Bacillus amyloliquefaciens and application thereof

Abstract

The application discloses a bacillus amyloliquefaciens and application thereof, and belongs to the technical field of microorganisms. The bacillus amyloliquefaciens 15 with high esterification enzyme yield is obtained by screening and separating from a mixture of Luzhou-flavor Daqu and fermented grains, and the preservation number is CGMCC No. 39292. The bacillus amyloliquefaciens 15 has good tolerance to pH and alcohol, and can adapt to the white spirit fermentation process. The bacillus amyloliquefaciens 15 can produce esterification enzyme with a yield of 674.36 U / mL. In addition, the bacillus amyloliquefaciens 15 can also produce saccharifying enzyme and protease at the same time, and has important potential in improving the white spirit fermentation effect and shaping the wine body flavor. Meanwhile, the bacillus amyloliquefaciens 15 can also produce ethyl acetate, ethyl butyrate and other ethyl esters, pyrazines, aldehydes, phenols and other flavor substances.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a Bacillus amyloliquefaciens and its applications. Background Technology

[0002] Baijiu is a traditional distilled spirit unique to China. Its brewing follows a solid-state, dual-fermentation process, using grains such as sorghum as the main raw material and large-grain, small-grain, or bran-based koji as saccharification and fermentation agents. Through a microbial community-driven process of simultaneous saccharification and fermentation, ethanol and flavor precursors are produced. Finally, through solid-state distillation and graded collection, a complex flavor system centered on ester compounds (such as ethyl hexanoate and ethyl acetate) is formed. Based on its unique brewing process and flavor characteristics, it can be further subdivided into sauce-aroma, strong-aroma, light-aroma, rice-aroma, and other aroma types. Different baijiu have their own unique flavor and taste. This is because specific microbial communities and enzyme systems during fermentation allow the starch and sugar in the raw materials to be converted into alcohol and various aroma substances.

[0003] Esterases, also known as esterases, mainly include lipases, ester synthases, phosphoesterases, and ester-degrading enzymes. As important industrial enzymes, esterases are widely used in food processing, biodiesel production, pharmaceutical synthesis, and the preparation of fine chemicals. They catalyze the formation and hydrolysis of ester bonds, significantly improving the efficiency and selectivity of related reactions and possessing significant economic value. In baijiu (Chinese liquor) production, esterases catalyze the synthesis of esters from acids and alcohols in microbial metabolism and fermentation processes, making them a major provider of ester aroma compounds in baijiu and playing a crucial role in the post-fermentation maturation of baijiu flavor.

[0004] Currently, the production of esterases during the fermentation of baijiu (Chinese liquor) mainly relies on microbial fermentation. Existing strains generally exhibit low esterase yields and insufficient enzyme activity stability, limiting their widespread application. To improve esterase yield, current technologies often employ strategies such as optimizing culture medium formulations or fermentation process parameters, but the improvement effects are limited.

[0005] Therefore, how to provide a strain with high esterase yield and stable enzyme activity is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a Bacillus amyloliquefaciens and its application.

[0007] This invention screened and obtained a high-yield esterase-producing Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens). Bacillus amyloliquefaciensThe strain exhibits an esterase activity of 674.36 U / mL. This invention also expands the application of this strain in the synthesis of flavor esters in food, providing an innovative solution for the efficient and low-cost industrial production of esterases. This strain possesses acid, alcohol, and temperature tolerance, and can be used in the fermentation of baijiu (Chinese liquor) to increase the ester content and improve the flavor of the liquor.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A type of Bacillus amyloliquefaciens, named 15, is classified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39292. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] The above-mentioned application of Bacillus amyloliquefaciens in the fermentation production of esterase.

[0011] The above-mentioned application of Bacillus amyloliquefaciens in the fermentation production of proteases and saccharifying enzymes.

[0012] The above-mentioned application of Bacillus amyloliquefaciens in the brewing of baijiu.

[0013] Furthermore, it is used to increase the content of total acids and total esters.

[0014] Furthermore, the grains used for brewing include at least one of sorghum, wheat bran, and five grains.

[0015] Furthermore, the aroma type of the baijiu includes at least one of the following: soy sauce aroma, strong aroma, light aroma, phoenix aroma, rice aroma, soy sauce aroma, mixed aroma, sesame aroma, special aroma, old baijiu aroma, rich aroma, and Dong aroma.

[0016] After solid-state fermentation with Bacillus amyloliquefaciens, volatile flavor compounds such as alcohols, aldehydes, acids, esters, and phenols were detected in the fermented liquid by GC-MS. Bacillus amyloliquefaciens can metabolize and produce these functional flavor compounds, and their application in fermented food production has a beneficial impact on product quality improvement and flavor shaping.

[0017] A microbial agent comprising the aforementioned Bacillus amyloliquefaciens.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention isolates and screens a high-yield esterase-producing strain of *Bacillus amyloliquefaciens* 15 from a mixture of strong-aroma daqu (fermentation starter) and mash, with the preservation number CGMCC No. 39292. This bacterium exhibits excellent tolerance to pH and alcohol content, enabling it to adapt to the baijiu fermentation process. This strain produces a high yield of esterase, at 674.36 U / mL. Furthermore, it can simultaneously produce saccharifying enzymes and proteases, demonstrating significant potential for enhancing baijiu fermentation and shaping its flavor. Simultaneously, this strain also produces high yields of ethyl esters such as ethyl acetate and ethyl butyrate, as well as various flavor compounds including pyrazines, aldehydes, and phenols. 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 description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is the α-naphthol standard curve in Example 1 of the present invention.

[0021] Figure 2 The images show the morphological identification results of strain 15 in Example 1 of this invention, where a is a physical image of the morphological characteristics of strain 15, b is the Gram staining result of strain 15, and c is an electron micrograph of strain 15.

[0022] Figure 3 This is an electrophoresis diagram of 16S rDNA of strain 15 in Example 1 of the present invention.

[0023] Figure 4 This is a phylogenetic tree diagram of strain 15 in Example 1 of the present invention.

[0024] Figure 5 The results show the ethanol tolerance of Bacillus amyloliquefaciens 15 in Example 2 of this invention.

[0025] Figure 6 The results show the acid and alkali tolerance of Bacillus amyloliquefaciens 15 in Example 2 of this invention.

[0026] Figure 7 The results show the temperature tolerance of Bacillus amyloliquefaciens 15 in Example 2 of this invention.

[0027] Figure 8 This is the tyrosine standard curve in Example 3 of the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The culture media used in the following examples are as follows: Screening medium (1 L): 3.00 g / L beef extract, 10.00 g / L peptone, 5.00 g / L NaCl, 20.00 g / L agar, 0.40% tretinoin, 1000 mL distilled water, sterilized at 121℃ for 20 min.

[0030] LB liquid medium (1 L): 10.00 g / L tryptone, 10.00 g / L sodium chloride, 5.00 g / L yeast extract, 1000 mL distilled water, sterilized at 121℃ for 20 min.

[0031] LB solid medium (1 L): tryptone 10.00 g / L, sodium chloride 10.00 g / L, yeast extract 5.00 g / L, agar 20.00 g / L, distilled water 1000 mL, sterilized at 121℃ for 20 min.

[0032] Fermentation medium (1 L): beef extract 20.00 g / L, NaCl 5.00 g / L, K2HPO4 1.00 g / L, (NH4)2SO4 1.00 g / L, MgSO4·H2O 1.00 g / L, FeSO4·7H2O 0.01 g / L, glucose 20.00 g / L, sterilized at 115 ℃ for 30 min.

[0033] Example 1 Screening and identification of strain 15 I. Screening of strain 15 1) Initial screening: Take 5 g of strong-aroma Daqu and 5 g of strong-aroma mash (both samples were taken from a winery in Yibin City, Sichuan Province) and add them to an Erlenmeyer flask containing 100 mL of sterile physiological saline. Incubate at 30℃ and 180 r / min for 24 h, then let stand for 30 min. Take 1 mL of the enriched supernatant into a centrifuge tube containing 9 mL of sterile physiological saline and perform serial dilutions to obtain 10... -2 10 -3 10 -4 10 -5 10 -6 Dilute the bacterial solution. Take 10 -3 10 -4 10-5 Three gradients of bacterial culture, 100 μL each, were spread onto selection medium, with three replicates for each dilution. The cultures were incubated at 30°C for 48 h. The ratio of the clear zone diameter (D) to the colony diameter (d) (HC) was calculated. Colonies with varying morphological characteristics and larger HC values ​​were selected and streaked onto LB medium for isolation. This process was repeated until single colonies appeared.

[0034] 2) Secondary screening: Seed culture of each purified strain was prepared and inoculated into fermentation medium at an inoculum of 1%, and cultured at 30℃ for 48 h. Parallel experiments were performed simultaneously. After centrifuging the fermentation broth at 10000 r / min for 10 min, the supernatant was collected, and the esterification enzyme activity was determined by the α-naphthol colorimetric method. The strain with the highest enzyme activity was selected for streak subculturing until a stable enzyme-producing strain 15 with high enzyme activity was obtained (Table 1).

[0035] Table 1. HC values ​​and enzyme activities of the screened strains

[0036] The α-naphthol colorimetric method for determining esterase activity is as follows: Seven test tubes were labeled 0, 1, 2, 3, 4, 5, and 6. 3.0 mL of pH 6.0, 0.2 mol / L phosphate buffer was added to each tube, followed by 0.00 mL, 0.01 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, and 0.10 mL of 1.5 mmol / L α-naphthol standard ethanol solution, respectively. The tubes were incubated at 37°C with shaking for 15 min. Each tube was then removed, and 0.40 mL of Gulan B salt solution was added to each. The mixture was shaken and incubated at 37°C for 10 min. The absorbance was measured at 528 nm, with tube 0 used as the zero point. A standard curve was plotted with absorbance on the ordinate and α-naphthol concentration on the abscissa. The results are shown below. Figure 1 As shown, its regression equation is y = 0.0028x + 0.0039, and the correlation coefficient R0 is... 2 =0.999.

[0037] Esterase activity determination: Take 0.50 mL of the treated enzyme solution, add 3.00 mL of pH 6.0 phosphate buffer solution and 0.10 mL of α-naphthyl acetate, incubate at 37℃ for 15 min, add 0.40 mL of Solid Blue B salt, incubate at 37℃ for 10 min, and then measure the absorbance at 528 nm, using a blank tube without α-naphthyl acetate to zero the sample. Calculate the esterase activity of the strain based on the α-naphthol standard curve.

[0038] Enzyme activity is defined as the amount of enzyme required to hydrolyze α-naphthyl acetate to produce 1.0 nmol of α-naphthol at 37℃ for 15 min, which is one enzyme activity unit.

[0039] II. Identification of strain 15 1) Morphological identification: The above-mentioned strain 15 was inoculated into LB solid medium and cultured at 30℃ for 48 h. The colony morphology was then observed. The colonies of strain 15 were pale yellow and transparent, with a moist and raised surface, relatively neat edges, and a relatively regular shape. They had low viscosity and were easily picked up. Figure 2 a). Gram staining is purple ( Figure 2 b). Scanning electron microscopy reveals that the cells are rod-shaped. Figure 2 c).

[0040] 2) Molecular biological identification: DNA was extracted from strain 15 using the TSINGKE Plant DNA Extraction Kit (Universal). PCR amplification was performed using universal primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2) and 1492R (5'-TACGGCTACCTTGTTA-3', SEQ ID NO.3). The amplification system was as follows: 45 µL of 1×TSE101 Gold Mix, 2 µL of universal primers 27F (10P) and 1492R (10P), and 1 µL of DNA template; the amplification program is shown in Table 2.

[0041] Table 2 Amplification Procedure

[0042] The extraction efficiency of the amplified products was detected using a 1.5% agarose gel electrophoresis. Figure 3 After meeting the requirements, the amplified product was sent to Qingke Biotechnology Co., Ltd. for gene sequencing. The sequencing results are shown in SEQ ID NO.1. Then, the sequence was submitted to the National Center for Biotechnology Information (NCBI) database for sequence alignment using the Basic Local Alignment Search Tool (BLAST) to construct a phylogenetic tree. Figure 4 ).

[0043]

[0044] Strain 15 was identified as Bacillus amyloliquefaciens through morphological and molecular biological analysis. Bacillus amyloliquefaciens .

[0045] III. Preservation Strain 15, classified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39292. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0046] Example 2 Acid resistance, ethanol resistance and temperature tolerance of Bacillus amyloliquefaciens 15 I. Ethanol Tolerance Fermentation medium was added to 250 mL Erlenmeyer flasks at concentrations of 97 mL, 96 mL, 95 mL, 94 mL, 93 mL, and 92 mL, respectively. The flasks were then autoclaved at 121 °C for 20 minutes and cooled to room temperature. Subsequently, under aseptic conditions, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, and 8 mL of anhydrous ethanol were added to each flask, respectively, to achieve final alcohol content (V / V) of 3%, 4%, 5%, 6%, 7%, and 8%. The activated Bacillus amyloliquefaciens 15 culture was inoculated at a 1% inoculum, and the cultures were incubated at 30 °C with shaking at 180 r / min for 24 h. The OD value of the culture at 600 nm was then measured to determine the ethanol tolerance of the strain.

[0047] like Figure 5 As shown, the growth of *Bacillus amyloliquefaciens* 15 is inhibited with increasing ethanol content, and the inhibitory effect is significant when the ethanol content reaches 7%. When the ethanol content is greater than 7%, the strain hardly grows. *Bacillus amyloliquefaciens* 15 has a maximum alcohol tolerance of 7%, indicating potential application in baijiu (Chinese liquor) production.

[0048] II. Acid-base tolerance The activated Bacillus amyloliquefaciens 15 bacterial suspension was inoculated into 100 mL of sterilized fermentation medium at an inoculation rate of 1%. The pH of the medium (adjusted with hydrochloric acid and sodium hydroxide solution) was 3, 4, 5, 6, 7, 8, 9, and 10, with three replicates for each pH. After shaking culture at 30℃ and 180 r / min for 24 h, the absorbance of the bacterial suspension at a wavelength of 600 nm was measured to determine the acid and alkali tolerance of the strain.

[0049] Depend on Figure 6 It can be seen that within the pH range of 3 to 7, as pH increases, OD...600 The higher the pH, the better the growth of Bacillus amyloliquefaciens 15; within the pH range of 8-10, growth gradually declines with increasing pH. Therefore, Bacillus amyloliquefaciens 15 can grow at pH 3-10, with good growth at pH 5-9; its optimal growth pH is 7; and its minimum tolerance pH is 3. It can adapt to the acidic fermentation environment of baijiu (Chinese liquor).

[0050] III. Temperature Tolerance The activated Bacillus amyloliquefaciens 15 bacterial suspension was inoculated into 100 mL of sterilized fermentation medium at an inoculation rate of 1%, and placed in shakers at 24℃, 27℃, 30℃, 33℃, 36℃, 39℃, and 42℃ respectively. After shaking and culturing at 180 r / min for 24 h, the OD value of the bacterial suspension at a wavelength of 600 nm was measured to determine the temperature tolerance of the strain.

[0051] like Figure 7 As shown, Bacillus amyloliquefaciens 15 exhibits good growth within the temperature range of 24℃ to 39℃, with an OD of [value missing] at 30℃. 600 It reaches its maximum. At 42℃, it decreases significantly, and the growth of the bacteria is somewhat inhibited. Its optimal growth temperature is 30℃.

[0052] Example 3 Other enzyme-producing characteristics of Bacillus amyloliquefaciens 15 The activated Bacillus amyloliquefaciens 15 bacterial suspension was inoculated into 100 mL of sterilized fermentation medium at a 1% inoculum rate and cultured at 30℃ and 180 r / min for 48 h with shaking. Parallel experiments were performed simultaneously. After centrifuging the fermentation broth at 10000 r / min for 10 min, the supernatant was collected, and the enzyme activities of protease and saccharifying enzyme in the fermentation broth were determined.

[0053] The protease activity was determined according to the Folin-Ciocalteu colorimetric method in DB34 / T 3085-2018 "Operating Procedures for the Detection of Strong Aroma Daqu". A standard curve was plotted with tyrosine concentration (μg / mL) on the x-axis and absorbance at 680 nm on the y-axis, and a linear regression equation was calculated. Figure 8 As shown, its regression equation is y = 0.0077x + 0.0684, and the correlation coefficient R0 is... 2 =0.999. The protease activity of Bacillus amyloliquefaciens 15 was obtained as 117.65 U / mL through regression equation.

[0054] The saccharifying enzyme activity was determined according to the saccharifying power method in QB / T 4257-2011 "General Analytical Methods for Brewing Daqu". The saccharifying enzyme activity of Bacillus amyloliquefaciens 15 was determined to be 337.25 U / mL by titration.

[0055] Example 4 Flavor determination of Bacillus amyloliquefaciens 15 solid-state fermentation 15 single colonies of Bacillus amyloliquefaciens were picked and inoculated into 100 mL of LB liquid medium and cultured at 30℃ and 180 r / min for 24 h to prepare seed culture.

[0056] Take 1000g of sorghum, lightly crush it to the size of plum blossom petals, and soak it in 100℃ water for 6-8 hours, with the water just covering the sorghum by 1 cm. Steam for 1 hour, then steam at 70℃ for 1 hour, and then steam again for 1 hour. Add 5% rice bran and steam for 1 hour. Let it cool to 40-50℃. Add the steamed rice bran and mix well; add 0.4% activated commercial brewing koji and mix well. Stack and saccharify at 25℃ for 24 hours.

[0057] The seed culture was inoculated into sorghum that had undergone saccharification at a rate of 1%, with 1% sterile water added as a control. Fermentation was carried out at a constant temperature of 25℃ in a sealed container for 15 days, followed by distillation. Total acidity was determined according to the titration method in GB / T 12456-2021 "Determination of Total Acidity in Food", and total esters were determined according to GB / T 10345-2022 "Analytical Methods for Baijiu". Flavor compounds were determined using GC-MS.

[0058] GC-MS (Internal Standard Method): Take 5 mL of wine sample into a 10 mL headspace vial, add 20 μL of 2-acetylpyridine as an internal standard, tighten the vial, and equilibrate at 50 °C for 5 min. After aging the extraction needle at 210 °C for 30 min, insert it into the vial, and then insert a 50 μm / 30 μm VB / CAR on PDMS extraction needle. Extract for 50 min, and desorb for 5 min.

[0059] Gas chromatography conditions: DB-WAX capillary column (30 m × 320 μm × 0.25 μm); injection port temperature 250℃, splitless mode; temperature program: 40℃ for 3 min, 5℃ / min to 180℃, and finally 230℃ at 2.5℃ / min for 10 min, for a total of 50 min; carrier gas: high-purity helium, flow rate 1 mL / min.

[0060] Mass spectrometry conditions: Electron impact ionization (EI) ion source, electron energy 70 eV, emission current 200 A, voltage 350 V, ion source temperature 200%, interface temperature 250 °C, scanning mass range 33~450 u, scanning range 20~500 u.

[0061] Table 3. Results of total acid and total ester determination of Bacillus amyloliquefaciens 15

[0062] Table 4. Relative quantification results of major flavor compounds in solid-state fermentation of Bacillus amyloliquefaciens 15

[0063] As shown in Table 3, compared with the control group, the experimental group with added Bacillus amyloliquefaciens 15 showed an increase in the total acid and total ester content in the distilled liquor sample, especially the total ester content. This indicates that Bacillus amyloliquefaciens 15 can effectively promote the synthesis of esters and improve the quality of baijiu during the fermentation process.

[0064] As shown in Table 4, Bacillus amyloliquefaciens 15 has a high content of esters during fermentation, and can also produce acids, alcohols, guaiacol and other substances, which can enhance the flavor and improve the quality of baijiu.

[0065] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0066] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Bacillus amyloliquefaciens, characterized in that, Named 15, its classification is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39292. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of Bacillus amyloliquefaciens as described in claim 1 in the fermentation production of esterase.

3. The application of Bacillus amyloliquefaciens as described in claim 1 in the fermentation production of proteases and saccharifying enzymes.

4. The application of Bacillus amyloliquefaciens as described in claim 1 in the brewing of baijiu (Chinese liquor).

5. The application as described in claim 4, characterized in that, Used to increase the content of total acid and total ester.

6. A microbial inoculant, characterized in that, Includes Bacillus amyloliquefaciens as described in claim 1.

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