Two strains producing ferulic acid esterase

Abstract

This invention discloses two ferulic acid esterase-producing strains isolated from baijiu daqu (fermentation starter) makers: *Enterococcus faecalis* L-7 and *Burkholderia burgdorferi* BK100. These strains are deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC NO.M 20221555 and CCTCC NO.M 20221553, respectively. Optimization of the fermentation conditions for *Enterococcus faecalis* L-7 revealed that the optimal fermentation conditions were: 32℃, 160 r / min, and an inoculum size of 6%, for 96 h. During this period, the strain exhibited the highest ferulic acid esterase activity, reaching 44.68 U / L, a 68.92% increase compared to the unoptimized 26.45 U / L. *Burkholderia burgdorferi* BK100, characterized by high ferulic acid esterase activity and high-temperature resistance, is suitable for daqu preparation and baijiu brewing, and can increase ferulic acid content and improve baijiu quality.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to two strains that produce ferulic acid esterase: Enterococcus faecalis L-7 and Burkholderia BK100. Background Technology

[0002] Daqu, or saccharification and fermentation agent, is used in the brewing of Chinese baijiu (white liquor). It is rich in microorganisms, hydrolytic enzymes, and flavor compounds. Daqu provides a stable microbial community for baijiu brewing and also provides nutrients for microbial growth. Simultaneously, the various enzymes produced by the microorganisms in Daqu aid in the decomposition of raw materials during baijiu fermentation. Daqu plays a crucial role in baijiu fermentation; "qu is the bone of liquor," and high-quality qu is an important prerequisite for producing high-quality baijiu. The microorganisms in Daqu and the enzymes they produce provide important guarantees for subsequent production of baijiu and also have a significant impact on the unique style of various aroma types of baijiu.

[0003] Ferulic acid is a phenolic acid with strong antioxidant, anti-radiation, coronary heart disease prevention, and liver damage inhibition effects. It is widely used in the pharmaceutical, cosmetic, and food industries. As an organic phenolic acid, ferulic acid has a strong aroma-enhancing effect and is one of the aroma compounds in alcoholic beverages. Ferulic acid can also serve as a precursor to aroma compounds in alcoholic beverages. Under the action of microorganisms, it can be transformed into aroma components such as vanillin, 4-EG, and 4-vinylguaiacol, making a significant contribution to the unique style of baijiu (Chinese liquor). Ferulic acid is widely present in plant cell walls, forming a network structure with polysaccharides and lignin through ether or ester bonds, limiting the utilization of cellulose and the degradation of plant fibrous materials by microorganisms.

[0004] Ferulic esterase belongs to the carboxylesterase subclass and catalyzes the hydrolysis of ester or ether bonds between ferulic acid and polysaccharides, cellulose, or lignin to produce ferulic acid. Ferulic esterase can also be used in the synthesis of esters, such as hydroxycinnamate. Fungi, bacteria, and yeasts can all secrete ferulic esterase. Ferulic esterases from different sources exhibit significant differences in enzymatic properties, sequence homology, and spatial structure. Currently reported ferulic esterases are mainly derived from microorganisms, but the enzyme activity of ferulic esterases isolated from them is generally low. Microbial strains are mainly screened from soil, seawater, and the intestines of humans and animals; relatively few strains producing ferulic esterase have been screened from raw materials used in baijiu (Chinese liquor) brewing. Ferulic esterase has wide applications in many fields such as food, feed, medicine, papermaking, and textiles. In the baijiu brewing process, it can accelerate the degradation of raw material cell walls, making the microbial transformation reaction in the starter culture more rapid and thorough. In addition, during the brewing process of baijiu, as fermentation proceeds, the acidity and ethanol concentration in the fermentation system gradually increase, and most commercial strains cannot tolerate adverse conditions such as high acidity and high ethanol concentration.

[0005] Currently, prior art document (CN109468232A) discloses a *Sherlocks* species that produces ferulic acid esterase and its application in the production of baijiu daqu (Chinese liquor starter). Previous studies have shown that *Enterococcus faecalis* is widely present in fermented foods, affecting not only their aroma, flavor, and texture but also possessing probiotic effects such as regulating intestinal flora and enhancing immunity. However, there are no reports of its production of ferulic acid esterase or ferulic acid.

[0006] In addition, while Burkholderia has been found to produce lipase, ACC deaminase, and cellulase, there are no reports of its production of ferulic acid esterase. Currently, patent CN109468232A has been found, which discloses a strain isolated from Daqu (a type of starter culture) that can produce ferulic acid esterase, and the addition of this strain's powder to the production of Baijiu Daqu in a specific ratio. Patent CN113308424A has also been found, which discloses a Bacillus pumilus that produces ferulic acid esterase, which maintains over 80% enzyme activity during fermentation at 45℃-60℃. However, the aforementioned prior art does not address whether the screened strain remains suitable for growth during the brewing process, nor does it mention the optimization and application of the crude enzyme solution measurement conditions for the ferulic acid esterase-producing strain. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide two strains that produce ferulic acid esterase. Enterococcus faecalis L-7 possesses acid, alkali, salt, and ethanol resistance, making it suitable for use in the brewing process of baijiu (Chinese liquor) to increase the ferulic acid content and enhance its health benefits. Burkholderia BK100, on the other hand, exhibits high temperature and alkali resistance, making it suitable for later-stage high-yield ferulic acid metabolism engineering modifications.

[0008] The technical objective of this invention is achieved through the following technical solution:

[0009] Firstly, two ferulic acid esterase-producing strains are provided: Enterococcus faecalis L-7 and Burkholderia burgdorferi BK100. Enterococcus faecalis L-7 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M20221555 on October 10, 2022. Burkholderia burgdorferi BK100 is deposited at the same collection with accession number CCTCC NO. M 20221553 on October 10, 2022.

[0010] Furthermore, the Burkholderia BK100 has a temperature tolerance range of 20-57℃, and grows well at 25-40℃, with an optimal growth temperature of 30℃.

[0011] Furthermore, the pH tolerance of the Enterococcus faecalis L-7 is pH 4–11, with good growth at pH 7–10, and the strain grows and reproduces fastest at pH 8.

[0012] Furthermore, the Enterococcus faecalis L-7 can grow normally in the range of NaCl concentration of 1% to 3% and ethanol concentration of 1% to 7%.

[0013] Furthermore, the Enterococcus faecalis L-7 grows well at a NaCl concentration of 2% and an ethanol concentration of 5%.

[0014] Secondly, the screening process for Enterococcus faecalis L-7 includes the following:

[0015] 1) Sampling: Take 10g of sample from each corner and center of the finished Daqu, grind and mix evenly to obtain Daqu sample;

[0016] (ii) Enrichment: Weigh 2g of the Daqu sample obtained in step (i), put it into a 250mL Erlenmeyer flask, add 100mL of sterile physiological saline, shake well, and culture in a shaker at 37℃ and 180r / min for 30min to prepare a bacterial suspension.

[0017] (iii) Screening: The bacterial suspension prepared in step 2) was serially diluted with sterile physiological saline, with 100 μL of each diluted 10 times. -3 10 -4 10 -5 10 -6 The bacterial suspension was spread on the selection medium, with three replicates for each gradient. The spread petri dishes were incubated upside down in a 37°C incubator for 72 hours.

[0018] (iv) Purification: Observe the size of colonies and clear zones on the screening medium. Select colonies with larger clear zones and streak them on LB solid medium. Incubate at 37℃ for 24 hours, repeating three times to obtain single colonies of the target strain. Inoculate the obtained single colonies into LB liquid medium and incubate overnight. Take 1 ml of bacterial culture and inoculate it into fermentation medium. Incubate for 72 hours, end fermentation, centrifuge, collect the supernatant, filter it into a liquid chromatography vial, and determine the ferulic acid content in the fermentation broth by high performance liquid chromatography. Preserve the ferulic acid esterase-producing strain in glycerol tubes.

[0019] The screening culture medium in steps three and four is as follows: sodium nitrate 2 g / L, dipotassium hydrogen phosphate 1 g / L, potassium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 20 g / L, ultrapure water 1 L, sterilized at 121℃ for 20 min; cooled to about 70℃, 1.5% (v / v) ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution, sterilized by filtration through a 0.22 μm sterile filter membrane) is added.

[0020] In step four), the LB solid medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, 1 L ultrapure water, natural pH, sterilized at 121°C for 20 min.

[0021] In step four), the LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 1 L ultrapure water, natural pH, sterilized at 121℃ for 20 min.

[0022] The fermentation medium in step four is: wheat bran 10g / L, yeast extract 1g / L, potassium chloride 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, dipotassium hydrogen phosphate 0.1g / L, ultrapure water 1L, pH natural, sterilized at 121℃ for 20min. Magnesium sulfate heptahydrate is sterilized separately and then added to the medium.

[0023] Thirdly, the screening process for Burkholderia BK100 includes the following:

[0024] 1) Enrichment: Samples of Daqu (fermented starter culture) from a certain winery, fermented for 1-30 days and stored for one month and two months, were pulverized and mixed to obtain starter powder. This powder was then stored at -80°C for later use. 10g of starter powder was added to 90mL of enrichment medium to prepare 10... -1 The Daqu-enriched solution was then diluted to prepare 10... -2 -10 -7 Daqu dilution solution;

[0025] (ii) Screening of high-yield ferulic acid esterase strains

[0026] 1) Primary screening: Take four different gradients of Daqu dilution and inoculate them on primary screening medium plates. Incubate them upside down at 30℃ for 3 days. Then purify and culture the single colonies on the medium. Then inoculate them on the primary screening medium and incubate them at 30℃ for 5 days. Observe whether a clear zone is formed. Then inoculate the colonies with clear zones on the primary screening medium again to purify and separate them to obtain pure colonies.

[0027] 2) Secondary screening: a) The strains that produced a clear zone obtained in step 1) were isolated and purified multiple times, with Burkholderia showing the largest clear zone. Burkholderia BK100 was inoculated into 100 mL of LB medium and incubated at 30 °C for 48 h; b) Burkholderia BK100 was inoculated into 100 mL of secondary screening medium at a volume ratio of 2% and incubated at 30 °C and 180 r / min for 72 h. The ferulic acid content in the fermentation broth was then determined by high performance liquid chromatography, and a strain with a ferulic acid esterase activity of 41.6 U / L was obtained. This strain was preserved in glycerol tubes.

[0028] The enrichment medium in step one) is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L glucose, 1 L distilled water, natural pH, sterilized at 121℃ for 20 min.

[0029] The initial screening culture medium in step 1) was: potassium chloride 0.5 g / L, dipotassium hydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium nitrate 2.0 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 20 g / L, sucrose 30 g / L, distilled water 1 L, pH natural, sterilized at 121℃ for 20 min. After cooling to 60℃, 10 mL of ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution) was added.

[0030] In step 2), the LB medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1 L distilled water, natural pH, sterilized at 121℃ for 20 min.

[0031] The culture medium for the second screening in step 2) is: 10g yeast powder, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 1.0g dipotassium hydrogen phosphate, 20g agar, 1L distilled water, natural pH, sterilized at 121℃ for 20min, cooled to 60℃ and 10mL of ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution) was added.

[0032] Fourthly, the application of Enterococcus faecalis L-7, as described in the first aspect, in the fermentation production of ferulic acid esterase is provided.

[0033] Furthermore, the fermentation conditions for producing ferulic acid esterase by fermentation of Enterococcus faecalis L-7 are as follows: inoculum size 3-7%, rotation speed 140-220 r / min, culture temperature 22-42℃, and culture time 48-144 h.

[0034] Furthermore, the fermentation conditions for producing ferulic acid esterase by fermentation of Enterococcus faecalis L-7 are as follows: inoculum size 6%, rotation speed 160 r / min, culture temperature 32℃, and culture time 96 h.

[0035] Fifthly, the application of Burkholderia BK100 as described in the first aspect in the fermentation production of ferulic acid esterase is provided.

[0036] Furthermore, the enzyme activity determination conditions for the production of ferulic acid esterase by Burkholderia BK100 fermentation are as follows: the optimal reaction temperature for enzyme activity in the crude enzyme solution is 20-60℃, the optimal reaction pH for enzyme activity in the crude enzyme solution is 5.0-9.0, the optimal addition amount of crude enzyme solution is 250-350μL, and the optimal reaction time for enzyme activity in the crude enzyme solution is 15-30min.

[0037] Furthermore, the enzyme activity determination conditions for the production of ferulic acid esterase by Burkholderia BK100 fermentation are as follows: optimal reaction temperature of enzyme activity in crude enzyme solution is 42℃, optimal reaction pH of enzyme activity in crude enzyme solution is 7.1, crude enzyme solution addition amount is 320μL, and optimal reaction time of enzyme activity in crude enzyme solution is 22min.

[0038] The Enterococcus faecalis L-7 involved in this invention is classified as Enterococcus faecalis and is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCCNO.M 20221555 and deposit date of October 10, 2022.

[0039] The Burkholderia BK100 of this invention, classified as Burkholderia, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCCNO.M 20221553 and deposit date of October 10, 2022.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1) This invention screened a strain of Enterococcus faecalis L-7 from baijiu daqu. This strain has evolved to adapt to its growth environment and has the characteristics of acid and alkali resistance, salt resistance, and alcohol resistance. This is beneficial for later use of molecular biology methods such as genetic engineering, metabolic engineering, and synthetic biology to modify it to improve its ability to produce ferulic acid esterase, thereby further increasing the ferulic acid content in baijiu.

[0042] 2) The Enterococcus faecalis L-7 of this invention can grow normally in the range of 1% to 7% ethanol concentration. This bacterium can be used in the brewing of baijiu to increase the content of ferulic acid, a health factor in baijiu.

[0043] 3) The Enterococcus faecalis L-7 of this invention is derived from Baijiu Daqu (a type of starter culture used in traditional Chinese liquor production), and the results of virulence gene detection and harmful metabolite experiments were both negative;

[0044] 4) The Enterococcus faecalis L-7 of this invention can ferment to produce ferulic acid esterase to degrade the ferulic acid ester bonds in plant cell walls. Applying it to the brewing process of Baijiu can make the degradation of raw material cell walls more complete and the microbial transformation reaction in the system more thorough.

[0045] 5) By optimizing the fermentation conditions of Enterococcus faecalis L-7, it was shown that the optimal fermentation conditions were: when cultured at 32℃, 160r / min, and 6% inoculum for 96h, the enzyme activity of ferulic acid esterase produced by this strain was the highest, reaching 44.68U / L, which was 68.92% higher than the 26.45U / L before optimization.

[0046] 6) This invention screened a strain of Burkholderia BK100 from baijiu daqu. Due to the special fermentation environment of daqu, Burkholderia BK100 has the characteristics of high temperature resistance and alkali resistance, and is suitable for high-yield ferulic acid metabolism engineering modification in the later stage.

[0047] 7) Burkholderia BK100 has the characteristics of high ferulic acid esterase activity and high temperature resistance, making it suitable for Daqu preparation and Baijiu brewing. It can increase the ferulic acid content and improve the quality of Baijiu.

[0048] 8) Burkholderia BK100 has a wide range of acid and alkali tolerance, and can grow in the pH range of 5.0-9.0, showing alkali resistance.

[0049] 9) The enzyme activity determination conditions for the production of ferulic acid esterase by Burkholderia BK100 fermentation were optimized. The results showed that the optimal determination conditions were: the optimal reaction temperature of the crude enzyme solution was 42℃, the optimal reaction pH of the crude enzyme solution was 7.1, the amount of crude enzyme solution added was 320μL, and the optimal reaction time of the crude enzyme solution was 22min. Under these conditions, the enzyme activity of ferulic acid esterase was 85.08U / L, which was 104.52% higher than that of the strain before optimization.

[0050] 10) Chemical synthesis methods have strict process requirements and cause serious pollution, while plant enrichment methods have complex extraction processes and low yields. This invention uses microbial fermentation to obtain high-yield ferulic acid esterase, which has the advantages of simple operation, high yield and green environmental protection. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention, 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 This is the phylogenetic tree diagram of L-7 in this embodiment of the invention;

[0053] Figure 2 This is a standard curve diagram of ferulic acid in an embodiment of the present invention;

[0054] Figure 3 These are the chromatograms of ferulic acid standard (top) and enzyme reaction liquid chromatograms (bottom) in embodiments of the present invention;

[0055] Figure 4 This is the L-7 growth curve in the embodiment of the present invention;

[0056] Figure 5This is the electrophoretic pattern of L-7 virulence gene amplification in an embodiment of the present invention;

[0057] Figure 6 This is a graph showing the L-7 hemolysis test results in an embodiment of the present invention;

[0058] Figure 7 This is a physical image of the morphological features of L-7 in the embodiment of the present invention;

[0059] Figure 8 This is a Gram staining image of L-7 in an embodiment of the present invention;

[0060] Figure 9 This is a pH tolerance effect diagram of L-7 in the embodiments of the present invention;

[0061] Figure 10 This is a diagram illustrating the salt tolerance effect of L-7 in this embodiment of the invention;

[0062] Figure 11 This is a graph showing the ethanol tolerance effect of L-7 in the embodiments of the present invention;

[0063] Figure 12 This is the initial screening result of Burkholderia in the embodiments of the present invention;

[0064] Figure 13 This is a chromatogram of the crude enzyme solution of BK100 in an embodiment of the present invention;

[0065] Figure 14 This is the BK100 phylogenetic tree in this embodiment of the invention;

[0066] Figure 15 This is the growth curve of BK100 in the embodiments of the present invention;

[0067] Figure 16 This refers to the growth temperature tolerance of BK100 in the embodiments of the present invention;

[0068] Figure 17 This refers to the growth pH tolerance of BK100 in the embodiments of the present invention;

[0069] Figure 18 The transparent zone of strain BK100 in this embodiment of the invention;

[0070] Figure 19 This refers to the Gram staining results of BK100 in the embodiments of the present invention;

[0071] Figure 20 This is a scanning electron microscope image of BK100 in an embodiment of the present invention;

[0072] Figure 21 This is the ferulic acid standard curve of BK100 in the embodiments of the present invention;

[0073] Figure 22 is a chromatogram of the crude enzyme solution of BK100 and a chromatogram of ferulic acid standard in an embodiment of the present invention.

[0074] Figure 23 This illustrates the effect of the reaction temperature of BK100 on enzyme activity assay in this embodiment of the invention.

[0075] Figure 24 This illustrates the effect of BK100 reaction time on enzyme activity assay in this embodiment of the invention.

[0076] Figure 25 This illustrates the effect of the reaction pH of BK100 on enzyme activity assay in this embodiment of the invention.

[0077] Figure 26 This describes the effect of the amount of crude BK100 enzyme solution added on the enzyme activity assay in this embodiment of the invention. Detailed Implementation

[0078] To make the technical problem to be solved, the technical solution and the beneficial effects of the present invention clearer, the following description is provided in conjunction with the appendix. Figure 1-26 The present invention will be further described in detail with reference to Examples 1-7.

[0079] Example 1: Isolation and screening of strain L-7

[0080] 1) Sampling, enrichment and screening

[0081] Take 10g samples from each corner and center of the finished Daqu (fermented starter culture), grind and mix them evenly to obtain Daqu samples; weigh 2g of Daqu samples, put them into a 250mL Erlenmeyer flask, add 100mL of sterile physiological saline, shake well, and incubate on a shaker at 37℃ and 180r / min for 30min to prepare a bacterial suspension; perform serial dilutions of the enriched bacterial suspension with sterile physiological saline, taking 100μL of each dilution factor of 10. -3 10 -4 10 -5 10 -6 The bacterial suspension was spread on the selection medium, with three replicates for each gradient. The spread petri dishes were incubated upside down in a 37°C incubator for 72 hours.

[0082] The screening medium, expressed in g / L, contains 2g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 20g agar, and 1L ultrapure water. The medium is sterilized at 121℃ for 20min, cooled to approximately 70℃, and then 1.5% (v / v) ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution, sterilized by filtration through a 0.22μm sterile filter membrane) is added.

[0083] II) Separation and Purification

[0084] Observe the size of colonies and clear zones on the culture medium. Select 5 colonies with larger clear zones and streak them on LB solid medium. Incubate at 37℃ for 24 h, repeating three times to obtain single colonies of the target strain. Inoculate the obtained single colonies into LB liquid medium and incubate overnight. Take 1 ml of bacterial culture and inoculate it into fermentation medium, with 3 replicates per group. Incubate for 72 h, then stop fermentation. Centrifuge and collect the supernatant. Filter through a 0.22 μm filter membrane into a liquid chromatography vial. Determine the ferulic acid content in the fermentation broth using high performance liquid chromatography. The strain with the highest ferulic acid production is preserved in a glycerol tube and named L-7. Subsequently, Enterococcus faecalis L-7 will be used as the starting strain for optimizing enzyme production conditions.

[0085] LB solid medium, expressed in g / L: 1L of medium contains 10g tryptone, 5g yeast extract, 10g sodium chloride, and 20g agar, at natural pH, sterilized at 121℃ for 20min.

[0086] LB liquid medium, expressed in g / L: 1L of medium contains 10g tryptone, 5g yeast extract, 10g sodium chloride, natural pH, sterilized at 121℃ for 20min.

[0087] Fermentation medium (g / L): 1L of medium contains 10g wheat bran, 1g yeast extract, 0.5g potassium chloride, 0.1g dipotassium hydrogen phosphate, 1L ultrapure water, natural pH, sterilized at 121℃ for 20min, and then 0.5g magnesium sulfate heptahydrate was sterilized separately and added to the medium.

[0088] Example 2: Identification and biological characteristics study of L-7 related 16S rDNA

[0089] I) Identification

[0090] Initial morphological observation led to a preliminary assessment that the bacteria was Gram-positive. Further molecular biological identification and sequencing using 16S rDNA were performed, with sequence comparison against the NCBI database. A phylogenetic tree was then constructed using MEGA 7.0.26 software, confirming the strain as *Enterococcus faecalis* L-7. (See below) Figure 1 .

[0091] II) Plotting the Ferulic Acid Standard Curve

[0092] Accurately weigh 1.0 mg of ferulic acid standard and prepare a 0.1 g / L ferulic acid standard stock solution with 50% methanol. Then, use the stock solution to prepare ferulic acid standard solutions with concentrations (g / L) of 0.04, 0.03, 0.02, 0.01, 0.007, 0.004, and 0.001. Measure the peak areas using high-performance liquid chromatography (HPLC). Plot a standard curve with the standard solution concentration (g / L) on the x-axis and the peak area on the y-axis, and then perform a linear regression equation. Figure 2 As shown, its regression equation is y = 32281x + 24.844, and the correlation coefficient R is... 2 =0.9997, which indicates a good linear relationship between the two.

[0093] (iii) Determination of ferulic acid content

[0094] Single colonies of the target strain were picked and inoculated into 15 mL centrifuge tubes containing 5 mL of LB liquid medium and cultured overnight. A 1% inoculum was then added to 100 mL of fermentation medium, with three replicates per group. The cultures were incubated at 37 °C and 180 rpm for 72 h. After fermentation, 1 mL of the fermentation broth was collected and centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and analyzed by high-performance liquid chromatography (HPLC). Uninoculated medium served as a blank control. The strain producing the highest ferulic acid yield was preserved in glycerol tubes and named L-7. Enterococcus faecalis L-7 was subsequently used as the starting strain for optimizing enzyme production conditions.

[0095] (iv) Determination of ferulic acid esterase activity

[0096] 1) Preparation of L-7 fermentation broth

[0097] ① Pick a single colony of L-7 and inoculate it into a 15mL centrifuge tube containing 5mL LB liquid medium and incubate overnight. ② Take 1ml of bacterial culture and inoculate it into 100mL fermentation medium. Incubate at 37℃ and 180r / min on a shaker for 72h. After fermentation, take 1ml as fermentation broth.

[0098] 2) Preparation of crude enzyme solution: Place 1 ml of fermentation broth obtained in step 1) into a high-speed centrifuge, centrifuge at 10000 r / min for 15 min, and take 250 μl of supernatant as crude enzyme solution;

[0099] 3) Enzyme activity assay: Take 250 μl of the crude enzyme solution obtained in step 2) into a 1.5 mL centrifuge tube, and immediately add 750 μl of 0.003 mol / L methyl ferulic acid solution (accurately weigh 0.031 g of methyl ferulic acid, dissolved in 50 mL of 0.05 mol / L Tris-HCl (pH 8.0) buffer solution). The 250 μL crude enzyme solution and 750 μL methyl ferulic acid solution constitute the reaction system. Then, incubate at 50℃ for 15 min. After the reaction is complete, immediately boil for 10 min to terminate the reaction. Set up a blank control group. After the reaction system cools to room temperature, centrifuge at 10000 r / min for 10 min, filter through a 0.22 μm filter membrane, and use the resulting sample solution in a liquid chromatography vial for high-performance liquid chromatography (HPLC) analysis. Figure 3 It can be seen that the peak elution time of the enzyme reaction solution sample (bottom) at around 14 min is close to that of the ferulic acid standard (top), indicating that the enzyme reaction solution contains ferulic acid esterase and can decompose ferulic acid methyl ester to produce ferulic acid.

[0100] Enzyme activity is defined as the amount of enzyme required to hydrolyze methyl ferulic acid at 50°C per minute to produce 1 μmol of ferulic acid, which is one unit of enzyme activity (1U).

[0101] (v) Growth curve of Enterococcus faecalis L-7

[0102] During the shake-flask culture at 37℃ and 180r / min, samples were taken every 2 hours to measure OD. 600 Value, by Figure 4 It can be seen that the growth is slow in the first 2 hours of cultivation, enters the logarithmic growth phase after 2 hours, is in the stable growth phase from 16 to 18 hours, and enters the decline phase after 18 hours.

[0103] (vi) Detection results of virulence genes and harmful metabolites of Enterococcus faecalis L-7

[0104] The results of the detection of virulence genes and harmful metabolites of Enterococcus faecalis L-7 are shown in Table 1. Figure 5 It was found that *Enterococcus faecalis* L-7 tested negative for all six virulence genes, indicating it does not carry the six common virulence genes found in enterococci. Furthermore, *Enterococcus faecalis* L-7 tested negative for amino acid decarboxylase, nitrate reductase, indole, and hemolysis, demonstrating that it lacks amino acid decarboxylase and nitrate reductase activity and cannot break down tryptophan to produce indole compounds. Hemolysis is one of the important indicators for evaluating the safety of enterococci. Figure 6 It can be seen that Enterococcus faecalis L-7 did not produce a hemolytic zone after being cultured on a blood agar plate for 48 hours, indicating that this strain does not have hemolytic toxicity.

[0105] Table 1. Detection results of Enterococcus faecalis L-7 virulence gene and harmful metabolites.

[0106]

[0107] Note: "+" indicates positive, and "-" indicates negative.

[0108] 7) Morphological identification of Enterococcus faecalis L-7

[0109] like Figure 7 As shown, the colonies of this strain are round or oval, with a smooth, opaque surface, no spores or flagella, and are arranged singly, in pairs or in short chains.

[0110] Gram staining results in purple, such as Figure 8 As shown, it belongs to Gram-positive bacteria.

[0111] 8) pH tolerance

[0112] like Figure 9 As shown, Enterococcus faecalis L-7 has a pH tolerance of 4–11, grows well at pH 7–10, and exhibits the fastest growth and reproduction at pH 8. This bacterium can tolerate a minimum pH of 4, and can also grow under higher pH conditions (pH 8–10), classifying it as an alkali-tolerant microorganism.

[0113] 9) Salt tolerance

[0114] like Figure 10 As shown, the growth of Enterococcus faecalis L-7 was inhibited when the NaCl mass fraction exceeded 3%. When the NaCl mass fraction was 8%, the bacterial density of Enterococcus faecalis decreased significantly, but the OD... 600 The value is still higher than 0.6. This indicates that the Enterococcus faecalis has a strong salt tolerance.

[0115] 10) Ethanol tolerance

[0116] like Figure 11 As shown, Enterococcus faecalis L-7 can grow normally in the range of 1% to 7% ethanol content, and its highest tolerance ethanol content is 13%.

[0117] Example 3: Optimization of enzyme production conditions by Enterococcus faecalis L-7

[0118] 1) Single-factor experiment

[0119] Single-factor experiments were conducted using four factors: inoculum size of 3%, 4%, 5%, 6%, and 7%; rotation speed of 140, 160, 180, 200, and 220 r / min; culture temperature of 22, 27, 32, 37, and 42℃; and culture time of 48, 72, 96, 120, and 144 h. Each experiment was conducted in triplicate.

[0120] (ii) Orthogonal experiment

[0121] Design L9(3) 4 An orthogonal experiment was conducted, selecting four factors for investigation: rotation speed, inoculum size, culture temperature, and culture time. Each factor was tested at three levels, and each group of experiments was conducted in triplicate.

[0122] Based on single-factor experiments, orthogonal experiments were conducted to investigate the influence of various factors on enzyme production by *Enterococcus faecalis* L-7 fermentation. The orthogonal experimental design is shown in Table 2, and the results are shown in Table 3. Range analysis showed that the influence of each factor on enzyme production was in the order of A > B > C > D, i.e., culture time > culture temperature > inoculum size > rotation speed. The optimal experimental combination was determined to be A2B1C3D1, i.e., culture time 96 h, culture temperature 32℃, inoculum size 6%, and rotation speed 160 r / min. Since no optimal combination was found in the orthogonal array, a validation experiment was required. When the combination was A2B1C3D1, the ferulic acid esterase activity produced by the strain reached 44.68 U / L.

[0123] Table 2 Orthogonal Experimental Design Scheme

[0124]

[0125] Table 3 Results of Orthogonal Experiments

[0126]

[0127] Comprehensive analysis revealed that the optimal fermentation conditions for enzyme production by Enterococcus faecalis L-7 were: 96 h culture time, 32 °C culture temperature, 6% inoculum size, and 160 rpm rotation speed. Under these conditions, the ferulic acid esterase activity produced by the strain reached 44.68 U / L, an increase of 68.92% compared to the unoptimized 26.45 U / L.

[0128] Example 4: Screening of Burkholderia BK100

[0129] 1) Enrichment

[0130] A batch of fermented koji samples from a winery, fermented for 1-30 days and stored for one month and two months, was pulverized and mixed to obtain koji powder. This koji powder was stored at -80°C for later use. 10g of the koji powder was added to 90mL of enrichment culture medium to prepare 10... -1 The Daqu enriched solution was then diluted to prepare 10 -2 -10 -7 This yields a diluted Daqu (a type of starter culture);

[0131] Enrichment medium (g / L): 1L of medium contains 10g tryptone, 5g yeast extract, 10g sodium chloride, and 20g glucose.

[0132] (ii) Screening of Burkholderia BK100, a high-yield ferulic acid esterase strain

[0133] 1) Initial screening: Take 10 -2 10 -3 10 -4 10 -5 Four different gradients of Daqu (a type of starter culture) dilutions were inoculated onto primary screening medium plates using ethyl ferulic acid as the carbon source and incubated upside down at 30°C for 3 days. Single colonies on the medium were then purified and cultured, and subsequently transferred to the primary screening medium and incubated at 30°C in a water-jacketed incubator for 5 days. The results showed that 32 strains were obtained from the primary screening. Colonies with clear zones were then transferred to the primary screening medium for further purification and isolation to obtain pure colonies, such as... Figure 12 As shown.

[0134] The initial screening medium, expressed in g / L, contained 0.5 g KCl, 1.0 g K₂HPO₄·3H₂O, 0.5 g MgSO₄·7H₂O, 2.0 g NaNO₃, 0.01 g FeSO₄·7H₂O, 20 g agar, 30 g sucrose, and 1 L distilled water at natural pH. The medium was sterilized at 121°C for 20 min. After cooling to 60°C, 10 mL of ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution) was added.

[0135] 2) Secondary screening: The strains that produced a clear zone obtained in step 1) were isolated and purified multiple times. Burkholderia BK100 had the largest clear zone. After streaking Burkholderia BK100 several times on the primary screening medium, pure single colonies were obtained. Burkholderia BK100 was inoculated into 100 mL of LB medium and incubated at 30℃ for 48 h. b) Burkholderia BK100 bacterial suspension was inoculated into 100 mL of secondary screening medium at a volume ratio of 2%, and incubated at 30℃ and 180 r / min for 72 h. The ferulic acid content in the fermentation broth was then determined by high-performance liquid chromatography. Figure 13 As shown, a peak appeared in the fermentation broth at 13 min, which is similar to the peak time of the ferulic acid standard, proving that the sample solution contains ferulic acid esterase and can metabolize methyl ferulic acid to produce ferulic acid. Based on the results of high performance liquid chromatography, a strain producing ferulic acid esterase with an activity of 41.6 U / L was obtained. This strain was named Burkholderia BK100 and preserved in glycerol tubes. Burkholderia BK100 was used as the starting strain in subsequent experiments.

[0136] LB medium, expressed in g / L: 1L of medium contains 10g tryptone, 5g yeast extract, 10g NaCl, 1L distilled water, natural pH, and is sterilized at 121℃ for 20min.

[0137] The secondary screening medium, expressed in g / L, contains 10g yeast extract, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 1.0g dipotassium hydrogen phosphate, 20g agar, and 1L distilled water. The pH is natural. The medium is sterilized at 121℃ for 20min, cooled to 60℃, and 10mL of ethyl ferulic acid (10% w / v dissolved in N,N-dimethylformamide solution) is added.

[0138] Example 5:

[0139] I) Molecular biological identification

[0140] The target strain, Burkholderia BK100, was streaked onto LB agar plates. Colony morphology and color were observed according to Bergey's Manual of Systematic Bacteriology. Gram staining and individual strain morphology were also performed. Morphological identification preliminarily determined it to be a Gram-negative, short rod-shaped bacterium. The purified Burkholderia BK100 was inoculated into LB medium and cultured overnight. The bacterial culture was then sent to Chengdu Qingke Biotechnology for bacterial species identification. Sequencing data were analyzed using homologous sequence searching in the National Center for Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed using MEGA 6.0.26 software. Figure 14 As shown. The strain was identified as Burkholderia and named Burkholderia BK100.

[0141] II) Growth curve of Burkholderia BK100

[0142] Single colonies of Burkholderia BK100 were picked and inoculated into LB broth. The culture was activated at 30°C and 180 rpm for 16 hours using a shaker. The activated bacterial solution was then inoculated into LB broth at a 1% inoculation rate and cultured in a constant-temperature shaker at 30°C and 180 rpm. The OD value was measured at 600 nm every 2 hours. A blank control was prepared using uninoculated LB broth. Three parallel experiments were performed, and the data were recorded and analyzed. A growth curve was plotted with the measured OD value on the ordinate and time on the abscissa. Figure 15 As shown in the figure. The test results showed that Burkholderia BK100 grew slowly from 0 to 2 hours, and was in the stasis phase; from 2 to 16 hours, it entered the logarithmic growth phase, and the strain grew rapidly; after 16 hours, it entered the stationary phase.

[0143] (iii) Temperature tolerance of Burkholderia BK100

[0144] Single colonies of Burkholderia BK100 were picked and inoculated into LB liquid medium. The culture was activated for 16 hours at 30°C and 180 rpm on a shaker. The activated bacterial solution was then inoculated into LB liquid medium at a 1% inoculum size. The cultures were incubated for 24 hours at 20°C, 25°C, 30°C, 37°C, 42°C, and 47°C, all at 180 rpm. The OD value was measured at 600 nm. A blank control was prepared using uninoculated LB liquid medium. Three parallel experiments were performed, and the data were recorded and analyzed. A curve was plotted with the measured OD value on the ordinate and temperature on the abscissa. Figure 16 As shown in the figure. The test results show that the optimal growth temperature of Burkholderia BK100 is 30℃, and it grows well in the range of 30-37℃. Growth is slow below 25℃ and above 42℃, and it hardly grows below 20℃.

[0145] (iv) Burkholderia bacillus BK100 pH tolerance

[0146] Single colonies of Burkholderia BK100 were picked and inoculated into LB liquid medium. The culture was activated for 16 h at 30°C and 180 rpm on a shaker. The activated bacterial solution was then inoculated at a 1% inoculum into LB liquid medium at pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The cultures were incubated at 30°C and 180 rpm for 24 h. The OD value was measured at 600 nm. A blank control was prepared using uninoculated LB liquid medium. Three parallel experiments were performed, and the data were recorded and analyzed. A curve was plotted with the measured OD value on the ordinate and temperature on the abscissa. Figure 17 As shown in the figure. The test results show that Burkholderia BK100 grows best at pH 7.0, and exhibits relatively stable and good growth at pH 5.0-8.0.

[0147] (v) Morphological and physiological-biochemical identification of Burkholderia BK100

[0148] Based on colony morphology observation, Burkholderia BK100 on the primary screening medium was characterized by round, relatively regular, light yellow, opaque, moist and smooth surface, with relatively high raised colonies and smooth and regular edges. Figure 18 As shown. Gram staining followed by observation under a light microscope reveals a red color, indicating Gram-negative bacteria, such as... Figure 19 As shown in the image, electron micrographs reveal that this bacterium is a short rod-shaped structure, as... Figure 20 As shown.

[0149] The physiological and biochemical results of Burkholderia BK100 are shown in Table 1. The catalase test of this bacterium was positive, while the methyl red test, acetylmethylethanol test, catalase test and starch hydrolysis test were negative.

[0150] Table 4 Physiological and biochemical characteristics of the strains

[0151]

[0152]

[0153] Example 6: Determination of ferulic acid content and ferulic acid esterase activity in the fermentation broth of Burkholderia BK100

[0154] 1) Plotting the standard curve of ferulic acid

[0155] Accurately weigh 0.1 g of ferulic acid standard and dilute to 10 ml with 50% methanol to prepare a 0.1 g / L stock solution. Dilute the stock solution sequentially to obtain sample solutions of 0.001 g / L, 0.0015 g / L, 0.01 g / L, and 0.04 g / L. Measure the peak area of ​​the sample solutions using high-performance liquid chromatography (HPLC). Plot a standard curve with sample concentration (g / L) on the x-axis and peak area on the y-axis, and construct a linear regression equation, such as... Figure 21 As shown. Its regression equation is y = 31439X + 5.94, and the correlation coefficient R is... 2 =0.9999, indicating a good linear relationship between the two.

[0156] (ii) Determination of ferulic acid content

[0157] Single colonies of Burkholderia BK100 were picked and inoculated into a seed culture medium at pH 7.0. The culture was activated for 16 h in a shaker at 30℃ and 180 rpm. At an inoculation rate of 4%, the activated seed culture was added to the fermentation medium, and the mixture was thoroughly stirred. Fermentation was carried out at 30℃ for 72 h. Then, PBS buffer solution at pH 6.0 was added, and the mixture was further extracted for 24 h in a shaker at 30℃ and 180 rpm. 20 mL of the 24-h fermentation broth was centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm aqueous filter and analyzed by high-performance liquid chromatography (HPLC). The corresponding peak areas were then recorded. Figure 21 The regression equation shows that the ferulic acid content in the fermentation medium was 623.08 ± 0.79 ng / L.

[0158] (iii) Determination of ferulic acid esterase activity

[0159] 1) Preparation of Burkholderia BK100 fermentation broth: a) Pick a single colony of Burkholderia BK100 and inoculate it into a seed culture medium with pH 7.0. Activate it in a shaker at 30℃ and 180r / min for 16h. b) Inoculate the activated solution into 100mL of fermentation medium with 4% inoculation amount. Stir well and place it in a 30℃ incubator for constant temperature fermentation for 72h. Then add PBS buffer solution with pH 6.0 and place it in a shaker at 30℃ and 180r / min for 24h for further extraction. This is the fermentation broth.

[0160] 2) Preparation of crude enzyme solution: Take 1 mL of the fermentation broth from step 1) and centrifuge at 10000 r / min for 15 min. Take the supernatant as crude enzyme solution.

[0161] 3) Enzyme reaction test: Take 250 μL of the crude enzyme solution from step 2) and 750 μL of 0.003 mol / L methyl ferulic acid solution (dissolved in 0.05 mol / L Tris-HCl solution) and add them to a test tube. After shaking, the pH of the reaction solution is 6.0. Place the test tube in a water bath at 50℃ for 15 min. After the reaction is complete, transfer the test tube to boiling water and boil for 10 min. After cooling, centrifuge at 10000 r / min for 5 min, collect the supernatant, and filter it through a 0.22 μm aqueous filter membrane as the sample solution. Replace the crude enzyme solution with an equal volume of Tris-HCl buffer as a blank control. Perform high performance liquid chromatography (HPLC) analysis on the sample solution. The results are shown in Figure 22. The peak time of ferulic acid in the sample solution (22a) and the peak time of ferulic acid standard (22b) are both around 13 min, which proves that the sample solution contains ferulic acid esterase and can metabolize methyl ferulic acid to produce ferulic acid.

[0162] Enzyme activity is defined as the amount of enzyme required per minute to hydrolyze methyl ferulic acid to produce 1 μmol of ferulic acid at a specific temperature; one enzyme activity unit (1 U) is defined as this amount of enzyme.

[0163] Example 7: Optimization of enzyme activity assay conditions for ferulic acid esterase produced by Burkholderia

[0164] 1) Single-factor experiment

[0165] 1) Effect of reaction temperature on enzyme activity assay: With other enzyme reaction conditions unchanged, reaction solutions with reaction temperatures set at 20℃, 30℃, 40℃, 50℃, and 60℃ were used to measure enzyme activity and investigate the effect of reaction temperature on enzyme activity. Three parallel experiments were performed at each temperature.

[0166] 2) Effect of reaction time on enzyme activity assay: The reaction temperature was the temperature with the highest enzyme activity in the above single-factor experiments, while the reaction conditions of other enzymes remained unchanged. Reaction solutions with reaction times of 10 min, 15 min, 20 min, 25 min, and 30 min were used for enzyme activity assay to investigate the effect of reaction time on enzyme activity. Three parallel experiments were performed for each time point.

[0167] 3) Effect of reaction pH on enzyme activity assay: The reaction temperature and time were the temperatures and times with the highest enzyme activity in the single-factor experiments mentioned above, respectively. The reaction conditions for other enzymes remained unchanged. Reaction solutions with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0 were used for enzyme activity assay to investigate the effect of reaction pH on enzyme activity. Three parallel experiments were performed for each pH.

[0168] 4) Effect of crude enzyme solution addition on enzyme activity assay: The reaction temperature, time, and pH were the highest for enzyme activity observed in the single-factor experiments described above, respectively. Other enzyme reaction conditions remained unchanged. Reaction solutions with crude enzyme solution additions of 150 μL, 200 μL, 250 μL, 300 μL, and 350 μL were used for enzyme activity assay to investigate the effect of crude enzyme solution addition on enzyme activity. Three parallel experiments were performed for each addition amount.

[0169] Depend on Figure 23 It can be seen that the activity of ferulic acid esterase produced by Burkholderia is the highest when the reaction temperature is 40℃, but the enzyme activity decreases when the temperature exceeds 40℃. The enzyme activity is also very low when the reaction temperature is below 30℃. This is because the enzyme reaction rate increases when the temperature rises, but because enzymes are proteins, excessively high temperatures will cause the enzymes to denature and become inactive.

[0170] Depend on Figure 24 It can be seen that the activity of ferulic acid esterase produced by Burkholderia gradually increases with the increase of reaction time, and the activity of ferulic acid esterase reaches the maximum when the reaction time is 20 min. However, the activity of ferulic acid esterase decreases as the reaction time continues to increase.

[0171] Depend on Figure 25 It is known that the activity of ferulic acid esterase produced by Burkholderia is the highest at a reaction pH of 7.0, and the enzyme activity is relatively high in enzyme reaction systems with pH of 6.0-8.0. The activity of ferulic acid esterase is lower in excessively acidic or alkaline conditions. This may be because excessively acidic or alkaline conditions destroy the spatial structure of the enzyme, or it may affect the dissociation process of the groups that maintain the spatial structure of the enzyme molecule, thereby affecting the conformation of the active site of the enzyme and thus affecting the enzyme activity.

[0172] Depend on Figure 26It can be seen that the enzyme activity increases with the increase of crude enzyme solution, and the enzyme activity is low when the amount of crude enzyme solution added is small. The enzyme activity of ferulic acid esterase of Burkholderia reaches the maximum when the amount of crude enzyme solution added is 300 μL. The enzyme activity decreases when the amount of crude enzyme solution added is greater than 300 μL, which may be because the crude enzyme solution contains certain substances that can affect the determination of ferulic acid esterase activity. When the content of such substances exceeds a certain amount, it will affect the determination results of ferulic acid esterase activity.

[0173] (ii) Response Surface Design

[0174] The results of the single-factor experiments were analyzed. Based on this, Design-Expert V8.0.6 software was used, and the Box-Behnken experimental design principle was applied. Reaction temperature (A), reaction time (B), reaction pH (C), and crude enzyme solution addition (D) were considered as factors, and ferulic acid esterase activity (E) was used as the response value to design and optimize the conditions for ferulic acid esterase activity determination. The factors and their levels on the response surface are shown in Table 2. The Box-Behnken experimental design and results are shown in Table 3.

[0175] Table 5 Response Surface Factor Level Coding Table

[0176]

[0177] Table 6 Results of ANOVA for the Regression Model

[0178]

[0179]

[0180] 1) Establishment and analysis of the quadratic response surface regression model

[0181] Based on the experimental results in Table 3, the experimental data were fitted using Design-Expert V8.0.6 software, and the resulting bivariate regression equation is as follows:

[0182] E=83.49+6.63A+5.95B+1.6C+1.88D+0.41AB+0.072AC+1.05AD+1.42BC+0.013BD+0.99CD-15.07A 2 -9.85B 2 -8.58C 2 -3.93D 2

[0183] Table 4 shows the variance analysis of the optimized regression model for enzyme activity assay conditions.

[0184] Table 7 Results of ANOVA for the Regression Model

[0185]

[0186]

[0187] 2) Analysis of the effects of interactions among various factors on ferulic acid esterase activity

[0188] Response surface methodology (RSM) can visually reflect the influence of experimental factors on ferulic acid esterase activity. A steeper slope on the RSM indicates greater sensitivity to changes in operating conditions. Table 4 shows that A and B are highly significant (P < 0.01), indicating that reaction temperature and reaction time have a significant impact on ferulic acid esterase activity. 2 B 2 C 2 D 2 The results were highly significant (P < 0.01), indicating that the effects of the four enzyme activity assay conditions—reaction temperature, reaction time, reaction pH, and crude enzyme solution dosage—on ferulic acid esterase activity were not a simple linear relationship. The order of influence of each assay condition was: reaction temperature > reaction time > crude enzyme solution dosage > reaction pH.

[0189] The optimized enzyme activity assay conditions using Design-Expert V8.0.6 software were: reaction temperature: 42.35℃, reaction time: 21.59 min, reaction pH: 7.14, and crude enzyme solution addition volume: 314.43 μL. The theoretical enzyme activity of ferulic acid esterase was 85.5953 U / L. To verify this result, considering practical conditions, the optimal enzyme activity assay conditions were modified to: reaction temperature: 42℃, reaction time: 22 min, reaction pH: 7.1, and crude enzyme solution addition volume: 320 μL. Three parallel experiments were conducted under these conditions, and the average enzyme activity of ferulic acid esterase was 85.08 U / L, which is close to the software's prediction. Therefore, optimizing the enzyme activity assay conditions of ferulic acid esterase using response surface methodology is feasible.

[0190] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Burkholderia (a type of bacteria) Burkholderia sp.) BK100, characterized in that, Burkholderia BK100 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 20221553 on October 10, 2022.

2. Burkholderia BK100 according to claim 1, characterized in that, Burkholderia BK100 grows well at 25-40℃, with the optimal growth temperature being 30℃.

3. The application of Burkholderia BK100 as described in claim 1 in the fermentation production of ferulic acid esterase.

4. The application according to claim 3, characterized in that, The conditions for determining the activity of the ferulic acid esterase are as follows: the enzyme activity reaction temperature in the crude enzyme solution is 20-60℃, the enzyme activity reaction pH in the crude enzyme solution is 5.0-9.0, the amount of crude enzyme solution added is 250-350μL, and the enzyme activity reaction time in the crude enzyme solution is 15-30min.

5. The application according to claim 4, characterized in that, The optimal conditions for determining the activity of ferulic acid esterase were: an optimal reaction temperature of 42°C, an optimal reaction pH of 7.1, an optimal amount of crude enzyme solution added (320 μL), and an optimal reaction time of 22 min.

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