Bacillus licheniformis DY-1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses bacillus licheniformis and application thereof. The strain is specifically bacillus licheniformis DY-1, is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 1, 2024, and has the preservation number of CGMCC No.31125. The strain is screened from fermented grains, belongs to a food safety level strain, and can produce amylase and various flavor substances. The strain and a fermentation product thereof can be used for preparing amylase; the production of one or a combination of more of acetoin, 2, 3-butanediol, 4-ethyl guaiacol, pyrazine and phenethyl alcohol can be selectively controlled by changing a fermentation substrate, and edible flavors with different high added values can be selectively obtained; the method can be applied to production of white spirit, produced amylase is beneficial to decomposition of starch in grain raw materials in the white spirit brewing process, produced flavor substances are beneficial to improvement of the overall flavor of the white spirit, and the method has good application prospects and practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus licheniformis DY-1 and an application thereof. Background Art

[0002] Baijiu is a traditional Chinese distilled liquor, and its unique flavor mainly comes from the volatile flavor substances in it. Although the content of these substances is not high, they play a decisive role in the aroma and taste of baijiu. The exploration of volatile flavors in the baijiu production process will help improve the quality and diversity of baijiu.

[0003] Acetoin is a precursor of 2,3-butanediol and can be produced through the metabolic pathway of microbial fermentation. Acetoin itself has a specific aroma and can add a complex flavor layer to liquor. In some studies, the production of 2,3-butanediol by Saccharomyces cerevisiae can be increased by exogenous addition of acetoin, thereby improving the quality of liquor. 2,3-Butanediol, as an important organic compound, plays a role in flavoring liquor and can increase the sweetness and mellowness of liquor. It is converted from acetoin through the action of 2,3-butanediol dehydrogenase (BDH) during microbial metabolism. The yield of 2,3-butanediol is an important indicator for measuring the quality of liquor. In addition, both acetoin and 2,3-butanediol are important precursors for the synthesis of tetramethylpyrazine, an important flavor substance. Guaiacol compounds can be used as flavor additives to provide a unique aroma for liquor. Phenylethanol naturally exists in plants and essential oils such as roses and carnations. It is an aromatic alcohol with the scent of roses. It also plays a role in enhancing the flavor of white wine. It has the aroma of roses and can add a delicate floral note to the white wine.

[0004] In the process of liquor brewing, Bacillus is one of the most important bacterial genera, especially for traditional Chinese Maotai-flavor and Luzhou-flavor liquors. In the process of liquor brewing, Bacillus can degrade protein, produce amino acids and other nitrogen sources, but the currently used Bacillus has a single function and cannot meet the actual production needs. Therefore, it is of great practical value to screen out a strain of Bacillus with multiple functions, and its application in the liquor brewing process plays an important role in enhancing the utilization of raw materials and the construction of flavor substances in the wine. Summary of the invention

[0005] The present invention aims at the problems in the prior art and provides a strain of Bacillus licheniformis DY-1 and its application. The strain is a natural probiotic selected from fermented grains, can produce amylase and a variety of flavor substances, and can be used to produce amylase, flavor substances, liquor and edible spices.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a strain of Bacillus licheniformis DY-1, whose classification name is Bacillus licheniformis ( Bacillus licheniformis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on July 1, 2024, and its deposit number is CGMCC No.31125; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The 16S rDNA sequence of Bacillus licheniformis DY-1 (shown in SEQ ID No. 1) is as follows:

[0008] The Bacillus licheniformis DY-1 provided by the present invention has flat, light yellow colonies with a smooth surface and a diameter of 3 mm, and shows a positive result after Gram staining.

[0009] Compared with the prior art, the Bacillus licheniformis DY-1 provided by the present invention with a deposit number of CGMCC No. 31125 can simultaneously produce amylase and multiple flavor substances, wherein the flavor substances include one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol. When Bacillus licheniformis DY-1 uses sorghum as a fermentation substrate, it has the ability to simultaneously produce acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol.

[0010] In a second aspect, the present invention also provides a fermentation product of the above-mentioned Bacillus licheniformis DY-1 strain, wherein the fermentation product is obtained by fermenting the Bacillus licheniformis DY-1 strain.

[0011] The fermentation products are various valuable substances generated by Bacillus licheniformis DY-1 through metabolic activities, including primary metabolites and secondary metabolites of raw materials after metabolism by Bacillus licheniformis DY-1, and their existence forms include Bacillus licheniformis DY-1 cells, fermentation broth, solid fermentation products, etc.

[0012] Preferably, the fermentation product is amylase or an extract comprising amylase.

[0013] Preferably, the fermentation product is one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol, or an extract containing one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

[0014] In a third aspect, the present invention further provides a bacterial agent comprising the above-mentioned Bacillus licheniformis DY-1.

[0015] The active microorganisms contained in the microbial agent provided by the present invention may be only Bacillus licheniformis DY-1 or a fermentation microbial combination containing Bacillus licheniformis DY-1; and may also include other substances required for preparing the microbial agent such as carriers, nutrients, protective agents, fillers, etc. The dosage form of the microbial agent includes but is not limited to suspensions, powders, granules, etc.

[0016] In a fourth aspect, the present invention also provides the use of the above-mentioned Bacillus licheniformis DY-1 in the preparation of one or more combinations of amylase, acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

[0017] The Bacillus licheniformis DY-1, the microbial combination comprising Bacillus licheniformis DY-1 or the fermentation product of Bacillus licheniformis DY-1 provided by the present invention can be used to prepare one or more combinations of amylase, acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

[0018] The suitable temperature of the amylase produced by the Bacillus licheniformis DY-1 of the present invention is 50-70°C, preferably 65°C; the suitable pH of the amylase produced is 5-7, preferably pH 6.

[0019] In a fifth aspect, the present invention also provides the use of the above-mentioned Bacillus licheniformis DY-1, fermentation product or bacterial agent in decomposing starch.

[0020] In a sixth aspect, the present invention also provides a method for producing amylase, comprising: fermenting and culturing the above-mentioned Bacillus licheniformis DY-1 to obtain amylase in the fermentation broth, wherein the fermentation and culturing conditions are pH 5.0-8.0, the fermentation temperature is 34-40°C, and the culture medium used is a starch-containing culture medium.

[0021] Preferably, the fermentation culture conditions are pH 7.0 and the fermentation temperature is 36-38°C.

[0022] Preferably, the culture medium is a culture medium containing starch and 1% (w / v)-5% (w / v) sodium chloride.

[0023] Optionally, the culture medium is a potato dextrose agar medium containing 1% (w / v)-5% (w / v) sodium chloride or a potato dextrose medium containing 1% (w / v)-5% (w / v) sodium chloride.

[0024] In a seventh aspect, the present invention also provides a method for producing flavor substances using the above-mentioned Bacillus licheniformis DY-1, comprising: inoculating the above-mentioned Bacillus licheniformis DY-1 into a culture medium, and obtaining a fermentation product containing flavor substances after fermentation culture; the flavor substances are one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

[0025] Preferably, the inoculation amount of Bacillus licheniformis DY-1 is 7×10 7 -3.5×10 8 CFU / mL; the fermentation culture temperature is 34-40°C and the time is 24-72 h.

[0026] Preferably, the main raw material of the culture medium is one or more combinations of sorghum, sorghum hydrolyzate or sorghum extract; and the flavor substances are acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol.

[0027] More preferably, the culture medium is a sorghum solid culture medium, which includes sorghum flour and water, with a material-water ratio of sorghum flour to water of 1:1-2 and a pH of 6-8.

[0028] Illustratively, the present invention provides a method for preparing a sorghum solid culture medium, wherein the sorghum is crushed and sieved and then mixed with water, soaked for 18 hours at a material-water ratio of 1:1, and sterilized at 115° C. for 20 minutes to adjust the pH.

[0029] More preferably, the culture medium is a sorghum hydrolyzate culture medium, and the preparation method of the sorghum hydrolyzate culture medium comprises the following steps: S1: water and glutinous sorghum flour are evenly mixed and gelatinized to obtain a sorghum suspension; S2: performing a composite enzymatic hydrolysis on the sorghum suspension, and the enzymatic hydrolysis is terminated when the sugar content reaches 7-11° Brix and is stable and no longer changes, thereby obtaining a sorghum enzymatic hydrolyzate; the composite enzymatic hydrolysis is performed by sequentially performing enzymatic hydrolysis with α-amylase, saccharifying enzyme and acid protease; S3: After solid-liquid separation of the bran enzymatic hydrolysate, the liquid phase is collected, the sugar content is adjusted to 8-10° Brix, and then sterilized to obtain a sorghum hydrolysate culture medium.

[0030] Exemplarily, the present invention provides a method for preparing a sorghum hydrolyzate culture medium, comprising the following steps: uniformly mixing water and glutinous sorghum flour in a solid-liquid ratio of 1:3-4, gelatinizing in a 60°C water bath for 30 min, adding 1 μL / g of α-amylase and liquefying in a 90°C water bath for 20 min, immediately heating and boiling on an induction cooker for 20 min, cooling to 60°C immediately, adding 2 μL / g of saccharifying enzyme and keeping warm in a 60°C water bath for 4 h, adjusting the temperature of the water bath to 40°C, adding acidic protease (2 μL / g) and continuing to keep warm in a 40°C water bath for 2 h, and completing the enzymolysis when the sugar content reaches 7-11°Brix and is stable and no longer changes, filtering with gauze after completion, adjusting the sugar content to 8-10°Brix, and sterilizing at 115°C and 40°C for 20 min.

[0031] Preferably, the carbon source of the culture medium is glucose; and the flavor substances are acetoin, 2,3-butanediol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine and phenylethanol.

[0032] More preferably, the culture medium is yeast extract peptone glucose medium.

[0033] In an eighth aspect, the present invention also provides the use of the above-mentioned Bacillus licheniformis DY-1 in liquor brewing.

[0034] The Bacillus licheniformis DY-1 strain provided by the present invention is beneficial to the decomposition of starch in raw materials in liquor brewing, and can produce acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol, which is helpful to improve the flavor of liquor. The application of the strain in the liquor brewing process plays an important role in enhancing the utilization of raw materials and the construction of flavor substances in the liquor body.

[0035] Preferably, the liquor is liquor made from sorghum as the main raw material, including but not limited to sauce-flavor liquor, strong-flavor liquor and light-flavor liquor made from sorghum as the main raw material.

[0036] In a ninth aspect, the present invention also provides the use of the above-mentioned Bacillus licheniformis DY-1 in the preparation of edible flavors.

[0037] The Bacillus licheniformis DY-1 strain provided by the present invention can be used to produce different aromas by fermenting different substrates, and the production of one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol can be selectively controlled by selecting fermentation raw materials. These edible flavors can be directly used for tobacco flavoring or used as food additives after purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 This is a Lushi iodine staining image of the Bacillus licheniformis strain DY-1 of Example 1 of the present invention; Figure 2 This is the plate colony picture of Bacillus licheniformis DY-1; Figure 3 This is an optical microscope examination image of Bacillus licheniformis DY-1; Figure 4 is the phylogenetic tree of Bacillus licheniformis strain DY-1; Figure 5 The results of the effect of temperature on the enzyme production of Bacillus licheniformis strain DY-1 in Example 2 of the present invention; Figure 6 The results of the effect of pH on the enzyme production of Bacillus licheniformis strain DY-1 in Example 2 of the present invention; Figure 7 The results of the influence of different temperatures on the amylase activity of Bacillus licheniformis DY-1 in Example 3 of the present invention; Figure 8The results are as follows: The results of the effects of different pH values ​​on the amylase activity produced by Bacillus licheniformis DY-1 in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] Terminology Note: One unit of enzyme activity (U) is defined as: the amount of enzyme required to produce 1 μg of glucose per minute in 1 mL of crude enzyme solution at 65°C and pH 6, and is expressed in U / mL.

[0042] The term "U / g" means the specific activity of the enzyme, that is, the enzyme activity contained per gram of enzyme or bacteria.

[0043] In the above text of the present invention, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the value of each number may vary by less than ±10% or reasonable differences considered by those skilled in the art, such as ±1%, ±2%, ±3%, ±4% or ±5%.

[0044] The term "room temperature" refers to a temperature between about 18°C ​​and about 35°C, or between about 20°C and 30°C, or about 25°C.

[0045] The culture medium used in the following embodiments of the present invention is: Starch clearing zone medium (g / L): peptone 10, beef powder 5, NaCl 5, soluble starch 10, agar powder 20, pH 7.0, 121℃, high pressure steam sterilization for 20 min.

[0046] Starch fermentation medium (g / L): peptone 20, soluble starch 20, disodium hydrogen phosphate 5, magnesium sulfate 0.1, sodium chloride 0.1, high-pressure steam sterilization at 121℃ for 20 min.

[0047] LB medium (g / L): 10% peptone, 5% yeast extract powder, 5% sodium chloride. Add 15% agar powder to prepare solid medium and sterilize with high pressure steam at 121℃ for 20 min.

[0048] Yeast extract peptone glucose medium (YPD): 10 g yeast extract powder and 20 g peptone are dissolved in 900 mL water, sterilized by high pressure steam at 121°C for 20 min, and then 100 mL 20 g glucose is added (the glucose solution needs to be sterilized before addition).

[0049] The preparation method of sorghum hydrolyzate culture medium is as follows: water and glutinous sorghum are evenly mixed in a solid-liquid ratio of 1:3, gelatinized in a 60°C water bath for 30 min, 1 μL / g of α-amylase is added and liquefied in a 90°C water bath for 20 min, and then heated and boiled on an induction cooker for 20 min, immediately cooled to 60°C, 2 μL / g of saccharifying enzyme is added and kept in a 60°C water bath for 4 h, the temperature of the water bath is adjusted to 40°C, acidic protease (2 μL / g) is added and kept in a 40°C water bath for 2 h, filtered with gauze after completion, the sugar content is adjusted to 10 °Brix, and sterilized at 115°C at 40°C for 20 min.

[0050] The sorghum solid liquid culture medium is prepared by crushing the sorghum, soaking it for 18 hours at a material-water ratio of 1:1, and sterilizing it at 115° C. for 20 minutes.

[0051] The method for determining the activity of amylase in the following examples is as follows: The amylase activity was determined using the 3,5-dinitrosalicylic acid method (DNS). Under alkaline conditions, 3,5-dinitrosalicylic acid (DNS) undergoes a redox reaction with reducing sugars, and the resulting 3-amino-5-nitrosalicylic acid appears brown-red under boiling conditions. Within a certain concentration range, the color depth is proportional to the reducing sugar content, and the reducing sugar content can be determined by colorimetry.

[0052] Preparation of DNS reagent: weigh 10 g DNS in 600 mL distilled water, gradually add 10 g sodium hydroxide, heat (50°C water bath) to dissolve, add 200.0 g potassium sodium tartrate, 2.0 g phenol and 5.0 g anhydrous sodium sulfite respectively, wait until all are dissolved and cool to room temperature, dilute to 1 L with water, filter, and store in a brown reagent bottle.

[0053] Preparation of glucose standard curve: 3,5-dinitrosalicylic acid (DNS) reagent configuration. Draw the glucose standard curve as shown in Table 1: Take 25 mL stoppered test tubes, add 1 mg / mL glucose standard solution and DNS reagent according to the amount shown in the table, and shake well; heat the test tube in a boiling water bath for 5 min. Take it out and cool to room temperature, dilute to 10 mL with deionized water, shake well, use tube No. 1 as blank zero, measure the absorbance values ​​of tubes 2-7 at a wavelength of 540 nm, make three parallels for each gradient, and draw a graph with glucose concentration as the horizontal axis and absorbance value as the vertical axis to establish a standard curve between glucose concentration and absorbance value.

[0054] Table 1 Drawing of glucose standard curve

[0055] The method for determining the yield of flavor substances in the following examples is as follows: Headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to determine the production of flavor compounds.

[0056] The GC-MS / MS used was an Agilent 8890+5977C gas chromatograph-mass spectrometer with an HP-INNOWAx (60 m×320 um×0.5 um) column. The heating program was as follows: initial temperature of 40°C for 3 min, then heated to 120°C at a rate of 5°C / min and held for 3 min, then heated to 230°C at a rate of 3°C / min and held for 5 min. Inlet temperature: 250°C; column head pressure: 2.9 psi; carrier gas: helium (99.9%); carrier gas flow rate: 1.5 mL / min; mode: splitless; solvent delay time: 3 min.

[0057] Mass spectrometry conditions: EI source; 70 ev; temperature 230°C; quadrupole temperature: 200°C; full scan mode: scan range 35-400 amu.

[0058] Unless otherwise specified, the fermentation conditions used in the examples of the present invention are conventional fermentation conditions in the art. Unless otherwise specified, the reagents used in the examples of the present invention are commercially available.

[0059] Example 1 Isolation, identification and sequencing of strains 1. Screening of Bacillus licheniformis DY-1 1.1 Screening of fermented grain microorganisms In the clean bench, take 10 g of fermented grains (the fermented grains come from a Luzhou-flavor winery in Cangzhou, Hebei) and add them to 90 mL of 0.9% saline. Oscillate at 200 r / min for 30 min to make a suspension. Take it out and put it in the clean bench. Take 1 mL and put it into a 9 mL sterile water test tube to dilute it 10 times. Take the dilution as 10 -4 -10 -6 0.15 mL of the suspension was spread on LB medium plates, and 4 parallels were made for each dilution, and they were cultured at 37°C for 24 h. The colonies with different colony morphology and good growth were selected and streaked on the corresponding plates to obtain pure colonies. Ten strains were isolated and transferred to slant surfaces and stored at 4°C for later use.

[0060] 1.2 Initial screening of Bacillus licheniformis The clear zone diameter / colony diameter was used as the index for initial screening. Lugol's iodine solution was added to the starch clear zone medium, and the strains stored on the slant were inoculated on the starch medium. The strains were cultured in a 37°C incubator for 24 h, and the L value (clear zone diameter / colony diameter) was measured. Ten strains with an L value greater than 1.5 cm were selected and named DY-1~DY-10. Figure 1This is the Lu's iodine staining of strain DY-1, and its L value is 2.4 cm.

[0061] 1.3 Bacillus licheniformis rescreening The bacteria were inoculated into 5 mL LB liquid medium and cultured at 37°C with shaking at 180 rpm for 24 h. 600 When the concentration of 1.0 is reached, the seed solution is obtained; the seed solution is inoculated with 5% of the inoculation amount into the starch fermentation medium (100 mL / 250 mL) for fermentation, the shake flask is cultured at 38°C and 180 r / min for 36 h, 50 mL of the fermentation liquid is collected in an EP tube, and centrifuged at 5000 r / min and 4°C for 15 min. The supernatant obtained is the crude enzyme liquid, and the amylase activity is measured at 65°C and pH 6. The strain with the highest amylase activity was selected and named DY-1. The amylase produced by it has an enzyme activity of 248.7±12.2 U / mL at 65°C and pH 6.

[0062] 2. Identification of Bacillus licheniformis DY-1 2.1 Colony and bacterial morphology of DY-1 strain Observation of bacterial colony morphology: The colonies are flat, light yellow, smooth, and 3 mm in diameter; see Figure 2 .

[0063] Morphological characteristics of bacteria: Gram-positive, cell morphology and arrangement are rod-shaped, solitary; see Figure 3 .

[0064] 2.2 Sequencing analysis of 16S rDNA of DY-1 strain The DY-1 strain obtained by screening was enriched and cultured, and then centrifuged to collect the bacteria for colony PCR. The product was detected by agarose gel electrophoresis and sent to Sangon Biotechnology Co., Ltd. for 16S rDNA sequencing.

[0065] PCR primers: bacterial universal primers 27F and 1492R.

[0066] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; SEQ ID NO.2; 1492R: 5'-TACGGYTACCTTGTTACGACTT-3'; SEQ ID NO. 3.

[0067] PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 15 s / 1 kb, 30 cycles; and full extension at 72°C for 5 min.

[0068] After obtaining the 16S rDNA sequencing results of the DY-1 strain, log in to the NCBI database, perform BLAST homology comparison and search on the 16S rDNA sequence of the strain with the database of the website, and construct a phylogenetic tree (such as Figure 4 ), obtained Gen Bank accession number (KC834069.1). Strain DY-1 and Bacillus licheniformis ( Bacillus licheniformis ) has the highest homology, with a similarity of 100%.

[0069] The 16S rDNA nucleotide sequence of strain DY-1 is as follows (SEQ ID NO: 1):

[0070] In summary, combined with 16S rDNA sequence analysis and colony morphology identification results, DY-1 was identified as Bacillus licheniformis ( Bacillus licheniformis ) strain and named it Bacillus licheniformis DY-1.

[0071] The Bacillus licheniformis DY-1 strain was deposited in the General Microbiology Center of China Culture Collection Administration on July 1, 2024, and its deposit number is CGMCC No.31125; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0072] Example 2 Application of Bacillus licheniformis DY-1 strain in amylase production This embodiment provides an application of Bacillus licheniformis DY-1 strain in producing amylase, and provides a method for producing amylase.

[0073] Bacillus licheniformis DY-1 was cultured until the bacterial solution OD 600 Reach about 1.0, and inoculate it into starch fermentation medium at a rate of 5%, control the pH value of the strain fermentation environment to be 5-8, and the fermentation temperature to be 34-38°C. Centrifuge the fermentation liquid and take the supernatant to obtain a crude enzyme liquid containing amylase.

[0074] Verification Example 1 Verification of the culture method for amylase production by Bacillus licheniformis DY-1 (1) Cultivation temperature of amylase production by Bacillus licheniformis DY-1 Inoculate Bacillus licheniformis DY-1 into 5 mL LB seed liquid medium and culture at 37°C with shaking at 180 r / min for 24 h. 600 When the inoculum concentration reached about 1.0, the seed solution was inoculated into the fermentation medium at a rate of 5%, and cultured at different temperatures (30, 32, 34, 36, 38, and 40°C) with shaking at 180 r / min for 36 h. The fermentation broth was collected and centrifuged at 5000 r / min and 4°C for 15 min. The supernatant was taken to obtain the corresponding crude enzyme solution. The amylase activity of the crude enzyme solution at 65°C and pH 6 was determined according to the DNS method. The results are as follows: Figure 5 shown.

[0075] As can be seen from the figure, as the culture temperature increases, the amylase activity in the obtained crude enzyme solution shows a trend of first increasing and then decreasing, that is, the amount of amylase produced by the corresponding Bacillus licheniformis DY-1 first increases and then decreases. The Bacillus licheniformis DY-1 provided by the present invention shows a good enzyme production effect at a culture temperature of 34-40°C, and the enzyme production effect is best at a culture temperature of 36-38°C.

[0076] (2) pH value of amylase produced by Bacillus licheniformis DY-1 Inoculate Bacillus licheniformis DY-1 into 5 mL LB seed liquid medium and culture at 37°C with shaking at 180 r / min for 24 h. 600 When the pH value of the fermentation medium reached about 1.0, the seed solution was inoculated with 5% of the inoculum into the fermentation medium with different pH values ​​(3, 4, 5, 6, 7, 8), and cultured at 38°C with shaking at 180 r / min for 36 h. The fermentation liquid was collected and centrifuged at 5000 r / min and 4°C for 15 min. The supernatant was taken to obtain the corresponding crude enzyme solution. The amylase activity of the crude enzyme solution at 65°C and pH 6 was determined according to the DNS method. The results are as follows: Figure 6 shown.

[0077] As can be seen from the figure, under acidic conditions, as the pH increases, the activity of amylase produced by Bacillus licheniformis DY-1 gradually increases, that is, the corresponding enzyme production of Bacillus licheniformis DY-1 gradually increases. When the pH is 7, the enzyme production reaches the maximum, and then as the pH value increases, the enzyme activity decreases, and the yield decreases. Therefore, the suitable pH for amylase produced by Bacillus licheniformis DY-1 provided by the present invention is 5.0-8.0, and the optimal pH is 7.0.

[0078] Example 3 Optimal reaction temperature of amylase produced by Bacillus licheniformis DY-1 strain A 1% soluble starch solution was prepared with a sodium hydrogen phosphate-citric acid buffer solution with a pH value of 6. A crude enzyme solution of Bacillus licheniformis DY-1 was prepared according to the method for preparing the crude enzyme solution in 1.3 of Example 1.

[0079] Take 1 mL of crude enzyme solution and add it to 1 mL of 1% soluble starch solution. According to the above-mentioned amylase activity determination method, the amylase activity at different temperatures (50℃, 55℃, 60℃, 65℃, 70℃, 75℃) was determined. The highest enzyme activity measured was 100%, and the enzyme activities measured under other temperature conditions were relative enzyme activities.

[0080] The results are as follows Figure 7 As shown, the enzyme activity is highest at 65°C, and the relative enzyme activity reaches more than 80% between 50-70°C. When the temperature is 75°C, the relative enzyme activity drops below 80%.

[0081] Example 4 Optimal reaction pH of amylase produced by Bacillus licheniformis DY-1 strain A 1% soluble starch solution with pH values ​​of 3, 4, 5, 6, 7, and 8 was prepared using disodium hydrogen phosphate-citric acid buffer. A crude enzyme solution of Bacillus licheniformis DY-1 was prepared according to the preparation method of the crude enzyme solution in 1.3 of Example 1. 1 mL of the crude enzyme solution was added to 1 mL of 1% soluble starch solution with different pH values, the reaction temperature was 65°C, and the highest enzyme activity measured was 100% according to the above-mentioned amylase activity determination method, and the enzyme activity measured under the remaining pH conditions was the relative enzyme activity.

[0082] The results are as follows Figure 8 As shown, the amylase activity produced by Bacillus licheniformis DY-1 can be maintained above 90% when the pH value is between 5 and 7, and the optimum pH value is 6.0. Below pH 6, the relative amylase gradually increases with the increase of pH, and above pH 6, the amylase activity gradually decreases.

[0083] Example 5 Application of Bacillus licheniformis DY-1 strain in producing flavor substances This embodiment provides an application of Bacillus licheniformis DY-1 strain to produce flavor substances, and provides a method for simultaneously producing acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol using sorghum hydrolyzate as a fermentation substrate.

[0084] The Bacillus licheniformis DY-1 strain was inoculated into LB liquid medium and cultured at 180 r / min for 24 h until the logarithmic culture phase. 8 The inoculum of CFU / mL was inoculated into sorghum hydrolysate and cultured at 37°C and 180 r / min for 24 h. After the fermentation, the volatile components in the sorghum hydrolysate were determined by headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 2.

[0085] Table 2 Flavor compound production of Bacillus licheniformis DY-1 in sorghum hydrolysate medium (mg·L -1 )

[0086] Note: “-” means no detection The results are shown in Table 1. Bacillus licheniformis DY-1 can simultaneously produce four flavor substances, namely acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol, in the sorghum hydrolysate medium, with the contents reaching 366.53±4.85 mg·L -1 、227.91±5.62 mg·L -1 、215.10±1.72 mg·L -1 、20.50±1.25 mg·L -1 .

[0087] Example 6 This embodiment provides an application of Bacillus licheniformis DY-1 strain to produce flavor substances, and provides a method for simultaneously producing acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol using sorghum hydrolyzate as a fermentation substrate.

[0088] The Bacillus licheniformis DY-1 strain was inoculated into LB liquid medium and cultured with shaking at 180 r / min for 24 h until the logarithmic culture phase. 7 The inoculum of 100 CFU / mL was inoculated into sorghum hydrolysate and cultured at 34°C and 180 r / min for 36 h.

[0089] Example 7 This embodiment provides an application of Bacillus licheniformis DY-1 strain to produce flavor substances, and provides a method for simultaneously producing acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol using sorghum hydrolyzate as a fermentation substrate.

[0090] The Bacillus licheniformis DY-1 strain was inoculated into LB liquid medium and cultured with shaking at 180 r / min for 24 h until the logarithmic culture phase. 8 The inoculum of 1000 CFU / mL was inoculated into sorghum hydrolysate and cultured at 40°C and 180 r / min for 48 h.

[0091] Example 8 Application of Bacillus licheniformis DY-1 strain in producing flavor substances This embodiment provides an application of Bacillus licheniformis DY-1 strain in producing flavor substances, and provides a method for simultaneously producing acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol using sorghum solid culture medium as a fermentation substrate.

[0092] The Bacillus licheniformis DY-1 strain was inoculated into sorghum hydrolysate at a 4% inoculum concentration and cultured at 180 r / min for 24 h until the logarithmic phase. 8 The inoculum of CFU / mL was inoculated into sorghum solid medium and cultured at 37℃ for 72 h. After the fermentation, the yield of flavor substances was determined by headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 3.

[0093] Table 3 Flavor compound yields of Bacillus licheniformis DY-1 in sorghum solid medium (mg kg -1 )

[0094] Note: “-” means no detection The results are shown in Table 3. Bacillus licheniformis DY-1 can simultaneously produce four flavor substances, namely acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol, in sorghum solid medium, with the contents reaching 27.03±1.26 mg·kg -1 , 17.04±0.62 mg·kg -1 , 2.25±0.11mg·kg -1 , 5.50±0.37mg·kg -1 .

[0095] Example 9 Application of Bacillus licheniformis DY-1 strain in producing flavor substances This embodiment provides an application of Bacillus licheniformis DY-1 strain to produce flavor substances, and provides a method for fermenting acetoin, 2,3-butanediol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine and phenylethanol using glucose as a single carbon source.

[0096] The Bacillus licheniformis DY-1 strain was inoculated into LB liquid medium and cultured with shaking at 180 r / min for 24 h until the logarithmic culture phase. 8 The inoculum of CFU / mL was inoculated into yeast extract peptone glucose medium and cultured at 37°C and 180 r / min for 48 h. After the fermentation, the flavor substance production was determined by headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 4.

[0097] Table 4 Flavor compound production of Bacillus licheniformis DY-1 in yeast extract peptone glucose medium (mg·L -1 )

[0098] Note: “-” means no detection The results are shown in Table 4. Bacillus licheniformis DY-1 can produce six flavor substances, including acetoin, 2,3-butanediol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine and phenylethanol, on the medium with glucose as the single carbon source, and their contents are 17.17±1.21 mg·kg -1 , 4.11±0.32 mg·kg -1 , 11.44±1.2 mg·kg -1 , 7.92±0.71 mg·kg -1 , 2.1±0.19 mg·kg -1 , 8.01±0.79 mg·kg -1 .

[0099] In summary, the Bacillus licheniformis DY-1 provided by the present invention can produce amylase and a variety of flavor substances. By controlling the fermentation substrate to selectively produce one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol, different high-value-added edible flavors can be obtained. The strain can improve the decomposition of starch in the raw materials during the liquor brewing process, and accompanies the production of acetoin, 2,3-butanediol, 4-ethylguaiacol and phenylethanol, thereby improving the flavor of the liquor, which is of great significance to improving the quality of the upper layer of fermented grains.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis DY-1, whose classification name is Bacillus licheniformis ( Bacillus licheniformis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on July 1, 2024, and its deposit number is CGMCC No.31125; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The fermentation product of Bacillus licheniformis DY-1 strain according to claim 1, characterized in that The fermentation product is obtained by fermenting the Bacillus licheniformis DY-1 strain.

3. A bacterial agent, characterized in that: The above-mentioned Bacillus licheniformis DY-1 is included.

4. Use of the Bacillus licheniformis DY-1 according to claim 1 in the preparation of one or more combinations of amylase, acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

5. Use of the Bacillus licheniformis DY-1 according to claim 1, the fermentation product according to claim 2 or the bacterial agent according to claim 3 in decomposing starch.

6. A method for producing amylase, characterized in that: include: The amylase in the fermentation liquid is obtained by fermenting the Bacillus licheniformis DY-1 according to claim 1, wherein the fermentation conditions are pH 5.0-8.0, the fermentation temperature is 34-40°C, and the culture medium used is a starch-containing culture medium.

7. The method for producing amylase according to claim 6, characterized in that The fermentation conditions are pH 7.0 and fermentation temperature 36-38° C.; and / or The culture medium is a culture medium containing starch and 1% (w / v)-5% (w / v) sodium chloride.

8. The method for producing flavor substances using the Bacillus licheniformis DY-1 according to claim 1, characterized in that: include: The Bacillus licheniformis DY-1 described in claim 1 is inoculated into a culture medium, and a fermentation product containing flavor substances is obtained after fermentation culture; the flavor substances are one or more combinations of acetoin, 2,3-butanediol, 4-ethylguaiacol, pyrazine and phenylethanol.

9. Use of the Bacillus licheniformis DY-1 according to claim 1 in liquor brewing.

10. Use of the Bacillus licheniformis DY-1 according to claim 1 in preparing edible flavors.

Images