Phytobacterium plantarum with high yield of feruloyl esterase and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and provides a high-yield feruloyl esterase plant lactobacillus and application thereof. The bacterial strain is lactobacillus plantarum Fs6, the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) No.36264, and the preservation date of the bacterial strain is October 20, 2025. According to the present invention, the fermentation performance of the dough can be significantly improved with the plant lactobacillus, and the good synergistic effect is represented during the co-fermentation with the yeast; and a good synergistic effect is shown during co-fermentation with yeast. When the mass concentration of bile salt is 0.3 and 0.6 g / L, the survival rate is relatively good, which shows that the lactic acid bacteria Fs6 has a certain probiotic function. The self-aggregation force (32.77%) and the hydrophobicity (32.43%) of the Fs6 are relatively low, and the adhesion of the Fs6 is relatively poor. And combined phenolic acid in the grains can be effectively released, so that the nutritional value of the grains can play a better health role in a human body.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus plantarum* that produces ferulic acid esterase and its applications. Background Technology

[0002] In rye, phenolic acids mainly exist in bound form, with free phenolic acids accounting for only 1%–5% of the total. Most bound phenolic acids are linked to cell wall components via ester bonds, and some are linked to lignin structures. Ferulic acidesterase (FAE) can effectively cleave ester bonds, releasing bound ferulic acid and other phenolic acids, thereby significantly enhancing the antioxidant and health benefits of rye products. Furthermore, the released phenolic acids can undergo non-covalent interactions or cross-linking with starch molecules, altering the gelatinization, retrogradation, and digestibility characteristics of starch, which helps improve the textural stability and nutritional properties of rye sourdough products. Therefore, screening for dominant strains with strong FAE production capacity and adaptability to fermentation environments is not only a key approach to releasing bound phenolic acids from rye but also an important foundation for developing high-quality, functional rye sourdough and its products. Summary of the Invention

[0003] This invention provides a high-yield ferulic acid esterase-producing Lactobacillus plantarum and its applications.

[0004] This invention is achieved by the following technical solution: a strain of *Lactobacillus plantarum* that produces ferulic acid esterase, specifically named *Lactobacillus plantarum* Fs6, with accession number CGMCC No. 36264, accession date October 20, 2025, and Latin name [missing information]. Lactiplantibacillus plantarum It has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The present invention also provides the application of the high-ferulate esterase-producing Lactobacillus plantarum in the preparation of a starter culture for fermented rye.

[0006] The *Lactobacillus plantarum* Fs6 described in this invention has been identified as significantly improving the fermentation performance of dough and exhibiting a good synergistic effect when co-fermented with yeast. It produces ferulic acid esterase with an activity of 13.51 ± 0.54 mU / mL. The enzyme is in the lag phase from 0-4 h, the logarithmic phase from 4-14 h, and reaches the stationary phase after 14 h. Combined fermentation with yeast (Ah4) significantly improves the fermentation performance of dough and exhibits a good synergistic effect when co-fermented with yeast. The survival rate is good at bile salt concentrations of 0.3 and 0.6 g / L, indicating that *Lactobacillus plantarum* Fs6 has certain probiotic functions. Fs6 exhibits low self-aggregation (32.77%) and hydrophobicity (32.43%), resulting in poor adhesion. It can effectively release bound phenolic acids from grains, allowing the nutritional value of the grains to exert a better health effect in the human body.

[0007] The specific name of the bacterial strain of this invention is *Lactobacillus plantarum* Fs6, with accession number CGMCC No. 36264, accession date October 20, 2025, and Latin name [missing information]. Lactiplantibacillus plantarum It has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0008] Figure 1 Results of the Oxford cup double screening transparent zone experiment for ferulic acid esterase-producing strain (Lactobacillus plantarum Fs6); Figure 2 For the ferulic acid content standard curve; Figure 3 The growth curve of the strain; Figure 4 The volume growth rate of dough fermented with different yeasts and lactic acid bacteria; Figure 5 The morphology of Lactobacillus plantarum Fs6 strain; Figure 6 Phylogenetic tree of Lactobacillus plantarum Fs6; Figure 7 This is a graph showing the results of the bile salt tolerance test. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but 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.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0011] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0012] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0013] I. Experimental Methods 1. The lactic acid bacteria strains used in this invention are isolated from traditional sourdough.

[0014] 2. Screening of lactic acid bacteria producing ferulic acid esterase Initial screening: After streaking lactic acid bacteria onto MRS solid medium, a single colony is picked up with an inoculation needle and transferred to screening medium. The culture is then incubated at 37°C for 72 hours, and the presence of a clear zone is observed. Strains producing clear zones can be preliminarily identified as producing ferulic acid esterase.

[0015] Secondary screening: For strains producing a clear zone, after overnight culture of the ferulic acid esterase-producing strain, bacterial cells were collected by centrifugation at 8000g for 5 min, washed with sterile phosphate buffer, and resuspended. 200 µL of the bacterial suspension was added to each well of a plate containing Oxford cups for the initial screening medium.

[0016] Preparation of primary screening medium: Remove glucose from MRS medium, sterilize and place at room temperature. Prepare a 10% ethyl ferulic acid solution with dimethylformamide. When the temperature of MRS medium drops to 55-63℃, add the ethyl ferulic acid solution at a volume ratio of 1% and mix well.

[0017] 3. Enzyme activity assay of crude ferulic acid esterase solution: Preparation of crude FAE enzyme solution: The FAE-producing strain was cultured overnight, centrifuged at 5000 rpm for 3 min, and the bacterial cells were collected. The bacterial cells were washed three times with sterile physiological saline and resuspended. The bacterial suspension was inoculated into liquid fermentation enzyme-producing medium at an inoculum size of 2%, and fermented at 37℃ for 48 h with a shaking frequency of 120 rpm. After fermentation, 10 mL of fermentation broth was centrifuged at 8000 rpm for 5 min, and the supernatant was collected to obtain the crude enzyme solution.

[0018] Ferulic acid esterase liquid fermentation enzyme production medium (for inducing lactic acid bacteria to produce ferulic acid esterase): Remove glucose from MRS liquid medium (without agar), sterilize and place at room temperature. Prepare a 10% methyl ferulic acid solution (prepared with dimethylformamide). When the temperature of the MRS medium drops to 55-63℃, add the methyl ferulic acid solution at a rate of 1.5% and mix well.

[0019] Determination of the ferulic acid standard curve: Accurately weigh 0.1000 g of trans-ferulic acid, dissolve it in an appropriate amount of ethanol, and dilute to 100 mL to obtain a 1.00 mg / mL trans-ferulic acid standard solution. Take 0.5, 0.7, 0.9, 1.1, and 1.3 mL of the ferulic acid standard solution respectively, and dilute to 100 mL with ethanol to obtain trans-ferulic acid standard solutions with concentrations of 5, 7, 9, 11, and 13 μg / mL respectively. Using ethanol as a blank, measure the absorbance of the solution at 320 nm. Plot a standard curve with absorbance on the ordinate and trans-ferulic acid concentration (μg / mL) on the abscissa, and calculate the linear regression equation.

[0020] Enzyme activity determination: The fermentation broth was centrifuged at 4000 r / min for 10 min. 5 mL of the supernatant diluted twice was taken, and 5 mL of citric acid buffer solution was added to a 50 mL test tube. The mixture was incubated in a 55℃ water bath for 5 min. 0.5 g of destarched wheat bran was added, and the mixture was kept at a constant temperature for 30 min. The enzyme was then inactivated by boiling water for 5 min to terminate the reaction. The reaction solution was centrifuged at 4000 r / min for 10 min. The supernatant was diluted with ethanol to an appropriate concentration, and the ferulic acid content was determined by spectrophotometer at 320 nm. Based on the ferulic acid esterase activity determined under the above conditions, one enzyme activity unit (U) was defined as the amount of enzyme required per milliliter of crude enzyme solution at 37℃ and pH 7.5 to degrade methyl ferulic acid ester per minute to produce 1 μmol of ferulic acid.

[0021] The calculation formula is: In the formula, U—feruloesterase activity, mU / mL; x—concentration of ferulic acid in the enzyme solution being tested, μg / mL; V1—total reaction volume, mL; N—dilution factor of crude enzyme solution; V2—volume of crude enzyme solution, mL; T—reaction time, min; 194.18—molecular mass of ferulic acid.

[0022] 4. Growth Curve: To screen for lactic acid bacteria with high ferulic acid esterase activity and vigorous growth and metabolism, their growth in liquid culture medium was further measured. The enzyme-producing bacteria were activated twice in MRS liquid medium and then inoculated into the liquid medium at a 2% (v / v) inoculum. The culture was incubated at 37℃ for 24 h. From the start of inoculation, the OD600 value of the bacterial suspension was measured every 2 h, with three repeated measurements. A growth curve was plotted with time on the x-axis and OD600 value on the y-axis.

[0023] 5. Fermentation Power Measurement: The dough volume method was used. 75g of flour, 25g of rye flour, 55g of water, 1% yeast, and 2% lactic acid bacteria were kneaded into a dough. 15g of dough was quickly placed into a 50mL graduated cylinder. A wooden stick with a diameter slightly larger than the cylinder radius and a smooth bottom was used to fill the bottom of the cylinder with dough, ensuring the top was horizontal. The time at this point was recorded as 0, and the dough volume was recorded. The cylinder was placed in a constant temperature and humidity chamber at 30℃ and 85% humidity. The dough volume reading was recorded every 2 hours until the 12th hour. The horizontal line on the convex surface of the dough was used as the reference when reading the volume.

[0024] 6. Bile salt tolerance characteristics of bacterial strains: Analysis of bile salt tolerance of lactic acid bacteria: Activated lactic acid bacteria were inoculated at a mass fraction of 2% into liquid culture media with bile salt concentrations of 0.3 g / L and 0.6 g / L, respectively, and cultured at 37°C for 24 h. The OD values ​​of each group of bacterial solutions were measured. 595 value.

[0025] 7. Determination of bacterial self-aggregation ability and hydrophobicity: Hydrophobicity of the strain: The bacterial cells were washed three times with sterile physiological saline, resuspended in deionized water, and the OD of the bacterial suspension was adjusted with PBS. 600 A live bacterial suspension was prepared at a concentration of 1.0-1.1. 5 mL of this suspension was then transferred to a test tube and inactivated at 120 °C to obtain an inactivated bacterial suspension. Both the live and inactivated bacterial suspensions were used as samples to measure their hydrophobicity, and the initial OD of the bacterial suspension was recorded. 600 For A1, take 3 mL of sample into a 5 mL centrifuge tube, add 1 mL of xylene solution, vortex to mix, and incubate at 37℃ for 3 h. Then, take the supernatant and measure its OD. 600 The hydrophobicity of the sample is calculated using the following formula: .

[0026] Self-aggregation capacity of the bacterial strain: A bacterial suspension was prepared, and its OD600 was adjusted to 1.0-1.1 with PBS to obtain a live bacterial suspension. 5 mL of this suspension was transferred to a sterile test tube and inactivated at 120 °C to obtain an inactivated bacterial suspension. The hydrophobicity of both the live and inactivated bacterial suspensions was measured, and the initial OD600 of the suspension was recorded as A1. The above bacterial suspension was dispensed into centrifuge tubes at 5 mL per tube and allowed to stand at room temperature for 2, 4, 6, and 8 h. The absorbance of the supernatant was then measured and recorded as A2. The self-aggregation rate of the sample was calculated using the following formula: .

[0027] II. Experimental Results 1. Screening of Ferulic Acid Esterase-Producing Strains: Developing edible strains capable of producing ferulic acid esterase can effectively release conjugated phenolic acids from grains, allowing the nutritional value of the grains to exert a better health effect on the human body. When a single colony was inoculated onto a screening plate, only one strain showed a clear zone after cultivation. This strain was then used in the Oxford Cup clear zone experiment. The clear zone formation after cultivation is shown below. Figure 1 As shown, the diameter of the transparent ring is 17.33 mm.

[0028] 2. Enzyme activity assay of crude ferulic acid esterase solution: A standard curve of ferulic acid was plotted, and the results showed that the concentration of ferulic acid in the range of 50~250 μg / ml had a good linear relationship with absorbance. Figure 2 ) Spectrophotometric determination Lactiplantibacillus plantarum The ferulic acid esterase activity of Fs6 was measured to be 13.51 ± 0.54 mU / mL.

[0029] 3. Growth curve and fermentation power of the strain: from Figure 3 It can be seen that, Lactiplantibacillus plantarum Fs6 is in a hysteresis phase from 0 to 4 hours, in a logarithmic phase from 4 to 14 hours, and reaches a stationary phase after 14 hours.

[0030] The fermentation power of three groups of bacteria was measured, and the fermentation status of different combinations of lactic acid bacteria and yeast Ah4 was observed. The fermentation rate of the dough was characterized by the dough volume growth rate. The results are shown in Figure 4. In the initial stage of fermentation (0-4h), the fermentation rate of the strains was slow, which may be because the yeast was not adapted to the unfamiliar environment in the early stage of fermentation, and its growth activity was not strong, and it was in the growth lag phase. After 4h, the dough volume increased rapidly, indicating that the yeast multiplied and grew vigorously in this stage. Compared with the mixed group, the fermentation rate of single yeast was relatively slow, and different lactic acid bacteria mixed with Ah4 showed different fermentation capabilities. The fermentation power of Fs6 was comparable to that of Fs1, and it could also significantly improve the fermentation performance of the dough, showing a good synergistic effect when co-fermented with yeast.

[0031] 4. The morphology of *Lactobacillus plantarum* Fs6 strain is as follows: Figure 5 As shown, *Lactobacillus plantarum* Fs6 is rod-shaped, with single colonies being round, milky white, with a matte surface, neat edges, and is opaque and immobile.

[0032] Depend on Figure 7 It was found that the growth of each strain was inhibited to varying degrees with increasing bile salt concentration. The survival rates of the three strains were all above 50% at concentrations of 0.3 and 0.6 g / L. Lactic acid bacteria Fs6 showed better survival rates at bile salt concentrations of 0.3 and 0.6 g / L, indicating that Fs6 may possess certain probiotic functions. Fs6 exhibited low self-aggregation (32.77%) and hydrophobicity (32.43%), indicating poor adhesion.

[0033] A phylogenetic tree was constructed using the neighbor-join method in MEGA 6.0 software. The 16S rRNA gene sequence of *Lactobacillus plantarum* Fs6 is shown in SEQ ID NO.1. The phylogenetic tree is as follows: Figure 6As shown. *Lactiplantibacillus plantarum* Fs6 is the most closely related to *Lactiplantibacillus plantarum* strain TMPC36211 in phylogenetic order. Therefore, this strain was identified as: *Lactiplantibacillus plantarum* (…). Lactiplantibacillus plantarum The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 36264 and deposit date of October 20, 2025.

[0034] This strain produces ferulic acid esterase with an activity of 13.51 ± 0.54 mU / mL. It is in the lag phase from 0 to 4 hours, in the logarithmic phase from 4 to 14 hours, and reaches the stationary phase after 14 hours. Co-fermentation with yeast Ah4 significantly improves the fermentation performance of dough, exhibiting a good synergistic effect. The survival rate is good at bile salt concentrations of 0.3 and 0.6 g / L, indicating that lactic acid bacteria Fs6 has certain probiotic functions. Fs6 has low self-aggregation (32.77%) and hydrophobicity (32.43%), resulting in poor adhesion. It can effectively release bound phenolic acids from grains, allowing the nutritional value of the grains to exert a better health effect in the human body.

Claims

1. A strain of *Lactobacillus plantarum* that produces ferulic acid esterase, characterized by: The specific name of this strain is *Lactobacillus plantarum* Fs6, with accession number CGMCC No. 36264, accession date October 20, 2025, and Latin name [missing information]. Lactiplantibacillus plantarum It has been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of a starter culture for fermented rye.

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