 was inoculated into seed culture medium and cultured at 37℃ and 180r / min for 24h. Then, it was inoculated into enzyme production medium at a 1% v / v inoculation rate and cultured under the same conditions. Samples were taken after 1d, 3d, 5d, 7d and 9d of culture, and the esterase activity was measured according to step (3) of Example 1. The results showed that the esterase activity reached its maximum value when the enzyme production time was 3d.

[0110] Routine inoculation: After culturing in the enzyme-producing medium for 3 days, 5 mL of rice-flavored Baijiu fermentation broth (16-day fermentation broth) was inoculated at inoculation rates of 1%, 5%, 10%, 15%, and 20% v / v, respectively. Fermentation was then intensified at 30℃ for 5 days, and the ethyl lactate concentration was measured. The results showed that under the routine inoculation conditions of 1%, 5%, 10%, 15%, and 20% v / v, the ethyl lactate contents were 0.135, 0.128, 0.127, 0.115, and 0.103 g / L, respectively. The ethyl lactate content reached its maximum value at an inoculation rate of 1%.

[0111] Concentrated inoculation: After culturing in the enzyme-producing medium for 3 days, inoculation volumes of 1%, 5%, 10%, 15%, and 20% v / v were taken respectively, centrifuged at 12000 rpm for 5 min, and the precipitate was collected. The precipitate was resuspended in 50 μL of PBS buffer to obtain concentrated inoculation solution, which was then added to 5 mL of rice wine fermentation broth (16-day fermentation broth) for reaction over 5 days. The results are as follows. Figure 4 As shown, a bacterial concentration of 15% yields the best results, increasing the concentration by 25-26% to 0.18 g / L. Compared to conventional inoculation, concentrated inoculation exhibits stronger ethyl lactate synthesis; therefore, concentrated inoculation with a bacterial concentration of 15% is preferred.

[0112] Fermentation time: After culturing the enzyme-producing medium for 3 days, the concentrated inoculum suspension (15% bacterial volume) obtained according to the above steps was inoculated into 5 mL of fermentation broth. The mixture was sealed and fermented at 30℃. Samples were taken daily to determine the ethyl lactate content. Results are as follows: Figure 5 As shown, the ethyl lactate content reached stability on the 7th day of fermentation.

[0113] Example 3 Application of Bacillus amyloliquefaciens FB13 in the late-stage fermentation broth of rice-scented Baijiu 1

[0114] (1) Seed culture preparation: Bacillus amyloliquefaciens FB13 was inoculated into the seed culture medium and cultured at 37℃ and 180r / min for 24h. Then, it was inoculated into the enzyme-producing medium at a 1% v / v inoculation rate and cultured under the same conditions for 3d.

[0115] (2) Preparation of concentrated inoculation suspension: Take the seed liquid obtained in step (1) and inoculate it with bacterial liquid at an inoculation volume of 15% v / v (i.e. 0.75 mL). Centrifuge at 4℃ and 12000 rpm for 5 min to collect the precipitate. Resuspend the precipitate with 50 μL PBS buffer to obtain concentrated inoculation suspension for later use.

[0116] (3) Inoculation and fermentation: The suspension obtained in step (2) was inoculated into 5 mL of late-stage fermentation broth (16-day fermentation broth) for further enhanced fermentation. The fermentation time was 7 days and the temperature was 30℃. After the fermentation was completed, the ethyl lactate content was measured.

[0117] Example 4: Application of Bacillus amyloliquefaciens FB13 in the late-stage fermentation broth of rice-scented Baijiu (2)

[0118] (1) Seed culture preparation: Bacillus amyloliquefaciens FB13 was inoculated into the seed culture medium and cultured at 37℃ and 180r / min for 24h. Then, it was inoculated into the enzyme-producing medium at a 1% v / v inoculation rate and cultured under the same conditions for 3d.

[0119] (2) Preparation of concentrated inoculation suspension: Take 33 mL of the culture medium from step (1), centrifuge at 4℃ and 12000 rpm for 5 min to collect the precipitate, suspend the precipitate in 2.2 mL of PBS buffer to obtain concentrated inoculation suspension for later use.

[0120] (3) Inoculation and fermentation: The suspension obtained in step (2) was inoculated into 220 mL of late-stage fermentation broth for further enhanced fermentation. The fermentation time was 7 days and the temperature was 30℃.

[0121] (4) Distillation: After enhanced fermentation, 80 mL of water was added to 220 mL of fermentation mash, and the base wine was obtained by distillation. Various flavor substances such as ethyl lactate were determined.

[0122] Example 5: Effect of different temperature treatments on the synthesis of ethyl lactate in the late-stage fermentation broth of rice-scented Baijiu

[0123] Take 5 mL of rice wine fermentation broth from day 16 into a vial, purge with nitrogen, and seal to prevent loss of volatile components such as ethanol. React at 30-100℃ for 4, 6, and 8 hours, respectively, and determine the ethyl lactate content. Results are as follows: Figure 6 As shown, the ethyl lactate content increases with increasing temperature. The best results were observed at 6h and 8h at 90℃ and 100℃, respectively, with no significant difference between the two. Considering the economic benefits, 90℃-6h was chosen.

[0124] Example 6: Application of temperature-controlled reaction at 90℃ for 6 hours in the late-stage fermentation liquid of rice-fragrant liquor 1

[0125] Take 5 mL of rice-flavored baijiu late-stage fermentation liquid (fermentation liquid on day 16) into a vial, blow it with nitrogen, seal it, and react it at 90℃ for 6 h. Then determine the ethyl lactate content.

[0126] Example 7: Application of temperature-controlled reaction at 90℃ for 6 hours in the late-stage fermentation liquid of rice-fragrant liquor 2

[0127] Take 220 mL of the late-stage fermentation liquid of rice-fragrant baijiu (fermentation liquid on day 16) into a microsphere, seal it after nitrogen blowing, and react it at 90℃ for 6 h. Take 220 mL of the fermentation liquid after the reaction is completed, add 80 mL of water, and distill to obtain the base liquor. Determine various flavor substances such as ethyl lactate.

[0128] Example 8: Application of Bio-enhanced Fermentation with Synergistic Temperature Control in the Late Fermentation Broth of Rice-Flavored Baijiu 1

[0129] (1) Seed culture preparation: Bacillus amyloliquefaciens FB13 was inoculated into the seed culture medium and cultured at 37℃ and 180r / min for 24h. Then, it was inoculated into the enzyme-producing medium at a 1% v / v inoculation rate and cultured under the same conditions for 3d.

[0130] (2) Preparation of concentrated inoculation suspension: Take 0.75 mL of the culture solution from step (1), centrifuge at 4℃ and 12000 rpm for 5 min to collect the precipitate, suspend the precipitate in 50 μL PBS buffer to obtain concentrated inoculation suspension for later use.

[0131] (3) Inoculation and fermentation: The suspension obtained in step (2) was inoculated into 5 mL of late-stage fermentation broth for further enhanced fermentation at a temperature of 30°C for 7 days.

[0132] (4) Temperature control treatment: After fermentation, the reaction was carried out at a controlled temperature of 90℃ for 6 hours. The ethyl lactate content was measured after the reaction was completed.

[0133] Example 9: Application of Bio-enhanced Fermentation and Synergistic Temperature Control in the Late Fermentation Broth of Rice-Flavored Baijiu 2

[0134] (1) Seed culture preparation: Bacillus amyloliquefaciens FB13 was inoculated into the seed culture medium and cultured at 37℃ and 180r / min for 24h. Then, it was inoculated into the enzyme-producing medium at a 1% v / v inoculation rate and cultured under the same conditions for 3d.

[0135] (2) Preparation of concentrated inoculation suspension: Take 33 mL of the culture medium from step (1), centrifuge at 4℃ and 12000 rpm for 5 min to collect the precipitate, suspend the precipitate in 2.2 mL of PBS buffer to obtain concentrated inoculation suspension for later use.

[0136] (3) Inoculation and fermentation: The suspension obtained in step (2) was inoculated into 220 mL of late-stage fermentation broth for further enhanced fermentation at a temperature of 30°C for 7 days.

[0137] (4) Temperature control treatment: After fermentation, a temperature control reaction of 90℃-6h is carried out.

[0138] (5) Distillation: After enhanced fermentation and temperature control, 80 mL of water was added to 220 mL of fermentation mash, and the base wine was obtained by distillation. Various flavor substances such as ethyl lactate were determined.

[0139] Comparative Example 1

[0140] 50 μL of PBS buffer containing Bacillus amyloliquefaciens FB13 without additive was added to 5 mL of late-stage fermentation broth and fermented for 7 days at 30°C, serving as a control for Example 3.

[0141] Comparative Example 2

[0142] The late-stage fermentation broth, which did not undergo microbial fermentation and temperature reaction, served as a control for Examples 6 and 8.

[0143] Comparative Example 3

[0144] The base liquor obtained by distillation of the late-stage fermentation broth without microbial fermentation and temperature reaction serves as a control for Examples 4, 7, and 9.

[0145] The ethyl lactate content of the above-described examples and comparative examples was detected, and the results are shown in Table 1.

[0146] Table 1. Comparison of ethyl lactate content (mg / L) in different embodiments

[0147]

[0148] Table 1 shows that strain FB13 has a higher ethyl lactate content in the late fermentation broth of rice-fragrant baijiu than in Comparative Example 1, increasing it by about 26%. In contrast, the effect on the base liquor of rice-fragrant baijiu obtained by distillation is even more significant, increasing it by 44% compared to Comparative Example 3.

[0149] The 90℃-6h temperature-controlled reaction had a stronger effect on the late-stage fermentation liquid of rice-fragrant liquor, namely Example 6, which could improve the effect by about 60-70% compared with Comparative Example 2. However, the effect on the rice-fragrant liquor base liquor obtained by distillation in Example 7 could only improve the effect by about 32% compared with Comparative Example 3.

[0150] The enhanced fermentation synergistic physical temperature-controlled reaction of strain FB13 at 90℃-6h improved the fermentation broth of rice-fragrant liquor by 95.5% (Example 8 compared to Comparative Example 2), and also achieved a synergistic improvement in the base liquor of rice-fragrant liquor obtained by distillation, with Example 9 showing an improvement of about 78% compared to Comparative Example 3.

[0151] In other words, whether it is the fermentation broth or the base wine obtained by distillation, the bio-enhanced fermentation synergistic physical treatment method provided by this invention can achieve a cumulative increase in ethyl lactate content.

[0152] Meanwhile, the relative abundance of each flavor compound in Comparative Example 3 and Example 9 was detected, and the results are as follows: Figure 7 As shown (C is Comparative Example 3, T is Example 7), it can be seen that the synergistic temperature control of microbial fermentation not only significantly increased the content of ethyl lactate and the alcohol yield, but also promoted the synthesis of other esters, increasing the total ester content by about 2 times. Most esters are aroma components of baijiu, and they are among the most crucial substances for baijiu flavor. Ethyl lactate, in particular, possesses fruity and waxy aromas, significantly enhancing the layering and complexity of the flavor profile, bringing a creamy, silky texture and a subtle fruity aroma, adding a smooth and balanced quality to the flavor spectrum. Its presence is especially crucial for neutralizing the irritation of volatile compounds, thereby optimizing the harmony of the aroma. Ethyl acetate, with its fruity aroma, imparts a delicate and pure fragrance. In addition to esters, the synergistic method provided by this invention can also increase the content of various flavor substances such as terpenes and aromatic compounds, while reducing the concentration of substances such as halogenated hydrocarbons that negatively impact flavor.

[0153] Therefore, the bio-fermentation synergistic physical treatment method provided by the present invention can significantly increase the total ester content of rice-fragrant liquor, especially the content of ethyl lactate, thereby improving the complex aroma of rice liquor without affecting its yield, thus improving the quality of the liquor and increasing the rate of premium liquor.

[0154] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens that produces ethyl lactate, characterized in that: The ethyl lactate-producing Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens FB13, with accession number GDMCC No: 67597, and was deposited on January 5, 2026 at the Guangdong Provincial Center for Microbial Culture Collection; the deposit address is No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The application of the ethyl lactate-producing Bacillus amyloliquefaciens according to claim 1 in the brewing of Baijiu (Chinese liquor).

3. The application according to claim 2, characterized in that: The liquor mentioned is rice-flavored liquor.

4. A method for increasing the ethyl lactate content of rice-flavored Baijiu through bio-enhanced fermentation and synergistic physical treatment, characterized in that... Includes the following steps: (1) Inoculate the seed culture of Bacillus amyloliquefaciens that produces ethyl lactate into the enzyme-producing medium and culture it. Centrifuge the resulting culture solution A and take the precipitate. (2) Add the precipitate obtained in step (1) to the rice-fragrant liquor post-fermentation liquid for enhanced fermentation; (3) Temperature control treatment is carried out after fermentation is completed; (4) After adding water to the mash obtained in step (3), distill to obtain the base liquor.

5. The method according to claim 4, characterized in that: The ethyl lactate-producing Bacillus amyloliquefaciens mentioned in step (1) is the ethyl lactate-producing Bacillus amyloliquefaciens mentioned in claim 1; The seed culture described in step (1) is obtained by the following steps: Bacillus amyloliquefaciens that produces ethyl lactate is inoculated into a seed culture medium and cultured with shaking at 35-40°C until the logarithmic growth phase to obtain the seed culture; The seed culture medium is a culture medium containing nitrogen and carbon sources; The inoculation amount of the seed solution mentioned in step (1) is 0.5% to 5% by volume; The enzyme-producing medium mentioned in step (1) is a nitrogen-containing medium; The culture conditions described in step (1) are: shaking culture at 35-40℃ for 48-96 hours; The centrifugation conditions described in step (1) are 10,000 to 15,000 rpm for 3 to 10 minutes.

6. The method according to claim 5, characterized in that: The seed culture medium is composed of the following: beef extract 2.5-3.5 g / L, peptone 8-12 g / L, glucose 4-6 g / L, and sodium chloride 4-6 g / L; The rotation speed of the oscillation culture is 150-250 rpm; The enzyme-producing culture medium has the following composition: 2.5-3.5 g / L beef extract, 8-12 g / L peptone, and 4-6 g / L sodium chloride.

7. The method according to claim 4, characterized in that: The precipitate mentioned in step (2) is the precipitate obtained by centrifugation of culture medium A, which is calculated as 5-20% of the volume of the fermentation liquid in the later stage of rice-fragrant liquor. The precipitate mentioned in step (2) is added to the rice-fragrant liquor fermentation liquid after the precipitate is evenly dispersed using rice-fragrant liquor fermentation liquid or PBS. The rice-scented liquor fermentation liquid mentioned in step (2) is the fermentation liquid after 15 days of rice-scented liquor fermentation; The enhanced fermentation conditions described in step (2) are fermentation at 28–32°C for 6–9 days under sealed conditions; The temperature control treatment conditions described in step (3) are 80-100℃ for 4-8 hours; The amount of water used in step (4) is 30 to 40% of the volume of the mash.

8. The method according to claim 7, characterized in that: The precipitate mentioned in step (2) is the precipitate obtained by centrifugation of culture medium A, which is calculated as 10-15% of the volume of the fermentation liquid in the later stage of rice-fragrant liquor. The amount of PBS used is 1-2% of the volume of the rice-fragrant liquor fermentation liquid in the later stage; The enhanced fermentation conditions described in step (2) are fermentation at 30°C for 7-8 days under sealed conditions; The temperature control treatment in step (3) is to treat at 90-100℃ for 6-8 hours.

9. The application of the method according to any one of claims 4 to 8 in the brewing of rice-scented liquor.

10. A rice-aroma baijiu, characterized in that: Obtained by the method described in any one of claims 4 to 8.

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