Process for producing vanillin by bioconversion of ferulic acid using a complex bacteria

Abstract

This invention belongs to the field of biofermentation technology. More specifically, it relates to a process for the biotransformation of ferulic acid by a compound microorganism to produce vanillin. The specific production steps of this invention include: strain activation: *Enterobacterium mulberryii*, *Enterobacter hominis*, and *Amylopectinobacterium pseudoclasticum* are inoculated into slant culture medium for activation culture to obtain activated strains; seed culture preparation: the activated strains are inoculated into seed culture medium for culture to obtain seed culture solutions of each strain; compound microorganism fermentation: the seed culture solutions of each strain are mixed, and then inoculated into fermentation medium at an inoculation amount of 10-20% by volume, and flake-shaped nano-alumina is added, followed by shaking culture to obtain fermentation broth; biotransformation: ferulic acid is added to the fermentation broth, and the mixture is cultured in the dark with shaking to obtain fermentation broth containing vanillin; separation and purification: the fermentation broth containing vanillin is separated and purified to obtain vanillin.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology. More specifically, it relates to a process for the biotransformation of ferulic acid by a complex microbial culture to produce vanillin. Background Technology

[0002] Vanillin is one of the most widely used food flavorings globally, with significant applications in food, beverages, tobacco, cosmetics, and pharmaceuticals. Currently, the main production methods for vanillin include plant extraction, chemical synthesis, and microbial transformation. Plant extraction produces natural products, but its output is limited by raw material supply, accounting for less than 2% of market demand. Chemical synthesis offers low cost and high yield, but suffers from severe environmental pollution and limited product aroma.

[0003] Microbial transformation has become a research hotspot due to its advantages of mild conditions, environmental friendliness, and natural and equivalent products. Several microbial strains have been disclosed in existing technologies that can be used to transform ferulic acid into vanillin, such as *Enterobacter mori*, *Enterobacter hormaechei*, and *Amycolatopsis sp.*, with vanillin yields reaching 1.5-13.4 g / L.

[0004] However, the above-mentioned single-strain transformation process still has the following technical problems: the single strain has limited tolerance to the substrate ferulic acid; the amount of by-products (such as vanillic acid, vanillyl alcohol, etc.) generated during fermentation is high, which increases the difficulty of separation and purification; the fermentation cycle is long and the process stability is poor; some strains have harsh requirements for fermentation conditions and are difficult to adapt to industrial scale-up production.

[0005] Synergistic metabolic interactions among bacterial strains can effectively improve substrate transformation efficiency, inhibit byproduct formation, and enhance system stability. However, there are currently no systematic reports on the use of synergistic bacterial transformation of ferulic acid as a substrate to produce vanillin. Therefore, developing a highly efficient, stable, and industrially suitable synergistic bacterial biotransformation process has significant application value. Summary of the Invention

[0006] The technical problem this invention aims to solve is the limited tolerance of existing single-strain fermentation methods for producing vanillin to the substrate ferulic acid, resulting in poor fermentation stability. Based on this challenge, this invention provides a process for the biotransformation of ferulic acid into vanillin using a compound microbial culture.

[0007] The purpose of this invention is to provide a process for producing vanillin by biotransformation of ferulic acid using compound bacteria.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: A process for the biotransformation of ferulic acid by a compound bacteria to produce vanillin, the specific production steps of which include: Strain activation: Enterobacter mulberryis, Enterobacter hominis, and Amyloliquefaciens were inoculated onto slant culture medium for activation culture to obtain activated strains; Seed culture preparation: The activated bacterial strains were inoculated into seed culture medium and cultured to obtain seed culture solutions for each strain; Compound microbial fermentation: The seed culture solutions of each strain are mixed, and then inoculated into the fermentation medium at an inoculation rate of 10-20% by volume. Flake nano-alumina is added, and the mixture is shaken and cultured to obtain the fermentation broth. The amount of the sheet-like nano-alumina added to the fermentation medium is 100-200 mg / L; Biotransformation: Ferulic acid was added to the fermentation broth, and the mixture was cultured in the dark with shaking to obtain a fermentation broth containing vanillin; During the light-protected shaking culture process, the concentration of ferulic acid in the fermentation broth is 10-15 g / L. Separation and purification: The fermentation broth containing vanillin is separated and purified to obtain vanillin.

[0009] The beneficial effects of the above technical solution are as follows: The above-mentioned technical solution utilizes the synergistic metabolism of three bacterial strains and their complementary metabolic pathways to form a highly efficient conversion pathway from ferulic acid to vanillin. Specifically, when the three strains are cultured together, their metabolic enzyme systems complement each other. *Enterobacter mulberryii* dominates the initial conversion, *Enterobacter hominis* continues the conversion in the alkaline environment of the later fermentation stage, and *Amylopectinobacter* selectively converts intermediate products into vanillin through a highly selective metabolic pathway, reducing the generation of byproducts vanillic acid and vanillyl alcohol. Based on this, sheet-like nano-alumina, through its two-dimensional planar structure, electrostatically adsorbs onto the bacterial cell wall, immobilizing the bacteria on its surface without damaging the cell membrane. Simultaneously, the sheet-like structure provides a large attachment surface area, capable of simultaneously supporting the three strains, forming a spatially "co-immobilized bacterial community."

[0010] Most importantly, the hydroxyl functional groups on the surface of the sheet-like nano-alumina can form hydrogen bonds with the carboxyl and phenolic hydroxyl groups in the ferulic acid molecule, creating a "micro-enrichment zone" for the substrate on the sheet surface. This local substrate concentration gradient reduces the concentration shock of direct exposure of the bacteria to high concentrations of ferulic acid, allowing the bacteria to gradually metabolize the substrate in a stable microenvironment. Simultaneously, the immobilized state enhances the metabolic activity of the bacteria, enabling the three strains to maintain normal growth and enzyme activity even at high substrate concentrations.

[0011] Furthermore, the compound microbial fermentation also includes: The seed culture solutions of each strain were mixed, and then inoculated into the fermentation medium at an inoculation rate of 10-20% by volume. Flake-shaped nano-alumina and spherical nano-alumina were added, and the mixture was shaken and cultured to obtain the fermentation broth. The amount of the sheet-like nano-alumina added to the fermentation medium is 100-200 mg / L; The amount of spherical nano-alumina added to the fermentation medium is 50-80 mg / L.

[0012] The beneficial effects of the above technical solution are as follows: The sheet-like nano-alumina provides a two-dimensional planar structure, serving as the main framework for bacterial attachment. Spherical nano-alumina, with its excellent three-dimensional flowability and dispersibility, fills the spaces between the sheet-like structures, forming a "sheet-sphere composite carrier network." The spherical nano-alumina is uniformly dispersed in the fermentation broth, continuously agitating the microenvironment through Brownian motion, promoting the rapid transfer of nutrients and the substrate ferulic acid from the culture medium to the bacteria on the sheet-like carrier surface. Simultaneously, vanillin products produced by bacterial metabolism can rapidly diffuse out of the bacterial microenvironment, mitigating product inhibition effects. The "microbial film" formed on the sheet-like carrier surface and the "mass transfer channels" formed by the spherical carrier together constitute a highly efficient mass transfer network.

[0013] Furthermore, the D50 of the sheet-like nano-alumina is 10-30 nm; the D50 of the spherical nano-alumina is 5-10 nm.

[0014] The beneficial effects of the above technical solution are as follows: Larger sheet-like structures (10-30 nm) serve as the framework, while smaller spherical structures (5-10 nm) act as fillers, forming an aggregate-fine particle gradation structure. The small spherical structures fill the gaps between the large sheet-like structures, increasing the packing density of the composite carrier and the number of bacterial attachment sites. At the same time, the higher specific surface area of ​​the small spherical structures further enhances the mass transfer capacity.

[0015] Furthermore, the sphericity of the spherical nano-alumina is 0.85-0.92.

[0016] The beneficial effects of the above technical solution are as follows: Nano-sized alumina particles with a sphericity of 0.85-0.92 exhibit high sphericity, enabling them to roll rather than slide when subjected to fluid shear forces in the fermentation broth. This rolling behavior creates a "micro-stirring" effect at the microscale, continuously renewing the diffusion boundary layer on the surface of the spherical particles. This maximizes the concentration gradient of nutrients and substrates around the particles, significantly enhancing mass transfer efficiency. Particles with a sphericity below 0.85 (irregular shapes) are prone to sliding or stagnation, resulting in decreased mass transfer efficiency; perfect spheres with a sphericity above 0.92 are costly and unnecessary.

[0017] Under shaking culture conditions (100-200 rpm), particles with a sphericity of 0.85-0.92 exhibit moderate fluid resistance, allowing them to disperse sufficiently with the culture medium without causing sedimentation due to excessive resistance. This hydrodynamic characteristic ensures that the spherical carrier remains in uniform suspension in the fermenter, guaranteeing consistent mass transfer throughout the entire reaction system.

[0018] Furthermore, the mixing of the seed culture solutions of each strain includes: The seed culture solutions of Enterobacter mulberry, Enterobacter hominis, and Amyloliquefaciens were mixed in a volume ratio of 1-3:1-2:1-2.

[0019] Furthermore, the fermentation medium is composed of: 5-20 g / L carbon source, 1-5 g / L nitrogen source, 0.5-1.5 g / L sodium chloride, 0.5-1.5 g / L ferrous sulfate heptahydrate, 1-5 g / L potassium dihydrogen phosphate, 0.2-1 g / L magnesium sulfate heptahydrate, and pH adjusted to 6-11. The carbon source is selected from one or more of wheat bran, rice husk, glucose, sucrose, and starch; the nitrogen source is selected from one or more of soybean meal, yeast extract, peptone, and beef extract.

[0020] Furthermore, the separation and purification includes: The fermentation broth containing vanillin was microfiltered using a ceramic membrane, and the dialysate was collected. The dialysate was adsorbed through a macroporous adsorption resin column and impurities were eluted with deionized water. Vanillin was then eluted with a 40-60% ethanol solution, and the eluent was collected. The organic solvent in the eluent is removed by evaporation, and then the concentration of vanillin is concentrated under reduced pressure to ≥40 g / L. The mixture is allowed to stand and separate into layers. The upper aqueous phase is collected, cooled and crystallized, filtered, and dried to obtain vanillin. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0022] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0023] Use kraft paper substrate with a moisture content of less than 6%; Example 1 Strain activation: Enterobacter mulberryis, Enterobacter hominis, and Amyloliquefaciens were inoculated onto slant culture medium for activation culture to obtain activated strains; The composition of the slant culture medium is as follows: The composition of the slant culture medium for Enterobacter mulberry is: 180 mL / L carrot extract, 18 g / L sucrose, and 12 g / L agar; The composition of the slant culture medium for Enterobacter hopnea is: 10 g / L peptone, 4 g / L beef extract, 4 g / L sodium chloride, and 14 g / L agar; The composition of the slant culture medium for non-branched acid bacteria is: glucose 4 g / L, yeast extract 4 g / L, malt extract 8 g / L, and agar 18 g / L. Seed culture preparation: The activated bacterial strains were inoculated into seed culture medium and cultured to obtain seed culture solutions for each strain; The seed culture medium is composed of: The seed culture medium for Enterobacter mulberry consists of: 5 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride. The seed culture medium for Enterobacter holmie consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The seed culture medium for amylopectin consists of: 26 g / L glucose, 9 g / L yeast extract, 0.7 g / L sodium chloride, 5 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 0.06 g / L calcium chloride. Compound bacterial fermentation: The seed culture broth of Enterobacter mulberry, Enterobacter hominis, and Amyloliquefaciens were mixed in a volume ratio of 1:1:1. Then, the mixture was inoculated into the fermentation medium at a volume percentage of 10%, and flake nano-alumina and spherical nano-alumina were added. The mixture was shaken and cultured at 25℃ and 100 rpm for 1 day to obtain the fermentation broth. The fermentation medium consists of: 5 g / L carbon source, 1 g / L nitrogen source, 0.5 g / L sodium chloride, 0.5 g / L ferrous sulfate heptahydrate, 1 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, and pH adjusted to 6. The carbon source is selected from wheat bran; the nitrogen source is selected from soybean meal; The amount of the sheet-like nano-alumina added to the fermentation medium is 100 mg / L; The amount of spherical nano-alumina added to the fermentation medium is 50 mg / L; The D50 of the sheet-like nano-alumina is 10 nm; the D50 of the spherical nano-alumina is 5 nm. The specific surface area of ​​the sheet-like nano-alumina is 150 m² / g.

[0024] The sphericity of the spherical nano-alumina is 0.85; Biotransformation: Ferulic acid was added to the fermentation broth, and the mixture was cultured in the dark with shaking at 25°C and 100 rpm for 12 days to obtain a fermentation broth containing vanillin. During the light-protected shaking culture process, the concentration of ferulic acid in the fermentation broth is 10 g / L. Separation and purification: The fermentation broth containing vanillin was microfiltered using a ceramic membrane with a pore size of 50 nm, and the dialysate was collected. The dialysate was adsorbed through a macroporous adsorption resin column and impurities were eluted with deionized water. Vanillin was then eluted with a 40% ethanol solution, and the eluent was collected. The organic solvent in the eluent is removed by evaporation, and then the vanillin is concentrated under reduced pressure at 60°C to a concentration ≥40 g / L. After standing and separating into layers, the upper aqueous phase is collected, cooled and crystallized, filtered, and dried to obtain vanillin.

[0025] Example 2 Strain activation: Enterobacter mulberryis, Enterobacter hominis, and Amyloliquefaciens were inoculated onto slant culture medium for activation culture to obtain activated strains; The composition of the slant culture medium is as follows: The composition of the slant culture medium for Enterobacter mulberry is: carrot extract 190 mL / L, sucrose 18 g / L, and agar 13 g / L; The composition of the slant culture medium for Enterobacter hopnea is: 11 g / L peptone, 4.6 g / L beef extract, 4.5 g / L sodium chloride, and 15 g / L agar; The composition of the slant culture medium for non-branched acid bacteria is: glucose 5g / L, yeast extract 5g / L, malt extract 9g / L, and agar 19g / L. Seed culture preparation: The activated bacterial strains were inoculated into seed culture medium and cultured to obtain seed culture solutions for each strain; The seed culture medium is composed of: The seed culture medium for Enterobacter mulberry consists of: 5 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride. The seed culture medium for Enterobacter holmie consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The seed culture medium for amylopectin consists of: 26 g / L glucose, 9 g / L yeast extract, 0.7 g / L sodium chloride, 5 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 0.06 g / L calcium chloride. Compound bacterial fermentation: The seed culture broth of Enterobacter mulberry, Enterobacter hominis, and Amyloliquefaciens were mixed in a volume ratio of 2:1:1.4. Then, the mixture was inoculated into the fermentation medium at a volume percentage of 15%, and flake nano-alumina and spherical nano-alumina were added. The mixture was shaken and cultured at 30℃ and 150 rpm for 2 days to obtain the fermentation broth. The fermentation medium consists of: 10 g / L carbon source, 3 g / L nitrogen source, 0.9 g / L sodium chloride, 0.8 g / L ferrous sulfate heptahydrate, 3 g / L potassium dihydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, and pH adjusted to 8. The carbon source is selected from rice husks; the nitrogen source is selected from yeast extract powder; The amount of the sheet-like nano-alumina added to the fermentation medium was 140 mg / L; The amount of spherical nano-alumina added to the fermentation medium was 60 mg / L; The D50 of the sheet-like nano-alumina is 20 nm; the D50 of the spherical nano-alumina is 8 nm. The specific surface area of ​​the sheet-like nano-alumina is 180 m² / g.

[0026] The sphericity of the spherical nano-alumina is 0.9; Biotransformation: Ferulic acid was added to the fermentation broth, and the mixture was cultured in the dark with shaking at 30°C and 140 rpm for 12 days to obtain a fermentation broth containing vanillin. During the light-protected shaking culture process, the concentration of ferulic acid in the fermentation broth is 12 g / L. Separation and purification: The fermentation broth containing vanillin was microfiltered using a ceramic membrane with a pore size of 100 nm, and the dialysate was collected. The dialysate was adsorbed through a macroporous adsorption resin column and impurities were eluted with deionized water. Vanillin was then eluted with a 50% ethanol solution, and the eluent was collected. The organic solvent in the eluent is removed by evaporation, and then the vanillin is concentrated under reduced pressure at 70°C to a concentration ≥40 g / L. After standing and separating the layers, the upper aqueous phase is collected, cooled and crystallized, filtered, and dried to obtain vanillin.

[0027] Example 3 Strain activation: Enterobacter mulberryis, Enterobacter hominis, and Amyloliquefaciens were inoculated onto slant culture medium for activation culture to obtain activated strains; The composition of the slant culture medium is as follows: The composition of the slant culture medium for Enterobacter mulberry is: 200 mL / L carrot extract, 20 g / L sucrose, and 14 g / L agar; The composition of the slant culture medium for Enterobacter hopnea is: 12 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, and 16 g / L agar; The composition of the slant culture medium for non-branched acid bacteria is: glucose 6 g / L, yeast extract 6 g / L, malt extract 10 g / L, and agar 20 g / L. Seed culture preparation: The activated bacterial strains were inoculated into seed culture medium and cultured to obtain seed culture solutions for each strain; The seed culture medium is composed of: The seed culture medium for Enterobacter mulberry consists of: 5 g / L beef extract, 10 g / L peptone, and 5 g / L sodium chloride. The seed culture medium for Enterobacter holmie consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The seed culture medium for amylopectin consists of: 26 g / L glucose, 9 g / L yeast extract, 0.7 g / L sodium chloride, 5 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, and 0.06 g / L calcium chloride. Compound bacterial fermentation: The seed culture broth of Enterobacter mulberry, Enterobacter hominis, and Amyloliquefaciens were mixed in a volume ratio of 3:2:2. Then, the mixture was inoculated into the fermentation medium at a volume percentage of 20%, and flake nano-alumina and spherical nano-alumina were added. The mixture was shaken and cultured at 40℃ and 200 rpm for 3 days to obtain the fermentation broth. The fermentation medium consists of: 20 g / L carbon source, 5 g / L nitrogen source, 1.5 g / L sodium chloride, 1.5 g / L ferrous sulfate heptahydrate, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, and pH adjusted to 11. The carbon source is selected from starch; the nitrogen source is selected from beef extract; The amount of the sheet-like nano-alumina added to the fermentation medium was 200 mg / L; The amount of spherical nano-alumina added to the fermentation medium was 80 mg / L; The D50 of the sheet-like nano-alumina is 30 nm; the D50 of the spherical nano-alumina is 10 nm. The specific surface area of ​​the sheet-like nano-alumina is 200 m² / g.

[0028] The sphericity of the spherical nano-alumina is 0.92; Biotransformation: Ferulic acid was added to the fermentation broth, and the mixture was cultured in the dark with shaking at 40°C and 200 rpm for 12 days to obtain a fermentation broth containing vanillin. During the light-protected shaking culture process, the concentration of ferulic acid in the fermentation broth is 15 g / L. Separation and purification: The fermentation broth containing vanillin was microfiltered using a ceramic membrane with a pore size of 200 nm, and the dialysate was collected. The dialysate was adsorbed through a macroporous adsorption resin column and impurities were eluted with deionized water. Vanillin was then eluted with a 60% ethanol solution, and the eluent was collected. The organic solvent in the eluent is removed by evaporation, and then the vanillin is concentrated under reduced pressure at 80°C to a concentration ≥40 g / L. After standing and separating the layers, the upper aqueous phase is collected, cooled and crystallized, filtered, and dried to obtain vanillin.

[0029] Example 4 The difference between this embodiment and Embodiment 1 is that spherical nano-alumina was not added, while all other conditions remained unchanged.

[0030] Example 5 The difference between this embodiment and Embodiment 1 is that the sphericity of the spherical nano-alumina is 0.8, while the other conditions remain unchanged.

[0031] Comparative Example 1 The difference between this comparative example and Example 1 is that spherical nano-alumina of equal mass is used instead of sheet-like nano-alumina, while the other conditions remain unchanged.

[0032] Comparative Example 2 The difference between this comparative example and Example 1 is that no sheet-like nano-alumina was added.

[0033] Comparative Example 3 The difference between this comparative example and Example 1 is that no flake-shaped nano-alumina or spherical nano-alumina was added, while all other conditions remained unchanged.

[0034] The vanillin products extracted in the above embodiments and comparative examples were tested, and the specific test methods and results are as follows: Using the fermentation methods described in the above examples or comparative examples, five batches of fermentation were carried out, and the yield of vanillin in different batches was counted to evaluate the fermentation stability. The quantitative analysis of vanillin was obtained by high performance liquid chromatography. The detailed results are shown in Table 1; Table 1: Statistical Results of Vanillin Yield in Different Batches As can be seen from the test results in Table 1, the fermentation process of the present invention exhibits higher fermentation stability in different batches.

[0035] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A process for the biotransformation of ferulic acid by compound bacteria to produce vanillin, characterized in that, The specific production steps include: Strain activation: Enterobacter mulberryis, Enterobacter hominis, and Amyloliquefaciens were inoculated onto slant culture medium for activation culture to obtain activated strains; Seed culture preparation: The activated bacterial strains were inoculated into seed culture medium and cultured to obtain seed culture solutions for each strain; Compound microbial fermentation: The seed culture solutions of each strain are mixed, and then inoculated into the fermentation medium at an inoculation rate of 10-20% by volume. Flake nano-alumina is added, and the mixture is shaken and cultured to obtain the fermentation broth. The amount of the sheet-like nano-alumina added to the fermentation medium is 100-200 mg / L; Biotransformation: Ferulic acid was added to the fermentation broth, and the mixture was cultured in the dark with shaking to obtain a fermentation broth containing vanillin; During the light-protected shaking culture process, the concentration of ferulic acid in the fermentation broth is 10-15 g / L. Separation and purification: The fermentation broth containing vanillin is separated and purified to obtain vanillin.

2. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to claim 1, characterized in that, The compound microbial fermentation also includes: The seed culture solutions of each strain were mixed, and then inoculated into the fermentation medium at an inoculation rate of 10-20% by volume. Flake-shaped nano-alumina and spherical nano-alumina were added, and the mixture was shaken and cultured to obtain the fermentation broth. The amount of the sheet-like nano-alumina added to the fermentation medium is 100-200 mg / L; The amount of spherical nano-alumina added to the fermentation medium is 50-80 mg / L.

3. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to claim 2, characterized in that, The D50 of the sheet-like nano-alumina is 10-30 nm; the D50 of the spherical nano-alumina is 5-10 nm.

4. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to any one of claims 2 or 3, characterized in that, The specific surface area of ​​the sheet-like nano-alumina is 150-200 m² / g. The sphericity of the spherical nano-alumina is 0.85-0.

92.

5. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to claim 1, characterized in that, The mixing of the seed culture solutions of each strain includes: The seed culture solutions of Enterobacter mulberry, Enterobacter hominis, and Amyloliquefaciens were mixed in a volume ratio of 1-3:1-2:1-2.

6. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to claim 1, characterized in that, The fermentation medium consists of: 5-20 g / L carbon source, 1-5 g / L nitrogen source, 0.5-1.5 g / L sodium chloride, 0.5-1.5 g / L ferrous sulfate heptahydrate, 1-5 g / L potassium dihydrogen phosphate, 0.2-1 g / L magnesium sulfate heptahydrate, and pH adjusted to 6-11. The carbon source is selected from one or more of wheat bran, rice husk, glucose, sucrose, and starch; the nitrogen source is selected from one or more of soybean meal, yeast extract, peptone, and beef extract.

7. The process for producing vanillin from ferulic acid by biotransformation of compound bacteria according to claim 1, characterized in that, The separation and purification process includes: The fermentation broth containing vanillin was microfiltered using a ceramic membrane, and the dialysate was collected. The dialysate was adsorbed through a macroporous adsorption resin column and impurities were eluted with deionized water. Vanillin was then eluted with a 40-60% ethanol solution, and the eluent was collected. The organic solvent in the eluent is removed by evaporation, and then the concentration of vanillin is concentrated under reduced pressure to ≥40 g / L. The mixture is allowed to stand and separate into layers. The upper aqueous phase is collected, cooled and crystallized, filtered, and dried to obtain vanillin.

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