Pseudomonas fluorescens aw10 and application thereof

Abstract

The application discloses Pseudomonas fluorescens Aw10 and application thereof, the strain Aw10 is preserved in China typical culture preservation center, the preservation number is CCTCC NO.M 20221544, and the preservation address is Wuhan, Wuhan University, China. Through fermentation medium optimization on the strain, the strain shake flask fermentation ferulic acid esterase enzyme activity is 73.5 U / L under the optimized condition, which is about 2 times higher than that of the strain before optimization. The strain has the advantages of high ferulic acid esterase yield and high temperature resistance, and can be applied to liquor brewing production, especially the application aspect of daqu production process, which helps to improve the content of ferulic acid in liquor, has a positive influence on the health attribute of liquor, and improves liquor body and flavor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to Pseudomonas fluorescens Aw10 and application thereof. BACKGROUND

[0002] The wine starter culture brewing wine is a brewing technology invented by China. The starter culture is a saccharification and fermentation agent for liquor brewing, and is referred to as the bone of liquor. High-quality starter culture is an important prerequisite for producing high-quality liquor. High-temperature starter culture, medium-temperature starter culture and low-temperature starter culture refer to starter culture with a bacteria cultivation temperature greater than 60 DEG C, between 50 DEG C and 60 DEG C and less than 50 DEG C, respectively. The bacteria in the finished product of high-temperature starter culture are dominant because of the excessively high bacteria cultivation temperature, and these bacteria are mostly Bacillus. The bacteria cultivation temperature of low-temperature starter culture is relatively low, so the microorganism species are relatively more, and include various yeast, mold and bacteria. The medium-temperature starter culture is mostly brick-shaped, and is suitable for the growth of saccharification bacteria because most raw materials are starch raw materials.

[0003] Ferulic acid is an important phenolic acid substance existing in plants. In cereals, most of the ferulic acid is connected with arabinoxylan by ester bond in addition to a small amount of free form, and has strong antioxidant activity and preservative effect, and is widely used in medicine, pesticide, health care product, cosmetic raw material and food additive and the like. Ferulic acid not only can quench free radicals, but also can regulate human physiological functions, inhibit enzymes producing free radicals and promote enzymes clearing free radicals. Ferulic acid and its derivatives have biological activities such as anti-blood, reducing blood fat, anti-inflammatory and anticancer. Ferulic acid is safe and non-toxic, and is easy to be metabolized by human body, and can be applied to the food industry as an antioxidant, function promoting substance and preservative. Japan and other countries have approved it as a food additive.

[0004] Ferulic acid esterase, also known as cinnamic acid esterase, is a sub-class of carboxylic acid esterases, and can catalyze the hydrolysis of cross-linking structures between ferulic acid and lignin, polysaccharide, cellulose and the like to obtain free phenolic acid substances such as ferulic acid and ferulic acid dimer. Ferulic acid esterase can catalyze the hydrolysis of ester bonds between ferulic acid and polysaccharide in plant cell walls to release free phenolic acid substances such as ferulic acid and ferulic acid dimer. Ferulic acid esterase causes the destruction of the complex network structure of plant cell walls, so that other hydrolytic enzymes can more easily approach the active center of the substrate, thereby improving the degradation efficiency of plant cell walls. Ferulic acid esterase has wide application value in many fields such as food, feed, medicine, papermaking and textile, and its application in liquor starter culture production can improve the degradation efficiency of raw material cell walls in the starter culture, so that the microbial conversion reaction in the starter culture is more thorough.

[0005] At present, the patent of the comparative file (CN110066757B) discloses a kind of pseudomonas that can produce ferulic acid esterase from wheat koji, and is applied to the brewing of yellow rice wine by preparing reinforced wheat koji.The comparative file (CN113308424A) discloses that the short bacillus ferulic acid esterase-producing bacteria can produce ferulic acid esterase, and the enzyme activity can still reach more than 80% at 45-60 DEG C.However, the above prior art does not mention whether the strain is still suitable for growth and aroma production during the process of liquor brewing, and neither does it mention the flavor produced by the strain under solid-state conditions, nor does it mention the optimization and application of the ferulic acid esterase-producing strain during the process of liquor brewing. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a kind of Pseudomonas fluorescens Aw10 and application.The present application is realized by the following technical solutions:

[0007] The first purpose of the present application is to provide a kind of Pseudomonas fluorescens Aw10, which is preserved in China Center for Type Culture Collection, with the preservation number CCTCC NO.M 20221544 and the preservation date October 10, 2022.

[0008] Preferably, the Aw10 temperature tolerance is 30-65 DEG C, and it grows well at 30-55 DEG C, and it grows and reproduces fastest at 35 DEG C.

[0009] The second purpose of the present application is to provide the Pseudomonas fluorescens Aw10 with the following separation and screening steps:

[0010] I) enrichment: use five-point sampling method to load the bulk koji into a crusher to crush it into powder, weigh the koji powder and add it into a conical flask containing glass beads and physiological saline, place it in a shaking bed and shake for 1 h, then take the suspension and add it into an enrichment medium and culture for 18 h.

[0011] The enrichment medium contains 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 1.5 g / L of ammonium sulfate and 1 g / L of magnesium sulfate heptahydrate.

[0012] II) separation and screening of high-yield ferulic acid esterase-producing strain

[0013] 1) separation and preliminary screening: in an ultraclean bench sterilized by ultraviolet radiation, shake the enriched bacterial solution, take the bacterial suspension into a sterile centrifuge tube, add sterile physiological saline for gradient dilution, and obtain gradient-diluted bacterial solution.Taking 3 different gradient-diluted bacterial solutions, spread them on separation medium for culture, and observe the colony situation and the size of the transparent circle on the plate.

[0014] The separation culture medium is as follows: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, 20 g of agar powder, and 15 mL of ethyl ferulate (15% W / V solution in N, N-dimethylformamide) is added when the temperature is cooled to 70 DEG C.

[0015] 2) Rescreening: a, the fluorescent pseudomonas strains obtained in step 1) are inoculated in centrifuge tubes containing seed culture medium and cultured for 22 h, b, inoculated in fermentation culture medium and cultured for 72 h, the fermentation is ended, the supernatant is obtained by centrifugation, and the filter membrane is put into a sample bottle, the fermentation liquor is determined by high performance liquid chromatography, the strain producing ferulate esterase is obtained, and the strain is preserved in a glycerol tube.

[0016] The seed culture medium is as follows: 2 g / L of sodium nitrate, 1 g / L of potassium phosphate dibasic, 0.5 g / L of potassium chloride, 0.5 g / L of magnesium sulfate heptahydrate, 0.01 g / L of ferrous sulfate heptahydrate, and 15 mL of ethyl ferulate (15% W / V solution in N, N-dimethylformamide) is added when the temperature is cooled to 70 DEG C.

[0017] The fermentation culture medium is as follows: 10 g / L of wheat bran, 1 g / L of yeast extract, 0.5 g / L of potassium chloride, 0.5 g / L of magnesium sulfate heptahydrate, and 0.1 g / L of potassium phosphate dibasic.

[0018] A third object of the present application is to provide the application of the fluorescent pseudomonas Aw10 in the fermentation production of ferulic acid.

[0019] A fourth object of the present application is to provide the application of the fluorescent pseudomonas Aw10 in the fermentation production of ferulic acid esterase.

[0020] The fermentation conditions for the fermentation production of ferulic acid esterase by the fluorescent pseudomonas Aw10 are as follows: pH 4.0-9.0, temperature 24-40 DEG C, inoculation amount 4-12%, rotation speed 140-220 r / min, 10-50 g / L of glucose as the carbon source in the fermentation culture medium, 30-70 g / L of yeast extract as the nitrogen source, 10-40 g / L of crushed wheat bran as the inducer, 0.05 g / L of KCl, 0.05 g / L of MgSO4·7H2O, and 0.1 g / L of K2HPO4.

[0021] Preferably, the fermentation conditions for the fermentation production of ferulic acid esterase by the fluorescent pseudomonas Aw10 are as follows: pH 6.0, temperature 30 DEG C, inoculation amount 6%, rotation speed 200 r / min, and the fermentation culture medium is as follows: 30 g / L of glucose, 60 g / L of yeast extract, 30 g / L of crushed wheat bran, 0.05 g / L of KCl, 0.05 g / L of MgSO4·7H2O, and 0.1 g / L of K2HPO4.

[0022] The fifth object of the present application is to provide the application of the Pseudomonas fluorescens Aw10 in solid-state fermentation, and the strain also produces flavor substances such as cyclobutanol, 2,3,5,6-tetramethylpyrazine, 2,3-butanedione, butyl isobutyrate, hexanoic acid and phenol in solid-state fermentation.

[0023] DEPOSIT DESCRIPTION

[0024] DEPOSIT ADDRESS: Wuhan, Wuhan University, China

[0025] DEPOSIT DATE: October 10, 2022

[0026] STRAIN NAME: Pseudomonas fluorescens

[0027] LATIN NAME: Pseudomonas fluorescens

[0028] STRAIN NUMBER: Aw10

[0029] DEPOSITING INSTITUTION: China Center for Type Culture Collection

[0030] DEPOSITING INSTITUTION ABBREVIATION: CCTCC

[0031] DEPOSIT CENTER REGISTRATION NUMBER: CCTCC NO.M 20221554

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1) The Aw10 is obtained by screening from Daqu, and has the characteristics of high temperature resistance due to long-term domestication of the growth environment, which is very beneficial to the later metabolic engineering modification of high yield of ferulic acid;

[0034] 2) Aw10 can grow at a fermentation temperature of 50 DEG C, and can be used in the application of Daqu production process in liquor brewing, to improve the content of ferulic acid in liquor and increase the health of liquor;

[0035] 3) The application of Aw10 in Daqu production process can improve the content of flavor substances such as cyclobutanol, 2,3,5,6-tetramethylpyrazine, 2,3-butanedione, butyl isobutyrate, hexanoic acid and phenol in liquor, and improve the taste and flavor;

[0036] 4) The strain has the advantages of high ferulic acid esterase enzyme activity and high temperature resistance, and can be used for the preparation of Daqu to improve the quality of liquor;

[0037] 5) Through the optimization of fermentation conditions of Aw10, the results show that the optimal fermentation conditions are: pH 6.0, temperature 30 DEG C, inoculation amount 6%, rotation speed 200r / min, fermentation medium: glucose 30g / L, yeast extract 60g / L, crushed wheat bran 30g / L, KCl 0.05g / L, MgSO4.7H2O 0.05g / L, K2HPO4 0.1g / L. Under the condition, the enzyme activity of strain shake flask fermentation of ferulic acid esterase is 73.5U / L, compared with the enzyme activity of 25.5U / L of the strain before optimization, it is increased by about 2 times.

[0038] 6) Since the chemical synthesis method has high preparation cost, is not environmental protection, the plant enrichment method has complex extraction process and low yield, the present application adopts the microbial fermentation method to obtain high yield ferulic acid esterase, and has the characteristics of safety, green, high efficiency, sustainability and the like. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0040] Figure 1 is the colony screening result of Aw10 in the embodiments of the present application;

[0041] Figure 2 is the initial enzyme activity comparison chart of Aw10 in the embodiments of the present application;

[0042] Figure 3 is the phylogenetic tree chart of Aw10 in the embodiments of the present application;

[0043] Figure 4 is the growth curve of Aw10 in the embodiments of the present application;

[0044] Figure 5 is the morphological feature actuality chart of Aw10 in the embodiments of the present application, a is the strain transparent circle, b is the gram staining result, and c is the scanning electron microscope chart;

[0045] Figure 6 is the alcohol tolerance effect chart of Aw10 in the embodiments of the present application;

[0046] Figure 7 is the temperature tolerance effect chart of Aw10 in the embodiments of the present application;

[0047] Figure 8 is the ferulic acid standard curve in the embodiments of the present application;

[0048] Figure 9 is the chromatogram of Aw10 enzyme reaction solution (upper) and ferulic acid standard (lower) in the embodiment of the present application;

[0049] Figure 10 is the single factor experiment result effect diagram of fermentation enzyme production of Aw10 in the embodiment of the present application, a is pH, b is temperature, c is inoculum amount, d is rotation speed, e is carbon source type, f is nitrogen source type, and g is inducer type;

[0050] Figure 11 is the effect of different concentrations of glucose on enzyme production of Aw10 in the embodiment of the present application;

[0051] Figure 12 is the effect of different concentrations of yeast extract on enzyme production of Aw10 in the embodiment of the present application;

[0052] Figure 13 is the effect of different concentrations of crushed wheat bran on enzyme production of Aw10 in the embodiment of the present application;

[0053] Figure 14 is the effect diagram of the interaction between factors in response surface analysis on the enzyme activity of ferulic acid esterase in the embodiment of the present application, a is glucose and yeast extract, b is glucose and crushed wheat bran, and c is yeast extract and crushed wheat bran. DETAILED DESCRIPTION

[0054] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the following will be further described in detail in combination with the accompanying drawings and embodiments 1-5. Figures 1-14 and embodiments 1-5.

[0055] Embodiment 1

[0056] Separation and screening of Aw10

[0057] I) Enrichment

[0058] The large lump of Daqu was ground into powder by a stirring crusher, 10.0 g of Daqu powder was weighed and added to a conical flask containing 100 mL of sterile normal saline with glass beads, which was placed in a 37℃, 180r / min shaking bed for 1h, and then 2 mL of the suspension was taken and added to the enrichment medium for 18h of culture to obtain a bacterial suspension.

[0059] Five-point sampling method: first determine the midpoint of the diagonal line of the Daqu lump as the center sampling point, and then select four points on the diagonal line with equal distance from the center sampling point as the sampling points;

[0060] In 1L of enrichment medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1.5 g of ammonium sulfate, and 1.0 g of magnesium sulfate heptahydrate.

[0061] II) Isolation and screening of high-yielding ferulic acid esterase strain Aw10

[0062] 1) Isolation and initial screening: In a clean bench sterilized with ultraviolet light, shake the bacterial suspension obtained in step 1) and pipette 0.1 mL into a sterile 1.5 mL centrifuge tube. Add 0.9 mL of sterile saline for gradient dilution to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 Take 10 times diluted bacterial solution. -3 , 10 -4 , 10 -5 0.2 mL of each of the three gradient dilutions was applied to the separation medium with ethyl ferulate as the carbon source. Single colonies with transparent circles were picked and streaked on the screening medium. The culture was then placed in a 30°C water-tight incubator until a single colony grew on the plate. This took about 1-3 days. The results showed that 54 strains were obtained in the initial screening, of which 5 strains produced transparent circles. Figure 1 As shown, they are A4, A5, D7, W10 and W11 respectively.

[0063] To 1 L of separation medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 20 g of agar powder, cool to 70°C and add 15 mL of ethyl ferulate (15% W / V dissolved in N,N-dimethylformamide solution).

[0064] 2) Rescreening: a. Inoculate the 5 strains that produced transparent circles obtained in the initial screening of step 1) into 8 mL of seed culture medium in a 15 mL centrifuge tube and culture in a constant temperature shaker at 30°C and 180 r / min for 20 h to obtain seed liquid. b. Inoculate 4% of the inoculum into 100 mL of fermentation medium and culture at 30°C and 180 r / min for 72 h. Then terminate the fermentation. Set up three replicates for each group and a blank control. Take 30 mL of the fermentation broth in a 50 mL centrifuge tube and centrifuge at 12000 r / min for 10 min. Take 1 mL of the supernatant and filter it through a 0.22 μm aqueous needle filter into a sample bottle. Determine the ferulic acid content in the fermentation broth by high performance liquid chromatography. Rescreen the fermentation by shaking flask fermentation. Figure 2 As shown, strain W10 had higher enzyme activity, and was named Aw10. The strain was preserved in a glycerol tube and Aw10 was used as the starting strain in subsequent experiments.

[0065] To 1L seed culture medium: 2g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, cooled to 70°C, add 15mL ethyl ferulate (15% w / v dissolved in N,N-dimethylformamide)

[0066] 1L fermentation medium: wheat bran 10 g, yeast extract 1 g, potassium chloride 0.05 g, magnesium sulfate heptahydrate 0.05 g, potassium phosphate dibasic 0.10 g.

[0067] Example 2

[0068] Molecular biology identification

[0069] I) Identification: morphological observation and identification preliminary judgment as gram-negative short rod-shaped bacteria, 16S rDNA molecular biology identification sequencing results and NCBI database were compared by BLAST, and MEGA-X software was used to construct phylogenetic tree based on Neighbor-Joining algorithm, and the strain was determined as Pseudomonas fluorescens Aw10 and named as Pseudomonas fluorescens Aw10, see Figure 3 .

[0070] II) Growth curve of Aw10

[0071] During the process of shake flask culture, the seed liquid was taken every 2h to measure OD 600nm value. The test results showed that the growth was slow before 6h of culture, entered logarithmic growth phase after 6h, was in stable growth phase at 18-24h, and entered decline phase after 24h, see Figure 4 .

[0072] III) Morphology and physiological and biochemical identification of Aw10

[0073] After colony morphology observation, the colony was yellow, the surface was wet and raised, the edge was irregular, the shape was relatively regular, the viscosity was low, and the colony was easily picked up. The colony was opaque, see 5a. After gram staining, the observation by optical microscope showed red, which was gram-negative bacteria, see Figure 5 b. It can be known from the electron microscope photograph that the bacteria were short rod-shaped, see Figure 5 c.

[0074] The physiological and biochemical results of the strain are shown in Table 1. The indole experiment and peroxidase test of the bacteria were positive, the methyl red test, VP test and starch hydrolysis test were negative, and the bacteria could utilize glucose, sucrose, sorbitol and xylitol to produce acid and gas, and could not utilize D-arabinose, lactose and cellulose disaccharide.

[0075] Table 1 physiological and biochemical characteristics of the strain

[0076]

[0077]

[0078] Note: “+” indicates that the reaction result is positive; “-” indicates that the reaction result is negative.

[0079] IV) Temperature tolerance

[0080] As shown in Figure 6 , with the increase of temperature, OD 600nm gradually increased, when 30-55℃, the strain normal growth, the optimum growth temperature was 35℃, showed that the strain can grow normally in the conditions of high temperature, with good high temperature characteristics.

[0081] V) Alcohol tolerance

[0082] As shown in Figure 7 , with the increase of alcohol content, OD 600nm gradually decreased, showed that the strain growth gradually deteriorated, when the alcohol content reached 6%, OD 600nm tended to 0, it can be known that the maximum tolerance of the strain alcohol content was 6%.

[0083] Example 3

[0084] Determination of ferulic acid content and ferulic acid esterase activity in Aw10 fermentation broth

[0085] I) Preparation of ferulic acid standard curve

[0086] 1.0 mg of ferulic acid (chromatographically pure grade) was weighed and dissolved in 50% methanol to 10 mL to obtain 0.1 g / L of ferulic acid standard solution. Then 0.1 g / L of ferulic acid standard solution was diluted to obtain 0.04 g / L, 0.03 g / L, 0.02 g / L, 0.01 g / L, 0.007 g / L, 0.004 g / L, 0.001 g / L of ferulic acid standard solution, and the peak area was determined by high performance liquid chromatograph. The standard curve was drawn with the standard solution concentration (g / L) as the abscissa and the peak area as the ordinate, and the linear regression equation was made. As shown in Figure 8 , the regression equation y = 37550x + 19.534, the correlation coefficient R 2 = 0.9998, indicating a good linear relationship.

[0087] II) Determination of ferulic acid content

[0088] A single colony of Aw10 from the separation medium was inoculated into a 15 mL centrifuge tube containing 8 mL of seed medium and incubated at 30°C, 180 r / min in a constant temperature shaker for 20 h to obtain a seed solution. Then, 4% of the seed solution was inoculated into 100 mL of fermentation medium and incubated at 30°C, 180 r / min in a constant temperature shaker for 72 h. After fermentation, the fermentation broth was obtained. Then, 30 mL of the fermentation broth was centrifuged at 12000 r / min for 10 min. Then, 1 mL of the supernatant was filtered through a 0.22 μm water needle filter and subjected to high performance liquid chromatography detection analysis. The medium without inoculation was used as a blank control. The content of ferulic acid in the fermentation medium inoculated with Aw10 was measured to be 559.34 ± 0.58 ng / L.

[0089] III) Determination of the enzyme activity of ferulic acid esterase

[0090] 1) Preparation of the fermentation broth of Aw10: a single colony of Aw10 from the separation medium was inoculated into a 15 mL centrifuge tube containing 8 mL of seed medium and incubated at 30°C, 180 r / min in a constant temperature shaker for 20 h to obtain a seed solution. Then, 4% of the seed solution was inoculated into 100 mL of fermentation medium and incubated at 30°C, 180 r / min in a constant temperature shaker for 72 h. After fermentation, the fermentation broth was obtained.

[0091] 2) Preparation of the crude enzyme solution: 1 mL of the fermentation broth in step 1) was centrifuged at 10000 r / min for 15 min. The supernatant was used as the crude enzyme solution.

[0092] 3) Enzyme reaction test: 0.25 mL of the crude enzyme solution in step 2) was immediately added with 0.75 mL of a 0.003 mol / L methyl ferulate solution (dissolved in a 0.05 mol / L Tris-HCl solution) to form a 1 mL enzyme reaction system. Then, the enzyme reaction system was placed in a 50°C constant temperature water bath for reaction for 15 min. After the reaction, the enzyme reaction system was immediately placed in a boiling water bath for inactivation for 10 min to terminate the enzyme reaction. The enzyme reaction solution was obtained. An equal amount of Tris-HCl buffer was used to replace the crude enzyme solution as a blank control. After the enzyme reaction solution was cooled to room temperature, it was centrifuged at 12000 r / min for 10 min. Then, the enzyme reaction solution was filtered through a 0.22 μm water needle filter and subjected to high performance liquid chromatography detection analysis. The results are shown in Figure 9 The chromatogram of the enzyme reaction solution (top) and the chromatogram of the ferulic acid standard (bottom) both showed that ferulic acid was eluted at about 14 min, which proved that the enzyme reaction solution contained ferulic acid esterase. The enzyme activity was determined to be 25.5 U / L, and the enzyme could metabolize methyl ferulate to generate ferulic acid.

[0093] Definition of enzyme activity: 1 unit (1 U) of enzyme activity is the amount of enzyme required to hydrolyze 1 μmol of methyl ferulate to generate ferulic acid per minute under the conditions of 30°C and natural pH.

[0094] Example 4

[0095] Fermentation condition optimization of high-yield ferulic acid esterase strain

[0096] I) Single factor experiment

[0097] Select pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, fermentation temperature 24, 27, 30, 33, 37, 40℃, inoculation amount 4, 6, 8, 10, 12%, rotation speed 140, 160, 180, 200, 220r / min, 10g / L sucrose, glucose, maltose, lactose, fructose, xylan, soluble starch as carbon source, 10g / L peptone, yeast extract, urea, sodium nitrate, ammonium nitrate, ammonium sulfate, soybean as nitrogen source, 5g / L wheat bran, crushed wheat bran, starch-free wheat bran, corn meal as inducer, etc. 7 factors do single factor experiment, each group do 3 parallel.

[0098] From Figure 10 a can be known, the initial pH of the fermentation medium is 6.0, the enzyme activity of ferulic acid esterase reaches the maximum value, with the increase of pH, the enzyme activity decreases, which may be related to the domestication of acidic environment of Daqu and the adaptability of the strain, so Aw10 has strong growth and reproduction and ferulic acid esterase accumulation ability at pH 6.0.

[0099] From Figure 10 b can be known, when the fermentation temperature rises from 24℃ to 30℃, the enzyme activity increases, and when the temperature is 30℃, the enzyme activity reaches the maximum value, which may be because Aw10 is most suitable for its growth and reproduction at 30℃, and its key enzyme activity also increases. Subsequently, the enzyme activity decreases with the increase of temperature, it is speculated that the metabolic enzyme of Aw10 is inhibited at high temperature.

[0100] From Figure 10 c can be known, the enzyme activity of ferulic acid esterase increases with the increase of inoculation amount, when the inoculation amount is 6%, the enzyme activity of ferulic acid esterase reaches the maximum, and then decreases, which is because too large inoculation amount causes high cell concentration in the early stage of fermentation, and then the substrate is consumed in large quantities, causing insufficient supply of nutrients in the product synthesis stage.

[0101] From Figure 10 d can be known, the enzyme activity of ferulic acid esterase increases with the increase of rotation speed, when the rotation speed is 200r / min, the enzyme activity of ferulic acid esterase reaches the maximum value, and then begins to decrease. The accumulation of bacteria needs a lot of oxygen, low rotation speed is not conducive to the growth and reproduction of bacteria, but too high rotation speed will accelerate the decline of bacteria.

[0102] From Figure 10As shown in e-g, the enzyme activity reached a higher value when glucose was used as carbon source, followed by fructose, lactose, maltose and sucrose, and soluble starch and xylanase activity was lower; among different nitrogen sources, the enzyme production effect of yeast extract was better, followed by peptone, soybean and urea, and sodium nitrate and ammonium nitrate were poorer; since ferulic acid esterase is an induced enzyme, wheat bran contains ferulic acid ester bond which can induce the production of ferulic acid esterase, thereby improving enzyme activity. Among different inducer species, the enzyme production effect of crushed wheat bran was better, followed by de-starch bran and wheat bran, and corn flour was poorer.

[0103] As Figure 10 As shown in a-g, the single factor test results showed that the optimal fermentation conditions were pH 6.0, temperature 30℃, inoculum size 6%, rotation speed 200r / min, carbon source glucose, nitrogen source yeast extract, and inducer crushed wheat bran.

[0104] II) Steepest ascent experiment

[0105] The optimal carbon source concentration was set to 10, 20, 30, 40, 50 g / L, the nitrogen source concentration was set to 30, 40, 50, 60, 70 g / L, and the inducer concentration was set to 10, 20, 30, 40 g / L, to study the effects of different concentrations of carbon source, nitrogen source and inducer on enzyme production, as shown in Figures 11-13 The best enzyme production effect was obtained when the glucose concentration was 30 g / L, the yeast extract concentration was 50 g / L, and the crushed wheat bran concentration was 30 g / L.

[0106] III) Response surface test design

[0107] Box-Behnken Design response surface test was designed using software Design Expert 8.0.6. According to the results of the climbing test, glucose, yeast extract and crushed wheat bran were used as variables, and enzyme activity production was used as response value, to conduct a 3-factor 3-level response surface analysis test.

[0108] 1) Establishment and analysis of quadratic response surface regression model

[0109] A response surface analysis test of 3 factors and 3 levels (total of 17 tests) was conducted for A (glucose), B (yeast extract) and C (crushed wheat bran). The response surface factor level coding is shown in Table 2, and the response surface result analysis is shown in Table 3. Through multiple quadratic regression fitting, a ferulic acid esterase fermentation condition optimization model was established, and the regression equation was: Y = 78.60 + 0.71A + 2.78B + 2.01C - 0.80AB + 0.075AC + 1.65BC - 6.61A 2 -13.59B 2 -6.06C 2The regression model variance analysis table 4 shows that the model p<0.01, reaches the extremely significant level, indicating that the predicted value of the model is very consistent with the actual value. The p of the misfit term is 0.0845>0.05, and the difference of the misfit term is not significant, proving that the model has good fitting degree with the experiment, and has statistical significance. The primary items A, B and C in the model have extremely significant effect on the enzyme activity, and the primary and secondary order of the influence factors of the primary items is C>B>A. The interaction of A, B and C in the fermentation process is shown in the figure, and the primary and secondary relationship of the mutual influence between the three factors is AB>AC>BC.

[0110] Table 2 response surface factor level coding

[0111]

[0112] Table 3 response surface experiment result analysis

[0113]

[0114] Table 4 regression model variance analysis

[0115]

[0116]

[0117] Note: significant difference (P<0.05), extremely significant difference (P<0.01)

[0118] 2) Analysis of the influence of the interaction of each factor on the ferulic acid esterase activity

[0119] The response surface graph can directly reflect the influence of the test factors on the ferulic acid esterase activity, and the steeper the response surface slope, the more sensitive the response surface value to the change of the operating conditions. The interaction of glucose, yeast extract and crushed wheat bran in the fermentation process is shown in the figure Figure 14 , and the primary and secondary relationship of the mutual influence between the three factors is AB>BC>AC.

[0120] According to the response surface experiment result, the optimal fermentation conditions for the fermentation production of ferulic acid esterase by Aw10 are that the addition amount of carbon source, nitrogen source and inducer is respectively: glucose 30 g / L, yeast extract 60 g / L, and crushed wheat bran 30 g / L.

[0121] In summary, the optimal fermentation conditions for the fermentation production of ferulic acid esterase by Aw10 are as follows: inoculum amount 60 mL / L, rotation speed 200 r / min, pH 6.0, temperature 30 ℃, glucose 30 g / L, yeast extract 60 g / L, crushed wheat bran 30 g / L, KCl 0.05 g / L, MgSO4·7H2O 0.05 g / L, and K2HPO4 0.10 g / L. Under the above conditions, the maximum enzyme activity of the strain Aw10 in shake flask fermentation is predicted to be 76.8 U / L. Under the above conditions, the average yield of ferulic acid esterase obtained by repeating the fermentation experiment for three times is 73.5 U / L. Comprehensive analysis shows that the fermentation experiment result is very close to the result of the response surface experiment, and the enzyme activity of the strain before optimization is 25.5 U / L, which is increased by about 2 times.

[0122] Example 5

[0123] Comparison test of Aw10 fermentation

[0124] The Aw10 is subjected to solid-state fermentation of wheat, and the solid-state fermentation of wheat without inoculation of the bacteria is used as a control group. As shown in Table 5, the Aw10 produces flavor substances such as 2,3,5,6-tetramethylpyrazine, 2,3-butanedione, 2-methyl butyl butyrate, and isobutyl butyrate in the solid-state fermentation, as compared with the control group. The 2,3,5,6-tetramethylpyrazine has the aroma of nuts, toasted bread, roasted peanuts, hazelnuts, and cocoa beans; the 2,3-butanedione has the aroma of butter, fermentation, and milk fat; the 2-methyl butyl butyrate has the fruity aroma of apples; and the isobutyl butyrate has the strong fruity aroma of fresh apples and pineapples, etc., which shows that the strain has the application value in improving the taste and flavor of liquor in the production of Daqu.

[0125] Table 5 Comparison of flavor substances in the solid-state fermentation of Pseudomonas fluorescens Aw10

[0126]

[0127]

[0128] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A fluorescent Pseudomonas ( Pseudomonas fluorescens ) Aw10, characterized in that The Pseudomonas fluorescens Aw10 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.M 20221554, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the Pseudomonas fluorescens Aw10 according to claim 1 in the fermentation production of ferulic acid.

3. Use of the Pseudomonas fluorescens Aw10 according to claim 1 in the fermentation production of ferulic acid esterase.

4. The use according to claim 3, characterized in that: The fermentation conditions for producing ferulic acid esterase by fermenting Pseudomonas fluorescens Aw10 are as follows: pH 4.0-9.0, temperature 24-40°C, inoculation amount 4-12%, rotation speed 140-220 r / min, 10-50 g / L of glucose as a carbon source, 30-70 g / L of yeast extract as a nitrogen source, 10-40 g / L of crushed wheat bran as an inducer, 0.05 g / L of KCl, 0.05 g / L of MgSO4·7H2O, and 0.1 g / L of K2HPO4 in the fermentation medium.

5. The use according to claim 3 or 4, characterized in that: The fermentation conditions for producing ferulic acid esterase by fermenting Pseudomonas fluorescens Aw10 are as follows: pH 6.0, temperature 30°C, inoculum size 6%, rotation speed 200 r / min, and fermentation medium: 30 g / L glucose, 60 g / L yeast extract, 30 g / L crushed wheat bran, 0.05 g / L KCl, 0.05 g / L MgSO4·7H2O, and 0.1 g / L K2HPO4.

6. A use of the Pseudomonas fluorescens Aw10 according to claim 1 in the production of Daqu, characterized in that: The Pseudomonas fluorescens Aw10 also produces cyclobutanol, 2,3,5,6-tetramethylpyrazine, 2,3-butanedione, butyl isobutyrate, hexanoic acid, and phenol during solid-state fermentation.

Images