Bacillus licheniformis with high yield of 4-vinylguaiacol and application thereof

Abstract

The application discloses a bacillus licheniformis with high yield of 4-vinylguaiacol and application thereof, and belongs to the technical field of microbial fermentation. The bacillus licheniformis D1 with high yield of 4-vinylguaiacol is obtained by screening and separation from a mixture of Daqu and fermented grains, and the preservation number is CGMCC No. 39294. The bacillus licheniformis has good tolerance to pH and alcohol, and can adapt to the white wine fermentation process. The bacillus licheniformis can produce 4-vinylguaiacol with a yield of 1.255 g / L, and has important potential in the aspects of white wine fermentation effect improvement and wine body flavor shaping.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically to a Bacillus licheniformis that produces high levels of 4-vinylguaiacol and its applications. Background Technology

[0002] Chinese Baijiu is a traditional brewing product with a long history and rich cultural tradition. It is made primarily from sorghum, wheat, and rice through processes such as high-temperature cooking, saccharification, fermentation, and distillation. Chinese Baijiu is characterized by its strong aroma, mellow taste, and long finish, making it widely popular among consumers both domestically and internationally. As a uniquely Chinese alcoholic beverage, Chinese Baijiu is not only a focus of international academic and industrial attention but also holds significant importance for exploring its brewing techniques, chemical composition, and nutritional components. In an environment of rapid economic development and people's pursuit of quality and health, the Baijiu industry has shifted from initially focusing on "quantity" to "quality." The "high quality" of Baijiu is mainly reflected in two aspects: flavor quality and functional quality.

[0003] 4-Vinylguaiacol (4-VG), chemically named 2-methoxy-4-vinylphenol, is a pyranocyanin. Natural 4-VG is mainly found in the volatiles of corn alcoholic fermentation, appearing as a colorless or pale straw-yellow oily liquid with clove, fermented, and roasted peanut aromas. It is insoluble in water but soluble in oils. 4-VG is not only an important flavor compound in soy sauce, baijiu (Chinese liquor), wine, and beer, but also an indispensable characteristic component for the comprehensive flavor evaluation of these fermented products. It also possesses health benefits; studies have shown that 4-VG can arrest the cell cycle and induce apoptosis, inhibiting the growth of cancer cells. 4-VG is also a valuable high-grade flavoring agent and food additive in the food, daily chemical, pharmaceutical, and fragrance industries, with a market value approximately 40 times that of ferulic acid. It can be further converted into high-value compounds such as ethylguaiacol, vanillin, and vanillyl alcohol. 4-VG is playing an increasingly important role in our lives; however, the amount of 4-VG available in natural plants is limited and cannot meet the increasing consumer demand.

[0004] Currently, most 4-VG is produced through chemical synthesis. However, with increasing preference for more environmentally friendly and natural products, the preparation of 4-VG using microbial fermentation has become a research hotspot in recent years. Existing microbial fermentation methods for producing 4-vinylguaiacol suffer from problems such as low yield, low fermentation efficiency, poor environmental tolerance, and difficulty in adapting to the baijiu (Chinese liquor) brewing system, which limit its widespread application in the brewing industry.

[0005] Therefore, how to screen and obtain a functional strain with high 4-vinylguaiacol production capacity, good environmental tolerance, and applicability to brewing systems such as baijiu 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 licheniformis that produces high levels of 4-vinylguaiacol and its applications.

[0007] Bacillus licheniformis D1 has good resistance to acid, salt, and ethanol. In liquid fermentation, it produces 1.255 g / L of 4-vinylguaiacol. It can be used in the production of soy sauce-flavored liquor, rice wine, etc., to improve the flavor and quality of the liquor and benefit human health.

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

[0009] A Bacillus licheniformis species that produces a high amount of 4-vinylguaiacol, named D1, is classified as Bacillus licheniformis. Bacillus licheniformis It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39294. 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 Bacillus licheniformis is used in the fermentation production of 4-vinylguaiacol.

[0011] The above-mentioned application of Bacillus licheniformis in the preparation of brewed products.

[0012] Furthermore, the brewed products are baijiu (Chinese white liquor) and rice wine.

[0013] Furthermore, it is used to increase the content of flavor substances in the brewed product.

[0014] A microbial inoculant, comprising the aforementioned Bacillus licheniformis.

[0015] 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-yielding strain of Bacillus licheniformis D1 from a mixture of Daqu (fermentation starter) and mash, with the preservation number CGMCC No. 39294. This bacterium exhibits excellent tolerance to both pH and alcohol content, enabling it to adapt to the baijiu (Chinese liquor) fermentation process. This strain produces a high yield of 1.255 g / L of 4-vinylguaiacol, demonstrating significant potential for improving baijiu fermentation efficiency and shaping its flavor profile. Attached Figure Description

[0016] 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.

[0017] Figure 1 The results of morphological identification of strain D1 in Example 2 of the present invention are shown, where a is the colony morphology, b is the Gram staining result, and c is the scanning electron microscope image.

[0018] Figure 2 This is a phylogenetic tree diagram of strain D1 in Example 2 of the present invention.

[0019] Figure 3 This is the growth curve of Bacillus licheniformis D1 in Example 3 of the present invention.

[0020] Figure 4 This refers to the ethanol tolerance of Bacillus licheniformis D1 in Example 3 of the present invention.

[0021] Figure 5 This refers to the temperature tolerance of Bacillus licheniformis D1 in Example 3 of the present invention.

[0022] Figure 6 This refers to the NaCl tolerance of Bacillus licheniformis D1 in Example 3 of the present invention.

[0023] Figure 7 This refers to the pH tolerance of Bacillus licheniformis D1 in Example 3 of the present invention. Detailed Implementation

[0024] 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.

[0025] The culture medium formulations used in the following examples are as follows: Liquid growth medium: glucose 5.0 g / L, yeast extract 2.0 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 4.0 g / L, CaCl2 0.05 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L.

[0026] Solid growth medium: Agar is added to the liquid growth medium at a concentration of 20 g / L.

[0027] Screening medium: Ferulic acid was added to the liquid growth medium to achieve a concentration of 1.0 g / L.

[0028] Liquid fermentation medium: Ferulic acid was added to the liquid growth medium to achieve a concentration of 1.0 g / 100 mL.

[0029] Example 1 Screening of high-yielding 4-vinylguaiacol strains I. Initial Screening A total of 10 g of Daqu (fermented starter culture) and mash samples from a winery in Yibin were weighed and ground in a mortar. The processed sample powder was placed in a 250 mL Erlenmeyer flask and 100 mL of physiological saline was added. The mixture was shaken well. Enrichment culture was carried out at 37 ℃ and 180 r / min.

[0030] Take 1 mL of enriched culture medium at 12 h, 24 h, 36 h, and 48 h, and dilute it with sterile water to a concentration of 10:1. -3 ~10 -7 The bacterial suspensions were prepared by pipetting 100 μL of different gradients onto selection medium plates and incubating them upside down at 37°C for 48 h. Colonies of various sizes and morphologies were selected and streaked onto solid growth medium for isolation. This process was repeated until single colonies appeared.

[0031] After obtaining the pure strain, transfer the single colony to a solid growth medium (slant), number it, and store it for later use.

[0032] II. Secondary Screening The above-screened strains were selected, activated, and single colonies were inoculated into liquid growth medium to prepare seed culture. This seed culture was then inoculated into liquid fermentation medium at a rate of 3% (100 mL / 250 mL). Parallel experiments were performed simultaneously, and the cultures were incubated at 37℃ and 180 r / min for 72 h. After fermentation, the 4-vinylguaiacol content in the fermentation broth was measured. A high-yield 4-vinylguaiacol-producing strain was selected and named D1, with a 4-vinylguaiacol content of 1.125 ± 0.014 g / L in its fermentation broth. The strain was preserved in glycerol tubes.

[0033] The content of 4-vinylguaiacol in the fermentation broth was determined by high performance liquid chromatography, as follows: An Agilent C18 column was used; mobile phase A was 0.1% phosphoric acid, mobile phase B was methanol, and the ratio of mobile phase A to mobile phase B was 65:35; flow rate: 1 mL / min; column temperature: 30℃; detection wavelength: 280 nm; sample loading volume: 10 µL; elution time: approximately 8.9 min; standard was dissolved in methanol; elution time: 29 min.

[0034] The obtained standard curve is Y = 180266X - 3957.9, R0 2 =0.9997.

[0035] Example 2 Identification of strain D1 I. Morphological Identification Based on colony morphology observation, strain D1 colonies were white, round, with a wrinkled surface, a round bulge in the center, relatively dry, somewhat adhesive, and opaque. Figure 1 a. Gram-stained bacteria appear blue-purple under an optical microscope, indicating they are Gram-positive. (See...) Figure 1 b. Electron micrographs show that this bacterium is rod-shaped. Figure 1 c.

[0036] II. Molecular biological identification DNA was extracted from strain D1 using a bacterial genome extraction kit, and PCR amplification was performed using 16S universal primers. 15µL of the PCR amplification product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are as follows.

[0037]

[0038] The 16S rDNA molecular biological identification and sequencing results were compared with the NCBI database using BLAST, and a phylogenetic tree was finally constructed using MEGA-X software based on the Neighbor-Joining algorithm, confirming that the strain is Bacillus licheniformis. Bacillus licheniformis ,See Figure 2 .

[0039] Strain D1 was identified as Bacillus licheniformis through morphological and molecular biological analysis. Bacillus licheniformis .

[0040] III. Preservation Strain D1, classified as Bacillus licheniformis. Bacillus licheniformis It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39294. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0041] Example 3 Growth characteristics of Bacillus licheniformis D1 I. Growth Curve A single colony of activated Bacillus licheniformis D1 was picked and inoculated into a 10 mL centrifuge tube. The culture was prepared by incubating the culture at 37℃ and 180 r / min in a shaker for 24 h. 3 mL of the seed culture was then inoculated into liquid growth medium and incubated at 37℃ and 180 r / min in a constant temperature incubator. Three replicates were set up, with an equal volume of uninoculated liquid growth medium as a blank control. OD values ​​were measured every 2 h. 600 nm, and plot the growth curve.

[0042] The results showed that *Bacillus licheniformis* D1 strain adapted to the culture medium conditions from 0 to 4 hours, exhibiting slow growth and reproduction. After 4 hours of culture, it entered the logarithmic growth phase, resulting in rapid bacterial proliferation. After 18 hours of culture, the strain entered the stationary phase, where the mortality rate was equal to the growth rate. (See...) Figure 3 .

[0043] II. Ethanol Tolerance A single colony of activated Bacillus licheniformis D1 was picked and inoculated into a 10 mL centrifuge tube. The culture was prepared by incubating the culture at 37°C and 180 r / min for 24 h in a shaker. Three replicates were prepared at 3% inoculation in liquid growth media with ethanol concentrations of 1%, 3%, 5%, 7%, 9%, and 11%. Each ethanol concentration was incubated in triplicate. The cultures were then incubated at 37°C and 180 r / min for 18 h. An equal volume of uninoculated liquid growth media served as a blank control. The absorbance was measured at 600 nm.

[0044] like Figure 4 As shown, the growth of Bacillus licheniformis D1 was slightly inhibited as the amount of ethanol added to the fermentation medium increased. When the amount of ethanol added was 1-5%, the OD value was greater than 1, indicating that the strain was growing and reproducing well.

[0045] III. Temperature Tolerance A single colony of activated Bacillus licheniformis D1 was picked and inoculated into a 10 mL centrifuge tube. The culture was prepared by incubating the culture in a shaker at 37°C and 180 r / min for 24 h. A 3% inoculum was then added to liquid growth medium and incubated for 18 h in shakers at 25°C, 29°C, 33°C, 37°C, and 41°C at 180 r / min. Three replicates were set up for each temperature. An equal volume of uninoculated liquid growth medium served as a blank control. The absorbance was measured at 600 nm.

[0046] like Figure 5 As shown, with increasing culture temperature, the life activities of Bacillus licheniformis D1 accelerate, leading to a gradual increase in strain concentration, reaching its peak at 37℃. When the temperature reaches 41℃, the OD value is less than 1, the number of surviving bacteria decreases, and growth and reproduction decline.

[0047] The yield of 4-vinylguaiacol was highest at a fermentation temperature of 37°C, at 1.158 ± 0.05 g / L.

[0048] IV. NaCl tolerance A single colony of activated Bacillus licheniformis D1 was picked and inoculated into a 10 mL centrifuge tube. The culture was prepared by incubating the culture in a shaker at 37°C and 180 r / min for 24 h. Three replicates were prepared for each NaCl concentration, using a 3% inoculum in liquid growth media containing 1%, 3%, 5%, 7%, 9%, 11%, and 13% NaCl. The culture was incubated in a shaker at 37°C and 180 r / min for 18 h. An equal volume of uninoculated liquid growth media served as a blank control. The absorbance was measured at 600 nm.

[0049] like Figure 6As shown, although the growth trend of Bacillus licheniformis D1 generally decreased with the increase of NaCl concentration, it generally had good salt tolerance. Bacillus licheniformis D1 had good growth at NaCl concentrations of 1% to 9%, and was almost completely inactivated at 13%.

[0050] V. pH tolerance A single colony of activated Bacillus licheniformis D1 was picked and inoculated into a 10 mL centrifuge tube. The culture was prepared by incubating the culture in a shaker at 37°C and 180 r / min for 24 h. A 3% inoculum was then inoculated into liquid growth media at pH 3, 4, 5, 6, 7, 8, 9, 10, and 11, with three replicates for each pH value. The cultures were incubated in a shaker at 37°C and 180 r / min for 18 h. An equal volume of uninoculated liquid growth media served as a blank control. The absorbance was measured at 600 nm.

[0051] like Figure 7 As shown, the growth of Bacillus licheniformis D1 strain in fermentation medium was severely inhibited at pH less than 4.0 and greater than 9.0, with OD values ​​less than 0.1. When the pH range was 5.0 to 7.0, the OD value increased with increasing pH, reaching the highest OD value at pH 7.0, and then gradually decreased with further increases in pH.

[0052] The yield of 4-vinylguaiacol reached its maximum at pH 7.0, at 1.144 ± 0.03 g / L.

[0053] Example 4 Optimization of conditions for 4-VG production by Bacillus licheniformis D1 in liquid fermentation Before optimization: initial pH 7.0, inoculum size 3%, fermentation temperature 37℃, rotation speed 180 r / min, fermentation time 72 h, 100 mL / 250 mL.

[0054] After optimization: fermentation temperature 37℃, initial pH 7.0, inoculum size 5%, rotation speed 240 r / min, fermentation time 72 h.

[0055] The yield of 4-VG produced by Bacillus licheniformis D1 fermentation under the conditions before and after optimization was determined. The results showed that the yield of 4-vinylguaiacol by Bacillus licheniformis D1 after optimization reached 1.255 g / L, while the yield before optimization was 1.125±0.014 g / L, which was 11.56% higher than that before optimization.

[0056] Example 5 Application of Bacillus licheniformis D1 in the production of 4-vinylguaiacol in simulated solid-state fermentation Soak sorghum in water for 24 hours, then wash repeatedly until the water becomes clear and transparent. Mix sorghum and rice husks at a volume ratio of 2:1, then steam for 2 hours to crack the sorghum skin and expose the internal starch. After steaming, add water to bring the moisture content to 50%, then spread out to cool at 35℃. Divide into small portions of 500 g each, add 100 g of high-temperature Daqu (a type of starter culture) powder, and then add 5 mL of Bacillus licheniformis D1 bacterial suspension (prepared by 3% inoculum at 37℃, 180 r / min, and LB liquid medium for 24 hours). Ferment in a sealed fermentation tank for 30 days. After fermentation, collect the mash and use a distillation column to extract the liquor sample.

[0057] The content of 4-vinylguaiacol in the wine sample was determined by high performance liquid chromatography, and the result was 18.238 mg / L.

[0058] The method for taking wine samples and analyzing flavor compounds is as follows: Sample pretreatment: Take 5 mL of wine sample and add 1.5 g of sodium chloride into a 20 mL headspace glass bottle.

[0059] 20 µL of internal standard mixture was added to a headspace vial. The vial was incubated at 50 °C for 5 min, then a black needle guide was inserted into the vial, and an optical fiber was inserted for extraction at 50 °C for 45 min. Subsequently, analysis was performed in a split-mode, with the optical fiber inserted into the GC injection port at 230 °C for 5 min. Helium (99.9995% purity) was used as the carrier gas at a constant flow rate of 1.0 mL / min. The initial temperature was maintained at 45 °C for 3 min, then increased to 150 °C at 4 °C / min and held for 2 min, then increased to 200 °C at 6 °C / min, and finally increased to 230 °C at 10 °C / min and held for 10 min. The ion source temperature was 230 °C. The ionization energy of electron impact mass spectrometry was 70 eV, and 35–400 amu were obtained in the m / z scan range.

[0060] GC-MS detected 106 flavor compounds in the liquor sample, including 24 acids, 18 alcohols, 10 aldehydes, 10 amines, 9 ethers, 16 alkanes / olefins, 1 ketone, and 18 others. The specific categories of key baijiu flavor compounds, including acids, alcohols, and aldehydes, are shown in Tables 1-3.

[0061] Table 1 Acids

[0062] Table 2 Alcohols

[0063] Table 3 Aldehydes

[0064] Example 6 Application of Bacillus licheniformis D1 in rice wine fermentation process Soak glutinous rice in water for 12 hours, then rinse repeatedly until the water becomes clear and transparent. Drain the rinsed glutinous rice and steam it in a steamer for 30 minutes until it is fully cooked, without any hard core, loose, and not sticky. After steaming, let it cool naturally to 30℃, turning it occasionally to prevent clumping. Divide the rice into small portions of 500 g each, add 80 g of rice wine yeast powder, and then add 5 mL of Bacillus licheniformis D1 bacterial suspension (prepared by 3% inoculation at 37℃, 180 r / min, and LB liquid medium for 24 hours), and mix thoroughly. Ferment in a sealed fermentation tank at a constant temperature of 28℃ for 7 days. The fermented wine sample was analyzed using high-performance liquid chromatography (HPLC) to determine the 4-vinylguaiacol content, which was found to be 13.199 mg / L.

[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 Bacillus licheniformis species that produces high levels of 4-vinylguaiacol, characterized in that, Named D1, its classification is Bacillus licheniformis. Bacillus licheniformis It was deposited on June 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39294. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The use of Bacillus licheniformis as described in claim 1 in the fermentation production of 4-vinylguaiacol.

3. The use of Bacillus licheniformis as described in claim 1 in the preparation of brewed products.

4. The application as described in claim 3, characterized in that, The brewed products are baijiu (Chinese liquor) and rice wine.

5. The application as described in claim 3, characterized in that, Used to increase the content of flavor substances in the brewed products.

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

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