CN103215197B - Enterobacter mori and method for producing natural vanillin by biotransformation of ferulic acid by Enterobacter mori - Google Patents

Enterobacter mori and method for producing natural vanillin by biotransformation of ferulic acid by Enterobacter mori Download PDF

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CN103215197B
CN103215197B CN201210117028.4A CN201210117028A CN103215197B CN 103215197 B CN103215197 B CN 103215197B CN 201210117028 A CN201210117028 A CN 201210117028A CN 103215197 B CN103215197 B CN 103215197B
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natural vanillin
vanillin
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ferulic acid
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陈勇
魏敏
罗诚浩
宋旭艳
喻林
王洁
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China Tobacco Hunan Industrial Co Ltd
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Abstract

The invention discloses an Enterobacter mori and a method for producing natural vanillin by biotransformation of ferulic acid by the Enterobacter mori, which belongs to the microbial fermentation technical field. The method for producing natural vanillin by biotransformation of ferulic acid by Enterobacter mori CCTCC No: M2012020 comprises the following steps: 1)activating bacterial classification; 2) preparing a seed nutrient solution; 3) fermenting; 4) conversing, performing centrifugal treatment on a broth for 12 minutes under condition of 7000rpm/min, and collecting a supernatant to obtain the natural vanillin broth having light yellow color. The Enterobacter mori CCTCC No: M2012020 which is not reported has the advantages of low cost of carbon source and nitrogen source, and wide source, in the broth, the natural vanillin has the advantages of large concentration and high output, the production technology has the advantages of simple process, mild condition, friend environment and industrial prospect, and has good social benefit and economic benefit for changing waste into valuables.

Description

Mulberry enterobacteria and utilize its biologically transform ferulic acid to produce the method for natural vanillin
Technical field
The invention belongs to microbial fermentation technology field, relate to particularly the method for a strain mulberry enterobacteria and biologically transform ferulic acid production natural vanillin thereof.
Background technology
In prior art, vanillin food grade,1000.000000ine mesh (Vanillin), has another name called Vanillin; Vanillin; 4-hydroxyl-3-methoxylbenxaldehyde; Vanillin food grade,1000.000000ine mesh.White, needle-shaped crystals, is the distinctive aromatic odour of XIANGJIALANDOU, micro-sweet.Vanillin food grade,1000.000000ine mesh comes from Mexico at first, is the main component of vanilla, is a kind of high-grade spices of broad spectrum type, of many uses.Vanillin food grade,1000.000000ine mesh can be rated as the widest sweetener of use in the world, the vanilla such as XIANGJIALANDOU, vanilla bean concrete goods are widely used in ice-creams, beverage, chocolate, candy, pudding, bakery product and drinks, cigarette, except sweetener, vanillin food grade,1000.000000ine mesh is as antioxidant in the food that contains how unsaturated component etc.In addition the series compound such as vanillin food grade,1000.000000ine mesh and vanillin food grade,1000.000000ine mesh or important industrial chemicals.Vanillin food grade,1000.000000ine mesh has special odor type, is a kind of flavouring agent that various countries human consumer likes the most, has the laudatory title of " king of food spice ".In foodstuffs industry, Chang Zuowei spices makes an addition in the food such as cake, soymilk, refreshment drink, candy; Be widely used as the blending raw material of the luxury foods such as Brand Name Tobacco and Alcohol, cream, coffee, cocoa, chocolate; Because vanillin food grade,1000.000000ine mesh has certain antibacterial ability, can be used as preservation agent in addition.Approximately 1.2 ten thousand tons of world's year consumptions of vanillin food grade,1000.000000ine mesh, and the output of natural vanillin is only 1800 tons, therefore far can not satisfy the demands.
Most vanillin food grade,1000.000000ine mesh products is by chemosynthesis in the market, and conventional synthetic substrate comprises Eugenol, xylogen, oxoethanoic acid and methyl catechol etc.Domestic conventional be the Short-Cut Nitrification Process that adopts methyl catechol and urotropine reaction, the method road is long, sepn process complexity, yield is low, environmental pollution is serious, raw material consumption is high, is abroad eliminated already.External main methyl catechol and the oxoethanoic acid condensation process of adopting, this processing unit is simple, and product purity is high, raw material sources are extensive, but have serious defect, such as environmental pollution is serious, product fragrance is single, easily doping, and the security of synthesis of vanillin is also under suspicion.
Biotransformation method is the chemical reaction carrying out under the biocatalysis such as cell or enzyme, both referred to and utilized microorganism (resting cell of microbial growth cell, microorganism), enzyme (from microorganism or vegeto-animal pure enzyme or thick enzyme) or vegetable cell, prepared the method for desired substance by simple bio-transformation or biosynthesizing.Compared with chemical catalyst, biological catalyst specificity is stronger, catalytic activity is higher, condition is more easy to control.Can accept a large amount of external source complicated molecules as substrate, the catalytic substrate of chemo-selective, regioselectivity, stereoselectivity or enantioselectivity generates corresponding product.Bio-transformation is widely used in the biosynthesizing of natural product, the conversion of medicine precursor compound, the asymmetric synthesis of organic compound, the numerous areas such as fractionation, active ingredient screening and new drug development, drug metabolism study of optically active compound.The vanillin food grade,1000.000000ine mesh of producing with biotransformation method and the natural vanillin of plant extract are equal to (Natural-identical vanillin), are called as biological vanillin food grade,1000.000000ine mesh (Bilogic Vanillin).It transforms precursor the natural compoundss such as Eugenol, isoeugenol, VANILLYL ALCOHOL MIN 98, vanilla amine, lubanol, forulic acid, die aromatischen Aminosaeuren.For example utilize the method for microorganism fermentation, analogue enztme explain ferulic acid derivative in vanilla time, produce vanillin food grade,1000.000000ine mesh process and " natural vanillin ", meet EU and the FDA use safety requirements to natural essence.
Have been reported about forulic acid being converted into vanillin food grade,1000.000000ine mesh with multiple-microorganism fermentation method, medium component is comparatively complicated, and production cost is higher, and more vanillinated, vanillic alcohol and the methyl catechol etc. of comprising of by product extract and brought suitable difficulty to product separation.
Summary of the invention
For the above-mentioned deficiency of prior art, the method that the technical problem to be solved in the present invention is to provide a strain mulberry enterobacteria and produces natural vanillin with this mulberry enterobacteria biologically transform ferulic acid.
A strain mulberry enterobacteria Enterobacter mori VL4-3 of the present invention, be preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012, preserving number is CCTCC NO:M2012020, the 16SrRNA sequence of the bacterial strain VL4-3 of described mulberry enterobacteria, its sequence total length 1414bp, reaches 99.608% with the similarity of mulberry enterobacteria.
One strain mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid is produced the method for natural vanillin and is carried out according to the following steps:
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on slant medium, under 25～35 ℃, pH5～9 condition, leave standstill to cultivate after 24～72 hours and obtain activated spawn, be placed in 4 ℃ of refrigerators;
Described slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) prepare seed culture fluid: with the slant culture of step (1) gained, be inoculated into seed culture medium, under 25～35 ℃ of conditions, 100～150rpm/min shaking culture 24h～72h, transfers 1～4 time continuously, makes seed culture fluid;
Described seed culture medium: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: use the seed culture fluid of step (2) gained, the by volume inoculum size of per-cent 2～20% access fermention medium, under 30～40 ℃ of conditions, 100～200rpm/min shaking culture 1～3 day;
Described fermention medium: carbon source 5～20g/L, nitrogenous source 1～3g/L, sodium-chlor 0.5～1.5g/L, iron vitriol 0.5～1.5g/L, regulates pH3～10; Sterilizing 20 minutes under 115 ℃ of conditions;
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid of 2～10g/L, under 30～40 ℃ of conditions, 100～200rpm/min lucifuge shaking culture stopped after 5～12 days,
Transformation fermentation liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid; The natural vanillin fermented liquid concentration obtaining can reach 1.5～2.3g/L, and transformation efficiency is 18.25～28.87%.
Wherein:
Described carbon source in step (3) is that starch, glucose, sucrose, D-Fructose, maltose, Fructus Hordei Germinatus soak one or more the mixing among powder, wheat bran, husk.Wheat bran preferably in described carbon source.
The pH of the described fermention medium in step (3) is 8.
The nitrogenous source of the described fermention medium in step (3) is one or more among dregs of beans, soybean cake powder, extractum carnis, peptone (Arg), yeast powder, soy peptone, saltpetre, ammonium sulfate, ammonium nitrate, ammonium phosphate.The preferably dregs of beans of nitrogenous source of the described fermention medium in step (3).
Described leavening temperature in step (3) is 37 ℃.
Preferably 100rpm/min of described fermentation rotating speed in step (3).
Described inoculum size during step (4) transforms is 10%, and described forulic acid addition is 8.5g/L, and the described forulic acid interpolation time is 24h.
Advantage of the present invention is: bacterial strain mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 has no report, utilize the method for its biologically transform ferulic acid production natural vanillin also without report, preferred version of the present invention, carbon source, nitrogenous source cost are very low, wide material sources are easy to get, in its fermented liquid, natural vanillin concentration is large, output is higher, production process is simple, mild condition, environmental friendliness, there is industrial prospect, and possess the good Social benefit and economic benefit of turning waste into wealth.
Accompanying drawing explanation
The different carbon source through fermentation of accompanying drawing 1 are produced the output comparison diagram of natural vanillin;
The output comparison diagram of accompanying drawing 2 different nitrogen sources fermentative production natural vanillins;
Under accompanying drawing 3 different fermentations initial pH value of medium, produce the output comparison diagram of natural vanillin;
The output comparison diagram of natural vanillin at accompanying drawing 4 different fermentations temperature;
Accompanying drawing 5 different fermentations are cultivated the output comparison diagram of producing natural vanillin under rotating speed;
Accompanying drawing 6 fermenting process illumination/lucifuge is produced the output comparison diagram of natural vanillin;
The different shaking flask liquid amounts of accompanying drawing 7 are produced the output comparison diagram of natural vanillin
The affect schematic diagram of the activation number of times of accompanying drawing 8 bacterial classifications on natural vanillin output.
Biomaterial preservation information
One strain mulberry enterobacteria Enterobacter mori VL4-3 is preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012, and preserving number is CCTCC NO:M2012020.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Be necessary to point out that at this following specific embodiment just further illustrates of the present invention, does not represent limiting the scope of the invention.Some nonessential modification and adjustment that other people make according to the present invention, still belong to protection scope of the present invention.
The biological property of mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 is: this bacterial strain is direct rod shape, for Gram-negative bacteria, facultative aerobic, there is the enzymic activity of ONPG beta-galactosidase enzymes, LDC lysine decarboxylase, ADH arginine dihydrolase, ODC ornithine decarboxylase; Tryptophane desaminase, urine enzyme reaction, H2S generation, gelatine liquefication, indoles produce and are negative; Can utilize citric acid, glucose, N.F,USP MANNITOL, sorbyl alcohol, rhamnosyl, sucrose, melibiose, amygdaloside, pectinose, it is sour gentle to produce, and generates sour-sweet aroma substance.Can grow take multiple amino acids, most of monose, polysaccharide, ester class and amino acid as sole carbon source; With cyclodextrin, methylene-succinic acid, α-one valeric acid, quinic acid, adonitol, i-erythritol, L-fucose, succinamic acid, lactulose, hydroxyl-L-PROLINE, L-Leu, L-Glutimic acid, D, VBT, γ-aminobutyric acid, D-lactobionic acid lactone, α-ketoglutaric acid, sebacic acid, gamma-hydroxybutyric acid, phenylethylamine, butanediamine, 2-monoethanolamine, can not grow on the 2,3-butanediol substratum that is sole carbon source.
(1) different carbon source through fermentation are produced the output of natural vanillin
Take Gause I substratum as basic medium, fixing nitrogenous source and inorganic salt, soak powder, wheat bran and husk as carbon source using starch, glucose, sucrose, D-Fructose, maltose, Fructus Hordei Germinatus respectively, and concentration is 20g/L.As shown in Figure 1, different carbon sources are that wheat bran, husk, sucrose, starch, Fructus Hordei Germinatus soak powder, maltose, glucose and fructose to vanillin food grade,1000.000000ine mesh yield effect from high to low successively.Wherein fructose is unfavorable for that forulic acid is converted into vanillin food grade,1000.000000ine mesh, and slow carbon source is easier to the generation of vanillin food grade,1000.000000ine mesh than quick-acting carbon sources, and the wheat bran in slow carbon source and husk may be easier to owing to wherein containing somatomedin the generation of vanillin food grade,1000.000000ine mesh than single carbon source.During wherein take wheat bran as carbon source, the concentration of Vanillin in Fermentation Broth is the highest, and wheat bran low price, and wide material sources are convenient.
Further again, described nitrogenous source is yeast powder, soybean cake powder, extractum carnis, soy peptone, animal proteinum peptone, ammonium sulfate, ammonium nitrate, ammonium phosphate and the optional one of dregs of beans, the preferred dregs of beans of described nitrogenous source.
(2) output of different nitrogen sources fermentative production natural vanillin
Take Gause I substratum as basic medium, fixing carbon source and inorganic salt, using yeast powder, soybean cake powder, extractum carnis, soy peptone, animal proteinum peptone, ammonium sulfate, ammonium nitrate, ammonium phosphate and dregs of beans as nitrogenous source, concentration is 1g/L respectively.
As shown in Figure 2, to have high to Low on the impact of vanillin food grade,1000.000000ine mesh output be dregs of beans, soybean cake powder, extractum carnis, peptone (Arg), yeast powder, soy peptone, saltpetre, ammonium sulfate, ammonium nitrate, ammonium phosphate to different nitrogen sources successively.Wherein organic nitrogen source is converted into vanillin food grade,1000.000000ine mesh than the inorganic nitrogen-sourced forulic acid that is more conducive to.The vanillin food grade,1000.000000ine mesh producing than other nitrogenous sources during using dregs of beans as nitrogenous source is more; Value of the meal is cheap.
Further again, described pH is preferably 8.
(3) natural vanillin output under different fermentations initial pH value of medium
Use the substratum after optimizing, select pH to be optimized at the pH of 3～10 pairs of substratum.As seen from Figure 4, between medium pH 3～5, because acidity is stronger, vanillin food grade,1000.000000ine mesh output is lower; Under alkaline condition, between pH6～10, all there is relatively high output, illustrate that this bacterium is more suitable for forulic acid and is converted into vanillin food grade,1000.000000ine mesh under alkaline condition.Be wherein that the concentration of 8 o'clock vanillin food grade,1000.000000ine meshs is the highest in pH value.
Further again, described leavening temperature is preferably 37 ℃.
(4) output of natural vanillin at different fermentations temperature
Use the substratum of optimizing, regulate pH to 8.0, select temperature to be optimized between 30～40 ℃.When in the time that temperature is between 30 ℃～37 ℃, the output of vanillin food grade,1000.000000ine mesh raises gradually; And when temperature is during at 37～40 ℃, may be the activity decreased that transforms forulic acid while arriving 40 ℃ due to temperature and generate the enzyme of vanillin food grade,1000.000000ine mesh, so vanillin food grade,1000.000000ine mesh concentration reduces.As seen from Figure 5, vanillin food grade,1000.000000ine mesh concentration when leavening temperature is 37 ℃ in fermented liquid is the highest for result, and at this temperature, suitable bacterial enzyme is lived and growth.
Further again, fermentation rotating speed is preferably 100rpm/min.
(5) different fermentations is cultivated the output of producing natural vanillin under rotating speed
Be that the rotating speed that shaking table is cultivated is optimized on basis by following condition: fermention medium pH=8,37 ℃ of culture temperature, inoculum size is 10%, and substrate add-on is 8.5g/L, and the substrate joining day is the rear 24h of inoculation.Can find out according to Fig. 7, along with the increase of rotating speed, the yield reducation of vanillin food grade,1000.000000ine mesh, in the time that fermentation rotating speed is 100rpm, the concentration of Vanillin in Fermentation Broth is the highest.
Further again, fermenting process is preferably lucifuge condition.
(6) fermenting process illumination/lucifuge is produced the output of natural vanillin
Illumination and lucifuge while selecting fermentation are studied the impact of illumination condition on vanillin food grade,1000.000000ine mesh, can find out according to Fig. 6, higher than the vanillin food grade,1000.000000ine mesh under illumination condition under lucifuge condition, may be that part vanillin food grade,1000.000000ine mesh decomposes or the decomposition of part forulic acid under illumination condition, thereby affect the output of vanillin food grade,1000.000000ine mesh, produce vanillin food grade,1000.000000ine mesh and be more suitable for carrying out under lucifuge condition so mulberry enterobacteria transforms forulic acid.
Under different fermentations culture condition, produce the output of natural vanillin
(1) inoculum size, substrate addition and substrate add the impact of time on vanillin food grade,1000.000000ine mesh output
To optimize the substratum obtaining as basis, regulate pH=8, culture temperature is 37 ℃.Optimize inoculum size, substrate addition and substrate and add the time, three factors are forulic acid add-on > forulic acid joining day > inoculum size on the impact order of vanillin food grade,1000.000000ine mesh output, be forulic acid add-on to vanillin food grade,1000.000000ine mesh yield effect maximum, inoculum size is to vanillin food grade,1000.000000ine mesh yield effect minimum.Be that inoculum size is 10% by the known best of breed of experimental result, substrate forulic acid addition is 8.5g/L, and the substrate forulic acid interpolation time is 24h.
(2) different shaking flask liquid amounts are produced the output of natural vanillin
Rotating speed and shaking flask liquid amount are two important factors that affect dissolved oxygen, also affect redox reaction simultaneously.Investigate the impact of liquid amount on vanillin food grade,1000.000000ine mesh output take rotating speed as 100rpm.Can find out according to Fig. 7, along with the increase of liquid amount, the yield reducation of vanillin food grade,1000.000000ine mesh, when liquid amount is 10%, the concentration of Vanillin in Fermentation Broth is the highest, therefore transforms the best fermentation liquid amount of forulic acid production vanillin food grade,1000.000000ine mesh using 10% liquid amount (adding 50mL fermention medium in 500mL triangular flask) as this bacterial classification.
(3) impact of the activation number of times of bacterial classification on natural vanillin output
The activity influence of the various enzymes of the state of bacterial strain to conversion forulic acid generation vanillin food grade,1000.000000ine mesh is larger, the impact of the state that the actication of culture of 4 ℃ of preservations is studied to bacterial classification for 1～4 time on vanillin food grade,1000.000000ine mesh output, can find out according to Fig. 8, along with actication of culture number of times increases, vanillin food grade,1000.000000ine mesh output increases thereupon, when activation number of times is at 3 times and when above, it is stable that the conversion capability of bacterial classification and the output of vanillin food grade,1000.000000ine mesh keep.
Advantage of the present invention is: bacterial strain mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 of the present invention has no report, utilize the method for its biologically transform ferulic acid production natural vanillin also without report, the present invention is through optimizing, carbon source is preferably plant waste wheat bran, when nitrogenous source is preferably plant waste dregs of beans, cost is very low, wide material sources are easy to get, in its fermented liquid, natural vanillin concentration can reach 2.3g/L, maximum conversion rate is 28.87%, output is higher, production process is simple, mild condition, environmental friendliness, there is industrial prospect, and possesses the good Social benefit and economic benefit of turning waste into wealth.
Embodiment 1
The method of microorganism strains mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on ready slant medium, in constant incubator, under 30 ℃, pH7 condition, leave standstill and cultivate 36h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VL4-3 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 30 ℃, rotating speed 100rpm/min shaking culture 24h; Activate 1 time, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: cultured VL4-3 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 10% (V/V), 40 ℃, 100rpm/min shaking culture 24h,
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid 25%(volume ratio of 3.5g/L), 40 ℃, rotating speed 100rpm/min, lucifuge is cultivated after 8 days and is stopped fermenting.
Consisting of of fermention medium: wheat bran 20g/L, dregs of beans 3g/L, sodium-chlor 1.0g/L, FeSO 47H 2o0.5g/L, pH9.Sterilizing 20 minutes under 115 ℃ of conditions.It is more than 10 orders that dregs of beans is pulverized through pulverizer.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 8000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid.
Embodiment 2
The method of microorganism strains mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on ready slant medium, in constant incubator, under 35 ℃, pH9 condition, leave standstill and cultivate 72h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VJ4-1 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 25 ℃, rotating speed 150rpm/min shaking culture 24h; Activate 2 times, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: cultured VJ4-1 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 40% (V/V), 35 ℃, 150rpm/min shaking culture 36h,
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid 25%(V/V of 5g/L), 30 ℃, rotating speed 200rpm/min, lucifuge is cultivated after 12 days and is stopped fermenting.
Consisting of of fermention medium: starch 5g/L, soybean cake powder 1g/L, sodium-chlor 1.5g/L, FeSO 47H 2o1.0g/L, pH5.Sterilizing 20 minutes under 115 ℃ of conditions.It is more than 10 orders that dregs of beans is pulverized through pulverizer.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 7000rpm/min condition, and 12min centrifugal treating, obtains faint yellow natural vanillin fermented liquid.
Embodiment 3
The method of microorganism strains mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on ready slant medium, in constant incubator, under 25 ℃, pH5 condition, leave standstill and cultivate 72h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VJ4-1 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 30 ℃, rotating speed 130rpm/min shaking culture 36h; Activate 4 times, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: cultured VJ4-1 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 30% (V/V), 30 ℃, 200rpm/min shaking culture 72h,
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid 25%(V/V of 8.5g/L), 40 ℃, rotating speed 100rpm/min, lucifuge is cultivated after 6 days and is stopped fermenting.
Consisting of of fermention medium: fructose 15g/L, yeast powder 2g/L, sodium-chlor 1.0g/L, FeSO 47H 2o1.5g/L, pH7.Sterilizing 20 minutes under 115 ℃ of conditions.It is more than 10 orders that dregs of beans is pulverized through pulverizer.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid.
Embodiment 4
The method of microorganism strains mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on ready slant medium, in constant incubator, under 30 ℃, pH7 condition, leave standstill and cultivate 36h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VJ4-1 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 30 ℃, rotating speed 100rpm/min shaking culture 36h; Activate 2 times, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: cultured VJ4-1 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 10% (V/V), 30 ℃, 100rpm/min shaking culture 36h;
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid 25%(V/V of 10g/L), 40 ℃, rotating speed 100rpm/min, lucifuge is cultivated after 8 days and is stopped fermenting.
Consisting of of fermention medium: husk 20g/L, extractum carnis 2g/L, sodium-chlor 1.5g/L, FeSO 47H 2o1.0g/L, pH6.Sterilizing 20 minutes under 115 ℃ of conditions.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid.
Embodiment 5
The method of microorganism strains food methyl alcohol bacillus methylotrophicus CCTCC M201200 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: take food methyl alcohol bacillus methylotrophicus VJ4-1CCTCC M2012004 and be inoculated on ready slant medium, in constant incubator, under 30 ℃, pH5 condition, leave standstill and cultivate 72h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VJ4-1 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 35 ℃, rotating speed 100rpm/min shaking culture 72h; Activate 1 time, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 50g/L, peptone 10g/L, sodium-chlor 5g/L.
(3) fermentation: cultured VJ4-1 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 10% (V/V), 35 ℃, 150rpm/min shaking culture 24h,
(4) transform: use the fermented liquid of step (3) gained, add the trans-ferulaic acid 25%(V/V of 5.0g/L), 30 ℃, rotating speed 150rpm/min, illumination cultivation stops fermentation after 8 days.
Consisting of of fermention medium: sucrose 20g/L, animal proteinum peptone 2g/L, sodium-chlor 1.5g/L, K 2hPO 40.5g/L, MgSO 47H 2o1.0g/L, FeSO 47H 2o0.05g/L, pH10.Sterilizing 20 minutes under 115 ℃ of conditions.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid.
Embodiment 6
The method of microorganism strains mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 fermentative production natural vanillin of the present invention is as follows:
1. microorganism fermentation
(1) actication of culture: get mulberry enterobacteria Enterobacter mori VL4-3CCTCC NO:M2012020 and be inoculated on ready slant medium, in constant incubator, under 30 ℃, pH5 condition, leave standstill and cultivate 72h, obtain activated spawn, be stored in 4 ℃ of refrigerators;
Slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) preparation seed culture fluid: encircle VJ4-1 to being equipped with in the 500mL triangular flask of 200mL liquid seed culture medium with transfering loop picking one, in constant-temperature table, 35 ℃, rotating speed 100rpm/min shaking culture 72h; Activate 1 time, make seed culture fluid;
Seed liquor substratum consists of: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: cultured VJ4-1 seed liquor is linked in fermention medium (liquid amount 10%) with the inoculum size of 10% (V/V), 35 ℃, 150rpm/min shaking culture 24h,
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid 25%(V/V of 5.0g/L), 30 ℃, rotating speed 150rpm/min, illumination cultivation stops fermentation after 8 days.
Consisting of of fermention medium: Fructus Hordei Germinatus soaks powder 20g/L, yeast powder 2g/L, sodium-chlor 1.5g/L, FeSO 47H 2o1.5g/L, pH8.Sterilizing 20 minutes under 115 ℃ of conditions.
2. the extraction purifying of natural vanillin in fermented liquid
Fermented liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid.

Claims (9)

1. a strain mulberry enterobacteria Enterobacter mori, it is characterized in that: mulberry enterobacteria VL4-3Enterobacter mori, be preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012, preserving number is CCTCC NO:M2012020, the 16SrRNA sequence of the bacterial strain VL4-3 of described mulberry enterobacteria, its sequence total length 1414bp, reaches 99.608% with the similarity of mulberry enterobacteria.
2. the method that a strain mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid as claimed in claim 1 is produced natural vanillin, the method described in it is characterized in that is carried out according to the following steps:
(1) actication of culture: get mulberry enterobacteria VL4-3Enterobacter mori CCTCC NO:M2012020 and be inoculated on slant medium, under 25～35 ℃, pH5～9 condition, leave standstill to cultivate after 24～72 hours and obtain activated spawn, be placed in 4 ℃ of refrigerators;
Described slant medium: Radix Dauci Sativae immersion liquid 200mL/L, sucrose 20g/L, agar 13g/L;
(2) prepare seed culture fluid: by the activated spawn of step (1) gained, be inoculated into seed culture medium, under 25～35 ℃ of conditions, 100～150rpm/min shaking culture 24h～72h, transfers 1～4 time continuously, makes seed culture fluid;
Described seed culture medium: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L;
(3) fermentation: use the seed culture fluid of step (2) gained, the by volume inoculum size of per-cent 2～20% access fermention medium, under 30～40 ℃ of conditions, 100～200rpm/min shaking culture 1～3 day;
Described fermention medium: carbon source 5～20g/L, nitrogenous source 1～3g/L, sodium-chlor 0.5～1.5g/L, iron vitriol 0.5～1.5g/L, regulates pH3～10; Sterilizing 20 minutes under 115 ℃ of conditions;
(4) transform: use the fermented liquid of step (3) gained, add the forulic acid of 2～10g/L, under 30～40 ℃ of conditions, 100～200rpm/min lucifuge shaking culture stopped after 5～12 days;
Transformation fermentation liquid is under 7000rpm/min condition, and 12min centrifugal treating, collects supernatant liquor, obtains faint yellow natural vanillin fermented liquid; The natural vanillin fermented liquid concentration obtaining can reach 1.5～2.3g/L, and transformation efficiency is 18.25～28.87%.
3. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 2, is characterized in that carbon source in the described fermention medium in step (3) fermentation is that starch, glucose, sucrose, D-Fructose, maltose, Fructus Hordei Germinatus soak one or more the mixing among powder, wheat bran, husk.
4. according to a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid described in claim 2 or 3, the carbon source in the described fermention medium of step (3) fermentation described in it is characterized in that is wheat bran.
5. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 2, the pH that it is characterized in that the described fermention medium in step (3) is 8.
6. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 2, the nitrogenous source that it is characterized in that the described fermention medium in step (3) is one or more among dregs of beans, soybean cake powder, extractum carnis, yeast powder, soy peptone, saltpetre, ammonium sulfate, ammonium nitrate, ammonium phosphate.
7. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 6, the nitrogenous source that it is characterized in that the described fermention medium in step (3) is dregs of beans.
8. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 2, the inoculum size access fermention medium that it is characterized in that the by volume per-cent 2-20% in step (3) is under 37 ℃ of conditions.
9. a kind of method of producing natural vanillin with mulberry enterobacteria CCTCC NO:M2012020 biologically transform ferulic acid according to claim 2, is characterized in that the described fermentation rotating speed in step (3) is 100rpm/min.
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