CN113583912B - Enterobacter cholerae and application thereof in vanillin production - Google Patents

Abstract

The invention discloses an enterobacter cholerae and application thereof in vanillin production, belonging to the field of biotechnology, wherein the enterobacter cholerae is named DQ and is preserved in China general microbiological culture collection center (China general microbiological culture collection center), the preservation date is 2021 and 07 and 26, and the preservation number is CGMCC No.22958. The enterobacter cholerae (DQ) can effectively utilize trans-ferulic acid to produce vanillin, can greatly improve the yield and the utilization efficiency of vanillin, has good tolerance of ferulic acid and vanillin, further improves the production and economic benefits, also satisfies the production under special environment, and effectively solves the defects of long fermentation period, poor condition universality, low strain conversion rate, intolerance to high-concentration ferulic acid and the like existing in the process of fermenting and producing vanillin by modern microorganisms by using trans-ferulic acid as a substrate.

Description

Enterobacter cholerae and application thereof in vanillin production

Technical Field

The invention relates to the field of biotechnology, in particular to enterobacter cholerae and application thereof in vanillin production.

Background

Vanillin is the third largest edible spice in the world, and the use amount of natural vanillin increases year by year with the increase of health consciousness of people. Modern methods for producing vanillin mainly comprise: chemical synthesis, plant extraction and microbial fermentation. Among them, the glyoxylate method is the main method for chemical synthesis, but it has the characteristics of complex product, great process pollution, low product purity, etc. The plant extraction method is to directly extract vanillin from vanilla plants by using a leaching extraction method, but meanwhile, the vanilla has the defects of limited planting area, large influence on yield, complex planting and processing treatment, complex operation and the like, so that the yield can not meet the market demand all the time. The microbial transformation production is a mode of microbial fermentation, and vanillin is directly extracted from metabolites through the biological metabolism of microorganisms. The strains for producing vanillin by using ferulic acid at present are as follows: aspergillus niger, mirabilis cinnabarinus and Streptomyces shaoxiensis. The production of vanillin by microbial fermentation conversion has received increasing attention in recent years.

In a plurality of microbial transformation methods, the effective fermentation conversion rate of the Aspergillus niger and the Mirabilitum cinnabarinus by a two-part combination method is 34.01 percent, and the conversion rate is lower. The yield of the strain of the streptomyces sarsasii is higher. However, the conversion rate of the strains is low, the required optimal fermentation pH is neutral, and the strains have the defect of intolerance to high-concentration ferulic acid. Therefore, the conditions required for the fermentation are relatively fixed, and are not applicable to some special fermentation conditions. Meanwhile, vanillin is phenolic acid substance, precursor substances in the synthetic route are vanillic acid, and vanillic acid and ferulic acid containing carboxylic acid functional groups and vanillin have certain toxicity to the strain, so that the function of the strain is affected, and the yield and the utilization efficiency of vanillin are reduced.

Disclosure of Invention

Aiming at the defects, the invention aims to provide the enterobacter cholerae and the application thereof in the production of vanillin, and can effectively solve the defects of long fermentation period, poor condition universality, low strain conversion rate, intolerance to high-concentration ferulic acid and the like in the process of fermenting and producing vanillin by using trans-ferulic acid as a substrate by modern microorganisms.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a bacillus cholerae (Enterobacter hormaechei), which can be used for efficiently producing vanillin by taking trans-ferulic acid as a substrate through fermentation, is named DQ and is preserved in China general microbiological culture collection center (CGMCC), the preservation address is 1 # 3 of North Chen West Lu in the Korean region of Beijing city, the preservation date is 2021, 07 month and 26 days, and the preservation number is CGMCC No.22958.

The invention also provides an application of the enterobacter cholerae (DQ) in vanillin production.

Further, the application of the enterobacter cholerae (DQ) in the production of vanillin takes trans-ferulic acid as a substrate, and the vanillin is produced by a microbial transformation method; the specific parameters in application are as follows: the pH is 9-11, the inoculation amount is 1.5-3 wt%, and the fermentation temperature is 30-35 ℃.

Further, the application of the enterobacter cholerae (DQ) in the production of vanillin is that the specific parameters in the application are as follows: the pH was 10.8, the inoculum size was 2wt%, and the fermentation temperature was 33 ℃.

A preparation for improving vanillin production efficiency comprises the above enterobacter cholerae (DQ) as effective component.

In summary, the invention has the following advantages:

1. the enterobacter cholerae (DQ) can effectively utilize trans-ferulic acid to produce vanillin, can greatly improve the yield and the utilization efficiency of vanillin, has excellent tolerance of ferulic acid and vanillin, further improves the production and economic benefits, also satisfies the production under special environment, and effectively solves the defects of long fermentation period, poor condition universality, low strain conversion rate and the like existing in the process of fermenting and producing vanillin by using trans-ferulic acid as a substrate by modern microorganisms.

Drawings

FIG. 1 is a standard graph drawn in the present invention;

FIG. 2 is a thin layer chromatography chromatogram of DQ in the present invention;

FIGS. 3-10 are population morphology and individual morphology diagrams of DQ-4, DQ, HS-2 and 6-6 according to the invention;

FIG. 11 is an electropherogram of PCR products of DQ-4, DQ, HS-2 and 6-6 according to the present invention;

FIGS. 12-15 are 16S rDNA sequence-based phylogenetic trees of 6-6, HS-2, DQ-4 according to the present invention;

FIG. 16 is a graph of growth curves for 6-6, HS-2, DQ-4 according to the invention;

FIG. 17 is a graph showing the results of substrate tolerance of 6-6, HS-2, DQ-4 according to the present invention;

FIG. 18 is a graph showing the growth of E.cholerae (DQ) in the present invention;

FIG. 19 is a graph showing fermentation of E.cholerae (DQ) in the present invention;

FIG. 20 is a graph showing the effect of substrate concentration on vanillin production by E.cholerae (DQ) in the invention;

FIG. 21 is a graph showing the effect of initial pH on vanillin production by Enterobacter cholerae (DQ) in the invention;

FIG. 22 is a graph showing the effect of inoculum size on vanillin production by Enterobacter cholerae (DQ) according to the invention;

FIG. 23 is a graph showing the effect of fermentation temperature on vanillin production by Enterobacter cholerae (DQ) in the invention;

FIGS. 24-29 are graphs showing the effect of interactions of factors on vanillin production by E.cholerae (DQ) in the invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.

Thus, the following detailed description of the embodiments of the invention, as provided, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.

Example 1 isolation, screening and identification of species

1. Sample processing

Weighing 10g of distilled grain and 10g of Daqu respectively, placing in a 250mL beaker, adding 90mL of sterile water, shaking uniformly, soaking for 30min, and filtering to obtain distilled grain filtrate and Daqu filtrate, numbering distilled grain sample liquid (JZ-1-6) and Daqu sample liquid (DH-1-6) for later use; 10mL Huang Shuiyu mL sterile water is measured to obtain yellow water sample liquid, no. Huang Shuiyang liquid (HS-1-6) for standby.

2. Isolation and purification of strains

Respectively taking 5mL of the three sample liquids by a pipette, inoculating the three sample liquids into a conical flask of a sterilized and cooled LB liquid culture medium (50 mL), and performing shake culture for 48h, wherein the culture rotating speed is 160r/min, and the culture temperature is 37 ℃; repeating the step, and carrying out enrichment culture again under the same conditions; after enrichment, diluting to 10-8 by adopting a 10-time serial gradient dilution method, respectively diluting 0.2mL of bacterial suspension with the multiple of 10-6, 10-7 and 10-8, coating the bacterial suspension on an LB solid culture medium, culturing at a constant temperature of 37 ℃ for 24 hours, picking single bacterial colonies with better growth and different bacterial colony forms, carrying out flat streaking on the single bacterial colonies in a culture medium with the single carbon source of trans-ferulic acid in a pH=7 environment by adopting an inoculating loop, and purifying for 2-3 times; after purification, the strain is inoculated in a slant culture medium for culturing for 24 hours at a constant temperature of 37 ℃, and then stored in a refrigerator at a temperature of 4 ℃ for standby.

3. Seed liquid preparation

And (3) using the burnt inoculating loop, picking a loop of single colony activated on the LB culture medium, inoculating the loop of single colony on the LB liquid culture medium filled with 50mL of the single colony, culturing at 160r/min and 37 ℃ until the absorbance OD value is within the range of 0.4-0.6, and simultaneously preparing the LB liquid culture medium without bacteria as a blank control to obtain the seed culture solution.

4. Primary screening results of vanillin-producing strains:

after enrichment culture, 32 single colonies were obtained in total by streaking on the isolation medium. Carrying out shaking table fermentation culture on all single colonies; through FeCl ₃ And (3) carrying out color development reaction to obtain 11 strains suspected to produce vanillin, wherein the color development results are shown in table 1-1. Wherein the strains numbered 2-4, 3-7 and 4-2 have weaker color development effect and almost no bluish purple; strains numbered 2-7 and 5-8 have weak and insignificant color development effects; the color development effect of the strains numbered DQ, DQ-4, HS-2, 4-1, 5-3 and 6-6 is obvious.

TABLE 1-1 FeCl ₃ Reaction color development Effect

Remarks: "+" indicates that the color development effect is weak, "++" indicates that the color development effect is weak, and "++" indicates that the color development effect is obvious

5. Vanillin producing strain re-screening results:

the vanillin content (mg/L) is taken as the abscissa X of a standard graph and OD is taken as the OD _440nm The value is the ordinate Y of the standard curve chartThe standard curve is shown in FIG. 1. The regression equation of the standard curve is y=0.002 x-0.0119, and the correlation coefficient R ² =0.9985, it can be stated that the OD at a wavelength of 440nm is linear with the vanillin content (mg/L).

And re-screening the strain suspected to produce vanillin in the primary screen by using a TBA photometry method, measuring an OD value at 440nm wavelength, and calculating the vanillin content in the fermentation liquor according to a standard curve, wherein the results are shown in tables 1-2. The screened vanillin-producing strains can be obtained, wherein the yield of the vanillin-producing strains is between 100.0mg/L and 1600.0mg/L, 5 strains are arranged between 100mg/L and 300mg/L, 6 strains are arranged between 300.0mg/L and 600.0mg/L, the numbers of the strains are DQ, DQ-4, HS-2, 4-1, 5-8 and 6-6, the suspected vanillin-producing strains have the strongest vanillin-producing capacity, the vanillin yield is 1482.62 +/-92.78 mg/L, the HS-2, 6-6 and DQ-4 are 864.28 +/-71.17 mg/L, 589.28 +/-80.98 mg/L and 550.95 +/-70.00 mg/L.

TABLE 1-2 screening of strains re-screening results

6. Thin layer chromatography chromatograms

Placing the bacterial liquid to be detected and the vanillin standard liquid on the activated thin plate, carrying out sample application and chromatography, taking out after chromatography, drying at 90 ℃ for 30min, and observing fluorescent strips under an ultraviolet irradiation lamp. Taking strain DQ as an example, a thin layer chromatogram is shown in FIG. 2 (wherein A-DQ broth; B-vanillin standard). Measured and calculated, vanillin R _f A value of 0.82; it was observed that on the bands of the fermentation broth there was one R _f Value and vanillin R _f The same value of fluorescent spots indicates that vanillin is generated in the fermentation product of the fermentation broth.

7. Morphological analysis

Strain morphology identification was performed on DQ-4, DQ, HS-2, 6-6 strains by observing the colony morphology of the strain on the plate and the individual morphology of the strain by gram staining (10×100-fold oleoscopy), DQ-4 colony morphology and individual morphology (10×100-fold oleoscopy) are shown in fig. 3 and 4, DQ-4 colony morphology and individual morphology (10×100-fold oleoscopy) are shown in fig. 5 and 6, HS-2 colony morphology and individual morphology (10×100-fold oleoscopy) are shown in fig. 7 and 8, 6-6 colony morphology and individual morphology (10×100-fold oleoscopy) are shown in fig. 9 and 10, and respective morphology analyses are shown in tables 1 to 3:

TABLE 1-3 morphological analysis of high-yield vanillin bacteria

8. Physiological and biochemical identification

The physiological and biochemical identification of DQ strain is carried out by referring to the experimental method described in the handbook of identification of common bacterial systems and the handbook of bacteriology of Berger's system, and the identification results are shown in tables 1-4.

TABLE 1-4 Bac1 bacterial physiological Biochemical characterization experiment results

The strain can grow on LB medium containing 0.5% NaCl, and according to the identification result, the strain is consistent with the characteristics of the enterobacter cholerae.

9. Identification of 16S rDNA Strain

The genome DNA of 4 high-yield vanillin strains is extracted, the bacterial 16S rDNA universal primers (27F and 1492R) are used for PCR amplification, and the obtained products are detected by 1% agarose electrophoresis, so that the PCR products of the high-yield strains are determined to be fragments with the size of about 1500bp, as shown in figure 11 (wherein 1 is DQ;2 is DQ-4;3 is 6-6;4 is HS-2), and the PCR products are consistent with the characteristics of target bacteria.

10. Phylogenetic tree constructed by high-yield strain gene sequence

Sequencing the PCR products obtained by DQ, DQ-4, 6-6 and HS-2 by a third party detection company, and performing BLAST homology comparison on the sequencing result in a GenBank database of NCBI, wherein the homology of the DQ-4 and a strain Cronobacter sakazakii with the number NR 114077.1 is 99.58% through comparison; homology of DQ to strain Enterobacter hormaechei numbered MH 542252 reaches 99.51%;6-6 shows a homology of 99.65% with strain Leclercia adecarboxylata numbered KX 959963; the homology of HS-2 with strain Serratia marcescens, numbered MN 396719, reached 99.44%.

By constructing phylogenetic Mega7.0 software, a visually distinct phylogenetic tree is constructed using the adjacency method. The results of the construction of the strains 6-6, HS-2, DQ-4 are shown in FIGS. 12-15. Each strain was collected in the closest relationship, and according to the results of molecular biological identification, the morphological characteristics of the strain were referenced and combined to preliminarily determine that DQ-4 was Cronobacter sakazakii of the genus Cronobacter (Enterobacter sakazakii), DQ was Enterobacter hormaechei of the genus Enterobacter (Enterobacter holoensis), 6-6 was Leclercia adecarboxylata of the genus Leclericia (non-decarboxylative Leercitrile), and HS-2 was Serratia marcescens of the genus Serratia (Serratia marcescens).

11. Growth curve determination of high-yield vanillin strain

The cell numbers of the 4 isolated strains (DQ, HS-2, 6-6, DQ-4) at 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h were measured at a wavelength of 660nm, with fermentation time (h) as abscissa and OD ₆₆₀ The values are on the ordinate and the growth curves of the resulting strains are shown in FIG. 16. From FIG. 16, it is evident that the logarithmic growth phase of HS-2 is 2 h-6 h, slow growth is performed at 6 h-10 h, and the growth is performed at the stationary phase after 10 h; 6-6 is in a stable state after 10 hours and 0 to 10 hours of longer logarithmic growth period; the adaptation period of DQ is relatively long, 6-8 h is a logarithmic growth period, and the growth is slow after 8 h; the DQ-4 strain has a logarithmic growth phase of 4-8 h, 8-10 h is in slow growth, and 10h later is in a stationary phase.

12. Determination of substrate tolerance of high-yield vanillin strains

DQ, HS-2, 6-6 and DQ-4 are respectively inoculated into trans-ferulic acid fermentation culture media containing 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L and 16g/L, and after the culture is carried out until the corresponding growth log phase, the content of vanillin in each fermentation broth is measured by utilizing a TBA photometry, and the conversion rate of each strain is calculated, as shown in figure 17 and tables 1-5. The results show that: DQ is subjected to constant temperature shake culture for 8 hours at 160r/min and 37 ℃ under the condition of pH=7 to the logarithmic phase, so that the maximum vanillin conversion rate of 76% can be obtained when the vanillin content in the fermentation broth is 4142.10mg/L under the concentration of 12g/L of the substrate addition; HS-2 is subjected to constant temperature shake culture for 6 hours to logarithmic phase at 160r/min and 37 ℃ under the condition of pH=7, so that the vanillin content in the fermentation broth is 2705.40mg/L and the vanillin conversion rate is 53% under the concentration of 12g/L of substrate addition; DQ-4 is subjected to constant temperature shake culture for 6 hours to logarithmic phase at 160r/min and 37 ℃ under the condition of pH=7, and the maximum vanillin content in fermentation liquor is 327.97mg/L under the condition of adding substrate with different concentrations, so that the conversion rate is low; 6-6, and culturing at 160r/min at 37 ℃ under the condition of pH=7 for 8 hours to logarithmic phase, wherein the maximum vanillin content in the fermentation broth is 572.40mg/L under the concentration of 8g/L of the substrate, and the maximum conversion rate is 42% under the concentration of 2g/L of the substrate.

Table 1-5 4 strain was compared in terms of Vanillin conversion ability at different substrate concentrations

Example 2 characterization of the target Strain

1. Growth characteristics of E.cholerae strain

As can be seen from FIG. 18, the curve shows little tendency at 0-4h of fermentation culture, indicating that Enterobacter cholerae is in the lag phase; after 4 hours of culture of the fermentation broth, the growth curve suddenly increases suddenly, which indicates that the enterobacter cholerae enters into the growth log phase; after 12h of fermentation culture, the curve again tended to plateau, when E.cholerae had entered the stationary phase.

2. Fermentation of enterobacter cholerae strains

As is clear from FIG. 19, the fermentation of E.cholerae was continued for 48 hours, and the highest peak was observed in the fermentation curve, indicating that the optimal fermentation time of E.cholerae was 48 hours.

3. Morphological characteristics of Enterobacter cholerae strains

The bacterial colony is large, slightly off-white and round and slightly convex, is about 2-3 mm in size, smooth and opaque in surface and neat in edge; the strain is dyed to be purple red and is gram negative bacteria.

Example 3 Single factor experiment of Strain fermentation

1. Effect of substrate concentration on vanillin production by enterobacter cholerae:

the contents of the immobilized peptone and the sodium chloride are unchanged, the content of the substrate (trans-ferulic acid) is changed, and the influence of different substrate concentrations on the vanillin production of the enterobacter cholerae is explored. As a result, as shown in FIG. 20, the concentration of the substrate was 2g/L to 12g/L, and the vanillin production amount by E.cholerae was increased. When the concentration of the substrate is 12g/L, the content of vanillin produced by the enterobacter cholerae is highest and reaches 5591.3 mg/L. After reaching 12g/L, the concentration of the substrate is increased again, and the vanillin production content of the enterobacter cholerae starts to be reduced continuously. Thus, a substrate concentration of 12g/L was chosen as the maximum tolerance of E.cholerae and on this basis a one-factor test of initial pH, inoculum size and fermentation temperature was performed.

TABLE 3-1 Effect of different substrate concentrations on Vanillin production by fermentation of Enterobacter cholerae

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2. Effect of initial pH on vanillin production by enterobacter johnsonii:

based on the determination of the optimal substrate concentration, the effect of different initial pH on the vanillin production of Enterobacter cholerae was investigated. As shown in FIG. 21, when the pH is between 2 and 10, the vanillin content is continuously increased under the condition of the rising pH, and the conversion of trans-ferulic acid into vanillin by the enterobacter cholerae is more facilitated under the alkaline condition; at pH 10, the vanillin content reaches the maximum value, namely 4887.3mg/L; when the pH exceeds 10, the vanillin content tends to decrease, and it is likely that the alkaline environment exceeds the environment in which the strain grows and propagates, resulting in a decrease in vanillin yield. So the initial pH values of 8, 10 and 12 are chosen to be appropriate for the levels under investigation.

TABLE 3-2 Effect of different initial pH on Vanillin production by fermentation of Enterobacter cholerae

3. Effect of inoculum size on vanillin production by enterobacter johnsonii:

based on the determination of the optimal substrate concentration, the effect of different inoculum sizes on vanillin production by enterobacter cholerae was investigated. As a result, as shown in FIG. 22, as the inoculum size (1 wt% to 2 wt%) was increased, the vanillin content was also increased continuously from 2200.2mg/L to 3314.8mg/L; on the other hand, when the inoculum size was changed from 2 to 12wt%, the vanillin content tended to decrease from 3314.8mg to 1653.8mg/L, indicating that the inoculum size was optimal at 2wt%, probably because the inoculum size in this experiment was 50mL and the nutrients were limited, and too much inoculum size was instead responsible for the decrease in vanillin content. Finally, it is appropriate to select inoculum sizes of 1wt%, 2wt% and 3wt% as the examined level.

TABLE 3 effects of different inoculum sizes on Vanillin production by fermentation of Enterobacter cholerae

4. Effect of fermentation temperature on vanillin production by enterobacter cholerae:

based on the determination of the optimal substrate concentration, the influence of different fermentation temperatures on the vanillin production of the enterobacter cholerae is explored. As shown in FIG. 23, the vanillin content tended to rise in the fermentation temperature range of 29℃to 33℃from 1470.7mg/L to 3145.0mg/L. When the fermentation temperature is above 33 ℃, the vanillin content generally tends to decrease, which means that too high temperature can destroy the normal metabolic environment of the thalli, and thus, the fermentation temperature is about 33 ℃, the vanillin content is highest, and the fermentation temperature can be selected to be 29-37 ℃ for examining the temperature.

TABLE 3-4 influence of different fermentation temperatures on Vanillin production by fermentation of Enterobacter cholerae

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5. Design and results of BOX-Behenken test

The response surface intuitively sees the influence of each factor on the vanillin production of the escherichia coli, and the theoretical values of different factors are calculated through a regression equation, so that the method is more practical to use. The following test schemes and results are shown in tables 3 to 5, which were obtained by analyzing 3 factors of pH, inoculum size, fermentation temperature by the response surface method.

Tables 3-5 response surface test design and results

The data from tables 3-5 were analyzed by Design-Expert 8.0.5 software and the results are shown in tables 3-6. As can be seen from tables 3 to 6: the pH has very remarkable influence on the vanillin content, the inoculum size has more remarkable influence on the vanillin content, and the fermentation temperature has relatively remarkable influence on the vanillin content. And (3) taking the vanillin content as a response value, and obtaining a regression equation after regression fitting:

vanillin content

＝6577.20+483.38A+105.75B+168.88C+43.00AB+27.25AC-39.50BC-573.98A ² -1180.73B ² -686.47 C ²

Tables 3-6 analysis of variance

According to the square difference results in tables 3-6, the regression equation describing the vanillin content describes a significant model of the relationship with the response values (P < 0.0002) and the mismatch term test is not significant (P > 0.7669), indicating that the test model adequately fits the test data, i.e., the regression equation can be used to determine the optimal fermentation conditions for vanillin production.

6. Response surface analysis for two-factor interactions

The response surface graph is a curved surface graph of a three-dimensional space formed by response values and each test factor, and the optimal parameters and interaction among the parameters can be visually seen from the response surface analysis graph. Response surface analysis charts of different factors are obtained according to regression equations of vanillin content, and the results are shown in fig. 24-29. The effect of the interaction of the factors on the vanillin content can be seen more intuitively from fig. 24-29, which shows that the steeper the curve, the greater the effect of the factor on the vanillin content. From FIGS. 24-29, it can be seen that pH has the greatest effect on vanillin content, and that fermentation temperature is secondary, consistent with the analysis of variance results of tables 3-6.

7. Verification experiment

Analysis by software determines that the optimal fermentation condition for producing vanillin by fermentation is pH=10.85, inoculum size of 2.05wt% and fermentation temperature of 33.52 ℃, and the theoretical value calculated by a formula under the condition is 6.69381g/L. Because the actual conditions of production conditions are required to be considered, the optimal theoretical value is adjusted to be: ph=10.8, inoculum size 2wt% and fermentation temperature 33 ℃. Three groups of verification experiments are carried out according to the obtained analysis data, the conditions of the verification experiments are that the pH=10.8, the inoculum size is 2wt% and the fermentation temperature is 33 ℃, the obtained vanillin content is 6.6924g/L, the measurement result is stable, the deviation is in a reasonable range, and the result is proved to be reasonable and reliable. Meanwhile, the higher the vanillin yield in the reaction system, the higher the tolerance of the strain to vanillin. The vanillin content obtained under the optimal condition in the invention is up to 6.6924g/L, which indicates that the enterobacter cholerae (DQ) has excellent tolerance to vanillin.

The foregoing is merely illustrative and explanatory of the invention as it is claimed, as modifications and additions may be made to, or similar to, the particular embodiments described, without the benefit of the inventors' inventive effort, and as alternatives to those of skill in the art, which remain within the scope of this patent.

Claims (3)

1. Enterobacter cholerae @Enterobacter hormaechei) The use of said E.cholerae in the production of vanillin, characterized in that said E.cholerae is namedEnterobacter hormaecheiDQ, the DQ is preserved in China general microbiological culture collection center (CGMCC) with a preservation date of 2021, 07 and 26 days and a preservation number of CGMCC No.22958; enterobacter choleraeEnterobacter hormaecheiDQ takes trans-ferulic acid as a substrate, and vanillin is produced by a microbial transformation method; the specific parameters in application are as follows: the pH is 9-11, the inoculation amount is 1.5-3 wt%, and the fermentation temperature is 30-35 ℃.

2. The enterobacter cholerae of claim 1Enterobacter hormaecheiThe application of DQ in the production of vanillin is characterized in that the specific parameters during the application are as follows: the pH was 10.8, the inoculum size was 2wt%, and the fermentation temperature was 33 ℃.

3. A preparation for improving the production efficiency of vanillin, which is characterized in that the effective component of the preparation is the enterobacter cholerae according to claim 1Enterobacter hormaechei DQ。

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