CN103451133B - Bacillus circulans and application for same in preparation for ferulic acid decarboxylase - Google Patents
Bacillus circulans and application for same in preparation for ferulic acid decarboxylase Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly discloses bacillus circulans and an application of the same in preparation for ferulic acid decarboxylase. The bacillus circulans is characterized in that a bacillus circulans HG12 strain is obtained by separating from a high-temperature yeast for brewing Maotai-flavour liquor in the white wine plants of China, and screening by virtue of ultraviolet mutation, as well as has the characteristics of being fast in growth and propagation speeds, capable of producing the ferulic acid decarboxylase under the induction of ferulic acid, stable in performance of producing the ferulic acid decarboxylase, and high in enzymatic activity. According to the bacillus circulans and the application disclosed by the invention, continuous and large-scale production for the ferulic acid decarboxylase can be realized, and industrialized production for the ferulic acid decarboxylase is facilitated; moreover, the enzyme is simple and convenient in production process, short in production cycle, and low in cost.
Description
(1) technical field
The invention belongs to technical field of bioengineering, particularly a kind of Bacillus circulans and preparing the application in ferulic acid decarboxylase.
(2) background technology
Ferulic acid decarboxylase (Ferulic acid decarboxyase, FAD) is the enzyme that catalysis forulic acid generation decarboxylic reaction produces 4-vinyl guaiacol.4-vinyl guaiacol is a kind of phenol compounds material that can produce peat-reek or cloves class fragrance, it can improve the organoleptic quality of food, the natural fragrance of the food such as drinks, soy sauce, cheese, beverage is had a significant impact, therefore, ferulic acid decarboxylase has good development prospect for food service industry.
Research shows, ferulic acid decarboxylase is present in many fungies, yeast and part bacterium, as bacillus pumilis (
bacillus pumilus), plant lactobacillus (
lactobacillus plantarum), subtilis (
bacillus subtilis), yeast saccharomyces cerevisiae (
saccharomycescerevisiae) etc., but the ferulic acid decarboxylase that mentioned microorganism produces is all different on aminoacid sequence, peptide chain structure, physico-chemical property and catalysis characteristics, and to produce the enzyme activity of ferulic acid decarboxylase not high yet.Therefore there is researchist to adopt genetic engineering technique to carry out breeding research to ferulic acid decarboxylase producing strains, construct ferulic acid decarboxylase genetic engineering bacterium, as Gu Wen (Yunnan University's Ph D dissertation, 2011) is carrying out bacterium
enterobacter sp.Px6-4the gene clone of ferulic acid decarboxylase, expression, crystallographic structural analysis and catalyst mechanism research time, the coding gene sequence of this enzyme clone is carried out abduction delivering in intestinal bacteria, is then obtained the ferulic acid decarboxylase of purifying by methods such as affinity chromatographys; As Chinese patent literature CN 101397568 A(application number 200810233517.X) disclose a kind of bacteria ferulic acid decarboxylase gene and crystalline structure thereof, this invention is obtained in bacterium by the method for gene clone decomposes the coding region sequence that forulic acid generates the key enzyme ferulic acid decarboxylase of 4-vinyl guaiacol, and by heterogenous expression technology intestinal bacteria (
e.colibL21) this enzyme of great expression in; As patent document CN 1206045(application number 98106195.8) disclose the yeast obtaining having the ferulic acid decarboxylase activity of enhancing by restructuring.But due to current, the bioelectric detecting mechanism understanding of ferulic acid decarboxylase is produced also not exclusively to microorganism, utilize gene engineering method to build ferulic acid decarboxylase production bacterial strain expend huge and be in conceptual phase, genetic engineering bacterium produces the requirement that ferulic acid decarboxylase does not also reach suitability for industrialized production, therefore adopts traditional breeding technology breeding high-yield ferulic acid decarboxylase bacterial strain to remain very necessary method.
Utilize the purebred cultivation of microorganism and beneficiation technologies, separation and purification is carried out to the bacterial strain producing ferulic acid decarboxylase under natural condition, the strain excellent producing ferulic acid decarboxylase can be screened, the dissociant of high yield ferulic acid decarboxylase can being obtained again by mutation breeding technologies, then obtaining production bacterial strain by carrying out genetic stability test to dissociant.Selection by mutation has extremely important practice significance, current fermentation industry produces the superior strain used, and almost all significantly improves its production performance by selection by mutation.Selection by mutation has the advantages such as simple and easy to do, economic, efficient, is still a kind of now widely used Microbial Breeding method.Selection by mutation is generally starting strain with wild strain, selects suitable mutagenic compound and dosage to carry out mutagenic treatment, then filters out the dissociant reaching target.Selection by mutation is divided into physical mutagenesis breeding and chemical mutation according to adopted physical mutagen and chemical mutagen, chemical mutagen has alkylating agent, base analogue and deamidization etc., and physical mutagen commonly uses ultraviolet, X-ray, gamma-radiation and fast neutron etc.Do not need special equipment owing to making mutagenic compound with ultraviolet, only need common sterilizing UV-lamp, and Mutagenic Effect is remarkable, is therefore widely used in industrial breeding.
It is reported, 4-vinyl guaiacol can be detected in the leavened prod of many microorganisms, this is the effect of the ferulic acid decarboxylase due to microorganism generation, in the high-temperature daqu of wherein equipment for making sauce aromatic white spirit, the content of 4-vinyl guaiacol is higher, this means in Daqu containing the microorganism producing relatively high ferulic acid decarboxylase vigor.High-temperature daqu take wheat as raw material, and make through strict and complicated production technique, it is rich in various microorganism and comprises bacterium, yeast and fungi.Bacillaceae (
bacillus sp.) microorganism because of its can when low moisture, high-temperature existence and the major microorganisms that becomes in Daqu microorganism species system, Bacillaceae (
bacillus sp.) it can be made to make full use of nutritive substance in environment and growth and breeding for microorganism produces amylase, proteolytic enzyme etc. and metabolism produces liquor flavor material.According to our research, in Daqu, most of ferulic acid decarboxylase producing strains is the sporiferous Bacillaceae of a class, but the ferulic acid decarboxylase vigor that these Institute of Micro-biology produce is lower, is thus difficult to carry out suitability for industrialized production.
Vigour at present due to the ferulic acid decarboxylase of wild-type microorganisms generation is low; ferulic acid decarboxylase genetic engineering bacterium is also in conceptual phase; the production of ferulic acid decarboxylase does not also form industrially scalable, therefore obtains high reactivity ferulic acid decarboxylase producing bacterial strain by mutation breeding technologies and the large-scale production that realizes ferulic acid decarboxylase is very necessary.
(3) summary of the invention
The present invention, in order to make up the deficiencies in the prior art, provides a kind of Bacillus circulans and is preparing the application in ferulic acid decarboxylase.
The present invention is achieved through the following technical solutions:
A kind of Bacillus circulans, is characterized in that: described Bacillus circulans (
bacillus circulans) HG12 bacterial strain is separated from the high-temperature daqu of Chinese distillery equipment for making sauce aromatic white spirit, and obtain through ultraviolet mutagenesis screening, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 19th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, culture presevation is numbered CGMCC No.8044.
Above-mentioned Bacillus circulans (
bacillus circulans) microbial characteristic of HG12 bacterial strain is: bacterium colony is creamy white, have fold, tarnish, rough; Thalline is shaft-like, straight or close to straight, single or become chain, and cell size is about (0.6-1.0) μm × (1.8-3.8) μm, produces gemma, sporangiocyst expands, gemma oval, raw to end life in gemma; Gram-positive, amphimicrobian, organic chemoheterotrophic bacteria, glucose, wood sugar, N.F,USP MANNITOL, pectinose is utilized to produce acid, V-P reaction negative, catalase is positive, nitrate reduction is positive, hydrolyzable casein, gelatin and starch, do not utilize Citrate trianion, propionic salt, grows at culture temperature is 55 DEG C; Can be fermented Testa Tritici, wheat, barley, the liquid nutrient medium made of one or more combination of Chinese sorghum and solid medium, produces the fragrant fragrance of obvious sauce.
Bacillus circulans of the present invention, preparing the application in ferulic acid decarboxylase, comprises the steps:
(1) choose 1-2 ring Bacillus circulans HG12 inoculation from test tube slant in liquid seed culture medium, 35-40 DEG C, under 120-160r/min condition, cultivate 12-16h, the liquid seeds after obtained activation;
(2) by liquid seeds by volume mark be that the inoculum size of 3-5% is inoculated in liquid fermentation medium, fermentation 10-14h, adds forulic acid, continues fermentation 36-48h, the obtained fermented liquid containing ferulic acid decarboxylase;
(3) by fermented liquid filtering with microporous membrane, collect filtrate, with ultra-filtration membrane by filtrate filtering and concentrating, collect trapped fluid, lyophilize, obtained ferulic acid decarboxylase product.
Its preferred technical characteristic is:
In step (1), the component of liquid seed culture medium is: glucose 10.0g/L, peptone 10.0g/L, extractum carnis 10.0g/L, NaCl 5.0g/L, pH 7.0, distilled water constant volume.
In step (2), the component of liquid fermentation medium is: glucose 80-120g/L, peptone 30.0g/L, extractum carnis 20.0g/L, dipotassium hydrogen phosphate 20.0g/L, forulic acid 0.5-1.0g/L, pH 7.0, distilled water constant volume.
In step (2), the fermentation condition of liquid seeds is 37-40 DEG C, 160-200r/min, and the consumption of forulic acid is 5.0-10.0g/L.
In step (3), the operating pressure of millipore filtration is 0.05-0.1MPa, and the aperture of millipore filtration is 0.2-0.4 μm.
In step (3), the operating pressure of ultra-filtration membrane is 0.2-0.5MPa, and its molecular weight cut-off is 10000-12000Dal.
The Bacillus circulans of seed selection of the present invention (
bacillus circulans) to have growth and breeding speed fast for HG12 bacterial strain, ferulic acid decarboxylase can be produced and produce the feature of ferulic acid decarboxylase stable performance under the induction of forulic acid.
The Bacillus circulans of seed selection of the present invention (
bacillus circulans) HG12 bacterial strain produces that forulic acid depickling enzyme activity is high, in its fermented liquid, enzyme activity can reach 202.6U/mL, and the ferulic acid decarboxylase preparation adopting it to produce can the flavour substances 4-vinyl guaiacol of production food service industry.
The present invention can realize serialization, the mass-producing of ferulic acid decarboxylase production, be convenient to its suitability for industrialized production, and the production technique of enzyme is easy, with short production cycle, cost is low.
(4) accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further illustrated.
Fig. 1 be Bacillus circulans of the present invention (
bacillus circulans) growth curve chart of HG12 bacterial strain in liquid seed culture medium;
Fig. 2 be Bacillus circulans of the present invention (
bacillus circulans) HG12 bacterial strain fermented liquid in the graph of a relation of forulic acid depickling enzyme activity and passage number.
(5) embodiment
Be specifically described below in conjunction with embodiment technical scheme of the present invention or be described further, object is that methods of this invention will be better understood, but protection scope of the present invention is not limited to following embodiment.
Bacillus circulans described in embodiment (
bacillus circulans) HG12 bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 19th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, culture presevation is numbered CGMCC No.8044.
in the present invention, the vitality test step of ferulic acid decarboxylase is as follows:
Get 9.71g forulic acid and be dissolved in 50 DEG C, in the deionized water of 1000mL, obtained concentration is the forulic acid solution of 50mmol/L; Getting ferulic acid decarboxylase sample is dissolved in deionized water, using Sodium phosphate dibasic (0.2mol/L)-citric acid (0.1mol/L) damping fluid (pH5.2) enzyme solution to be diluted to enzyme activity unit is again 0.1-0.2U/mL, obtained ferulic acid decarboxylase sample solution; Get 10.0mL forulic acid solution to join in the Sodium phosphate dibasic-citrate buffer solution (pH5.2) of 90.0mL, vibration shakes up, by solution dilution 10 times, and obtained substrate solution.
The substrate solution getting 2.0mL is heated to 40 DEG C, then adds 1.0mL ferulic acid decarboxylase sample solution, rapidly after mixing, at 40 DEG C, react 30min, then in boiling water bath, maintain 10min termination reaction immediately, collected by centrifugation supernatant liquor, cool under room temperature, obtained solution to be measured.High performance liquid chromatograph (purchased from Shanghai Tian Pu Analytical Instrument Co., Ltd) is adopted to measure the content of the 4-vinyl guaiacol reacting generation in solution to be measured.
The enzyme activity unit of ferulic acid decarboxylase is defined as: under condition determination, and the enzyme amount needed for 4-vinyl guaiacol that per minute hydrolysis forulic acid produces 1 μm of ol is 1U.
Raw material sources: forulic acid is purchased from Pu Zhen bio tech ltd, Shanghai.
Embodiment 1
Bacillus circulans (
bacillus circulans) HG12 bacterial strain has typical Bacillus circulans feature: bacterium colony is creamy white, have fold, tarnish, rough; Thalline is shaft-like, straight or close to straight, single or become chain, and cell size is about (0.6-1.0) μm × (1.8-3.8) μm, produces gemma, sporangiocyst expands, gemma oval, raw to end life in gemma; Gram-positive, amphimicrobian, organic chemoheterotrophic bacteria, glucose, wood sugar, N.F,USP MANNITOL, pectinose is utilized to produce acid, V-P reaction negative, catalase is positive, nitrate reduction is positive, hydrolyzable casein, gelatin and starch, do not utilize Citrate trianion, propionic salt, grows at culture temperature is 55 DEG C; Can be fermented Testa Tritici, wheat, barley, the liquid nutrient medium made of one or more combination of Chinese sorghum and solid medium, produces the fragrant fragrance of obvious sauce.
Embodiment 2
Bacillus circulans (
bacillus circulans) separation screening of HG12 bacterial strain, step is as follows:
(1) bacterium source that the present invention relates to is in the high-temperature daqu producing Maotai-flavor liquor " Guizhou peace wine ".Take and pulverize uniform high-temperature daqu powder 10g in 20mL stroke-physiological saline solution, 37 DEG C, vibrate under 150r/min 24h, obtained bacteria suspension; Bacteria suspension is inserted in 80 DEG C of water-baths, heating 20min; Then get in appropriate above-mentioned bacteria suspension aseptic technique access broth culture, 37 DEG C, cultivate 18h under 150r/min, obtain nutrient solution; By above-mentioned nutrient solution again through 80 DEG C, after 20min process, cooling, after suitably diluting, nutrient agar flat board is coated with 10 times of dilution methods, cultivates 48h for 37 DEG C.Picking grows better, that proterties is different colony inoculation in bouillon agar slant medium, as screening bacterial strain.
(2) above-mentioned bacterial strains is inoculated on primary dcreening operation culture medium flat plate, cultivates 48h for 37 DEG C.Select the colony inoculation of display white transparent circle in multiple sieve substratum from primary dcreening operation substratum, 37 DEG C, cultivate 48h under 150r/min and carry out multiple sieve, obtain a strain and produce the highest bacterial strain H1 of ferulic acid decarboxylase vigor, in its fermented liquid, ferulic acid decarboxylase vigor is 23.2U/mL after testing.Bacterial strain H1 is deposited on nutrient agar slant medium, and as mutagenesis starting strain.
(3) bacterial strain H1 is inoculated in liquid seed culture medium, cultivates 24h for 37 DEG C, obtained somatic cells suspension.Getting 10mL cell suspending liquid pours in sterilized petri dishes, is placed on apart from 15W ultraviolet lamp 30cm place in ultraviolet mutagenesis case, uviolizing 200s.Get the above-mentioned bacterium liquid of 1mL in sterile test tube, 30 DEG C of lucifuges cultivate 24h, obtain nutrient solution.Nutrient solution is diluted 10 times, 10 respectively
2doubly, 10
3doubly, 10
4doubly, 10
5doubly, 10
6doubly, obtain mycelium dilution liquid, then coat on primary dcreening operation culture medium flat plate by mycelium dilution liquid, 37 DEG C of lucifuges cultivate 24h.The colony inoculation of generation white clear circle is selected in multiple sieve substratum from primary dcreening operation culture medium flat plate, 37 DEG C, lucifuge is cultivated 48h and is carried out multiple sieve under 150r/min, the enzyme measuring ferulic acid decarboxylase in nutrient solution is lived, obtain a strain enzyme the highest bacterial strain alive, called after HG12, enzyme activity in its fermented liquid reaches 107.3U/mL, improves 3.63 times than the enzyme activity of starting strain H1.Bacterial strain HG12 is deposited on nutrient agar slant medium, for subsequent use.
(4) with transfering loop from picking 1 ring HG12 inoculation strain tube inclined-plane in liquid seed culture medium, at 37 DEG C, cultivate 22h, every 2h sampling measures the absorbance of strain cultured solution at 600nm place with 752 type spectrophotometers (purchased from Shanghai opticinstrument one factory), with incubation time (T) for X-coordinate, with absorbance (A) for ordinate zou, obtained somatic cells growth curve (result as shown in Figure 1), has the fireballing feature of growth and breeding by the HG12 bacterial strain of the known seed selection of growth curve.
Bouillon agar slant medium component in described step (1) following (g/L): extractum carnis 5.0, peptone 10.0, NaCl 5.0, agar 20.0, pH 7.0.
Nutrient agar component in described step (1), (2), (3) following (g/L): peptone 10.0, extractum carnis 3.0, NaCl 5.0, agar 20.0, pH 7.0.
Following (g/L): the K of primary dcreening operation nutrient media components in described step (2), (3)
2hPO
46.0, KH
2pO
44.0, MgSO
47H
2o 0.20, (NH
4)
2sO
42.0, NaCl 0.10, forulic acid 20.0, agar 20.0, pH 7.2.
Multiple sieve nutrient media components in described step (2), (3) following (g/L): glucose 20.0, peptone 10.0, forulic acid 5.0, K
2hPO
46.0, KH
2pO
44.0, MgSO
47H
2o 0.20, (NH
4)
2sO
42.0, NaCl 0.10, pH 7.2, distilled water constant volume.
Liquid seed culture medium component in described step (3), (4) following (g/L): glucose 5.0, peptone 10.0, extractum carnis 5.0, NaCl 5.0, pH 7.0, distilled water constant volume.
Embodiment 3
Bacillus circulans (
bacillus circulans) HG12 bacterial strain produces the stability experiment of ferulic acid decarboxylase, step is as follows:
The Bacillus circulans HG12 bacterial strain of seed selection is transferred on slant medium successively, goes down to posterity altogether 5 times, at 37 DEG C, cultivate 24h, the test tube slant of obtained different algebraically; Be inoculated in liquid seed culture medium from each 1 ring of picking bacterial strain the test tube slant of different algebraically respectively again, 37 DEG C, under 150r/min condition, cultivate 16h, the liquid seeds after obtained activation; By obtained liquid seeds by 5%(v/v) inoculum size be inoculated in respectively in liquid fermentation medium, 40 DEG C, under 200r/min condition, cultivation 48h.Measure the ferulic acid decarboxylase vigor (result as shown in Figure 2) in the fermented liquid of different algebraically bacterial strain, with bacterial strain H1 for contrast, the Bacillus circulans HG12 bacterial strain of seed selection has the performance of more stable generation ferulic acid decarboxylase.
Described liquid seed culture medium component following (g/L): glucose 5.0, peptone 10.0, extractum carnis 5.0, NaCl 5.0, pH 7.0, distilled water constant volume.
Described fermention medium component following (g/L): glucose 20.0, peptone 10.0, forulic acid 5.0, K
2hPO
46.0, KH
2pO
44.0, MgSO
47H
2o 0.20, (NH
4)
2sO
42.0, NaCl 0.10, pH 7.0, distilled water constant volume.
Embodiment 4
One utilize Bacillus circulans (
bacillus circulans) HG12 strain fermentation produces the method for ferulic acid decarboxylase, step is as follows:
(1) with transfering loop from picking 1 ring Bacillus circulans HG12 inoculation strain tube inclined-plane in the liquid seed culture medium of 10mL, 37 DEG C, under 150r/min condition, cultivate 14h, the liquid seeds after obtained activation.
(2) by liquid seeds obtained for step (1) according to 5%(v/v) inoculum size be inoculated in the liquid fermentation medium of 200mL, 40 DEG C, under 160r/min condition, fermentation 12h, add the forulic acid of 2.0g, 40 DEG C, under 200r/min condition, continue fermentation 48h, the obtained fermented liquid containing ferulic acid decarboxylase.
(3) be that the filtering with microporous membrane of 0.2 μm degerming with aperture by the fermented liquid containing ferulic acid decarboxylase obtained for step (2) under 0.1MPa pressure, collect filtrate, under 0.5MPa pressure, adopt molecular weight cut-off to be that the ultra-filtration membrane of 10000Dal is by filtrate filtering and concentrating, collect trapped fluid, lyophilize, obtained ferulic acid decarboxylase product 2.68g.
Ferulic acid decarboxylase vigor after testing in fermented liquid is 202.6U/mL.
Liquid seed culture medium component in described step (1) following (g/L): glucose 10.0, peptone 10.0, extractum carnis 10.0, NaCl 5.0, pH 7.0, distilled water constant volume.
Liquid fermentation medium component in described step (2) following (g/L): glucose 120, peptone 30.0, extractum carnis 20.0, dipotassium hydrogen phosphate 20.0, forulic acid 1.0, pH 7.0, distilled water constant volume.
Embodiment 5
One utilize Bacillus circulans (
bacillus circulans) HG12 strain fermentation produces the method for ferulic acid decarboxylase, step is as follows:
(1) with the step (1) of embodiment 4
(2) by liquid seeds obtained for step (1) by 3%(v/v) inoculum size be inoculated in the liquid fermentation medium of 200mL, 37 DEG C, under 160r/min condition, fermentation 14h, add the forulic acid of 2.0g, 37 DEG C, under 200r/min condition, continue fermentation 36h, the obtained fermented liquid containing ferulic acid decarboxylase.
(3) be that the filtering with microporous membrane of 0.4 μm degerming with aperture by the fermented liquid containing ferulic acid decarboxylase obtained for step (2) under 0.05MPa pressure, collect filtrate, under 0.2MPa pressure, adopt molecular weight cut-off to be that the ultra-filtration membrane of 12000Dal is by filtrate filtering and concentrating, collect trapped fluid, lyophilize, obtained ferulic acid decarboxylase product 2.42g.
Ferulic acid decarboxylase vigor after testing in fermented liquid is 184.2U/mL.
Liquid fermentation medium component in described step (2) following (g/L): glucose 80, peptone 30.0, extractum carnis 20.0, dipotassium hydrogen phosphate 20.0, forulic acid 0.5, pH 7.0, distilled water constant volume.
Embodiment 6
Utilize as described in Example 4 Bacillus circulans (
bacillus circulans) HG12 strain fermentation produces the method for ferulic acid decarboxylase, difference is:
In step (2), the add-on of forulic acid is 1.0g.
Obtained ferulic acid decarboxylase product 2.54g.
Ferulic acid decarboxylase vigor after testing in fermented liquid is 193.7U/mL.
Embodiment 7
Utilize as described in Example 5 Bacillus circulans (
bacillus circulans) HG12 strain fermentation produces the method for ferulic acid decarboxylase, difference is:
In step (2), the add-on of forulic acid is 1.0g.
Obtained ferulic acid decarboxylase product 2.41g.
Ferulic acid decarboxylase vigor after testing in fermented liquid is 178.3U/mL.
Claims (8)
1. a Bacillus circulans, is characterized in that: described Bacillus circulans (
bacillus circulans) HG12 bacterial strain is separated from the high-temperature daqu of Chinese distillery equipment for making sauce aromatic white spirit, and obtain through ultraviolet mutagenesis screening, its culture presevation is numbered CGMCC No.8044.
2. Bacillus circulans according to claim 1, is characterized by, described Bacillus circulans (
bacillus circulans) microbial characteristic of HG12 bacterial strain is: bacterium colony is creamy white, have fold, tarnish, rough; Thalline is shaft-like, straight or close to straight, single or become chain, and cell size is about (0.6-1.0) μm × (1.8-3.8) μm, produces gemma, sporangiocyst expands, gemma oval, raw to end life in gemma; Gram-positive, amphimicrobian, organic chemoheterotrophic bacteria, glucose, wood sugar, N.F,USP MANNITOL, pectinose is utilized to produce acid, V-P reaction negative, catalase is positive, nitrate reduction is positive, hydrolyzable casein, gelatin and starch, do not utilize Citrate trianion, propionic salt, grows at culture temperature is 55 DEG C; Can be fermented Testa Tritici, wheat, barley, the liquid nutrient medium made of one or more combination of Chinese sorghum and solid medium, produces the fragrant fragrance of obvious sauce.
3. Bacillus circulans according to claim 1 is preparing the application in ferulic acid decarboxylase, it is characterized by, comprise the steps: that (1) chooses 1-2 ring Bacillus circulans HG12 inoculation in liquid seed culture medium from test tube slant, 35-40 DEG C, under 120-160r/min condition, cultivate 12-16h, the liquid seeds after obtained activation; (2) by liquid seeds by volume mark be that the inoculum size of 3-5% is inoculated in liquid fermentation medium, fermentation 10-14h, adds forulic acid, continues fermentation 36-48h, the obtained fermented liquid containing ferulic acid decarboxylase; (3) by fermented liquid filtering with microporous membrane, collect filtrate, with ultra-filtration membrane by filtrate filtering and concentrating, collect trapped fluid, lyophilize, obtained ferulic acid decarboxylase product.
4. Bacillus circulans according to claim 3 is preparing the application in ferulic acid decarboxylase, it is characterized in that: in step (1), the component of liquid seed culture medium is, glucose 10.0g/L, peptone 10.0g/L, extractum carnis 10.0g/L, NaCl 5.0g/L, pH 7.0, distilled water constant volume.
5. Bacillus circulans according to claim 3 is preparing the application in ferulic acid decarboxylase, it is characterized in that: in step (2), the component of liquid fermentation medium is, glucose 80-120g/L, peptone 30.0g/L, extractum carnis 20.0g/L, dipotassium hydrogen phosphate 20.0g/L, forulic acid 0.5-1.0g/L, pH 7.0, distilled water constant volume.
6. Bacillus circulans according to claim 3 is preparing the application in ferulic acid decarboxylase, it is characterized in that: in step (2), and the fermentation condition of liquid seeds is 37-40 DEG C, 160-200r/min, and the consumption of forulic acid is 5.0-10.0g/L.
7. Bacillus circulans according to claim 3 is preparing the application in ferulic acid decarboxylase, it is characterized in that: in step (3), and the operating pressure of millipore filtration is 0.05-0.1MPa, and the aperture of millipore filtration is 0.2-0.4 μm.
8. Bacillus circulans according to claim 3 is preparing the application in ferulic acid decarboxylase, it is characterized in that: in step (3), and the operating pressure of ultra-filtration membrane is 0.2-0.5MPa, and its molecular weight cut-off is 10000-12000Dal.
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| CN107438672B (en) * | 2015-04-09 | 2021-09-28 | 雀巢产品有限公司 | Method for forming dihydroferulic acid |
| CN105002225A (en) * | 2015-09-02 | 2015-10-28 | 常州市长宇实用气体有限公司 | Method for preparing 4-ethenyl guaiacol by utilizing bagasse |
| CN105274086A (en) * | 2015-10-19 | 2016-01-27 | 量子高科(中国)生物股份有限公司 | High-throughput screening method for bacillus circulans |
| CN107217077B (en) * | 2017-07-12 | 2020-04-07 | 大连理工大学 | Method for co-producing rice bran protein and 4-vinyl guaiacol by using rice bran meal |
| CN112352497B (en) * | 2020-08-20 | 2022-05-10 | 扬州大学 | Barley malt rich in ferulic acid and preparation method and application thereof |
| CN114854797A (en) * | 2022-06-08 | 2022-08-05 | 四川轻化工大学 | Method for releasing phenolic acid substances by utilizing white spirit waste lees through microbial flora |
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| CN1206045A (en) * | 1997-02-07 | 1999-01-27 | 协和发酵工业株式会社 | Ferulic acid decarboxylase |
| CN101397568A (en) * | 2008-10-31 | 2009-04-01 | 云南大学 | Bacteria ferulic acid decarboxylase gene and crystal structure thereof |
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| CN1206045A (en) * | 1997-02-07 | 1999-01-27 | 协和发酵工业株式会社 | Ferulic acid decarboxylase |
| CN101397568A (en) * | 2008-10-31 | 2009-04-01 | 云南大学 | Bacteria ferulic acid decarboxylase gene and crystal structure thereof |
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| 崔晓斌 等."理论研究阿魏酸脱羧酶的反应机理".《中国化学会第28届学术年会第13分会场摘要集,2012年》.2012,第1页. * |
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