CN103911399A - Method for producing vanillin based on transformation of isoeugenol by microorganisms - Google Patents
Method for producing vanillin based on transformation of isoeugenol by microorganisms Download PDFInfo
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- CN103911399A CN103911399A CN201410142056.0A CN201410142056A CN103911399A CN 103911399 A CN103911399 A CN 103911399A CN 201410142056 A CN201410142056 A CN 201410142056A CN 103911399 A CN103911399 A CN 103911399A
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Abstract
The invention discloses a method for producing vanillin based on transformation of isoeugenol by microorganisms. According to the method, a microbial strain SW-B9 screened from soil is selected, resists high substrate concentration and transforms isoeugenol into vanillin, and the preserved number is CGMCC1347; the strain is cultured in a slant culture medium, then transferred into a seed culture medium for culturing and subsequently added into a fermentation culture liquid for fermentation, and 0.03%-0.20% w/v promoter is added into the fermentation liquid; the promoter comprises 40%-60% of sodium dodecyl sulfate and the balance of ferrous chloride. The method for producing vanillin based on transformation of isoeugenol by microorganisms of the invention has higher transformation rate, the content of the target product in the fermentation liquid is increased, and the fermentation time is reduced.
Description
Technical field
The present invention relates to the production method of vanillin food grade,1000.000000ine mesh, be specifically related to a kind of method of microbial transformation isoeugenol production vanillin food grade,1000.000000ine mesh.
Background technology
Isoeugenol is for preparing essence and prepare vanillin food grade,1000.000000ine mesh, can be used for preparing the essential oil such as Yilan, Semen Myristicae; Also can be in order to prepare the food flavour of immature fruit of Juteleaf Raspberry, peach, spicy, cloves type fragrance.It is the raw material of semi-synthetic vanillin food grade,1000.000000ine mesh.Vanillin food grade,1000.000000ine mesh, has another name called Vanillin, for the widely used edible spices of one, can in the seed of vanilla, find, also can synthetic, there is strong milk breath.Be widely used in the various blending food that need to increase milk breath, as cake, cold drink, chocolate, candy; Also can be used for perfumed soap, toothpaste, perfume, rubber, plastics, pharmaceuticals.Existing vanillin food grade,1000.000000ine mesh production method has chemical conversion to synthesize and microbial method, as the application number patent of invention that is 200510064494.0, bacterial classification and method that microbial transformation isoeugenol is produced Vanillin are disclosed, the biological preserving number of this bacterial classification is CGMCC1347, this patent of invention discloses and has a kind ofly transformed the microorganism of isoeugenol and utilize the method for this microbial transformation Vanillin, in this patent, disclosed preparation method is comparatively conventional, and the indexs such as its transformation efficiency still have larger room for promotion.
In order to solve above-mentioned deficiency of the prior art, the present invention proposes a kind of new solution.
Summary of the invention
The object of this invention is to provide a kind of microbial transformation isoeugenol and produce the method for vanillin food grade,1000.000000ine mesh.
For reaching above-mentioned purpose, the technical solution adopted in the present invention is: provide a kind of microbial transformation isoeugenol to produce the method for vanillin food grade,1000.000000ine mesh, choose the microbial strains SW-B9 that screening is obtained from soil, it can tolerate high concentration of substrate, isoeugenol can be converted into vanillin food grade,1000.000000ine mesh, preserving number is CGMCC1347; Bacterial classification proceeds in seed culture medium and cultivates after slant medium is cultivated, join again and in fermentation culture, carry out fermentation culture, then add in the bio-transformation substrate that contains isoeugenol and carry out conversion reaction, in bio-transformation substrate, add the promotor of 0.03% ~ 0.20%w/v; Described promotor comprises 40% ~ 65% sodium lauryl sulphate and the iron protochloride of surplus, and wherein the concentration of iron protochloride is 1mmol/L.
In sum, the present invention has the following advantages:
The method of utilizing microbial transformation isoeugenol to produce vanillin food grade,1000.000000ine mesh disclosed by the invention has higher transformation efficiency, and the content of the target product in fermented liquid increases, and has reduced fermentation time.
Embodiment
Choosing of bacterial classification
Obtain from China Committee for Culture Collection of Microorganisms's common micro-organisms preservation center, Chinese BeiJing ZhongGuanCun the bacillus fusiformis that preserving number is CGMCC 1347.
In one embodiment of the invention, the culturing process of microorganism comprises following process:
Slant culture: bacterial classification is inoculated on slant medium, and slant medium is glucose agar medium.
Seed culture: the bacterial classification after slant culture is linked in the seed culture medium of 25ml and continues to cultivate, seed culture medium comprises corn steep liquor 1% ~ 10%, potassium hydrogen phosphate 0.01% ~ 0.05%, potassium primary phosphate 0.01% ~ 0.5%, magnesium sulfate 0.01% ~ 0.5%, glucose 0.1% ~ 1%, ammonium sulfate 0.1 ~ 0.2% and yeast powder 0.2% ~ 0.5%.Bacterial classification at the culture condition of seed culture medium is: 38 DEG C of temperature, 180r/min shaking table shaking culture 12 hours, obtains seed culture fluid.
Fermentation culture: fermention medium comprises corn steep liquor 1% ~ 10%, potassium hydrogen phosphate 0.1% ~ 0.5%, magnesium sulfate 0.1% ~ 0.5%, peptone 0.2% ~ 0.5%, zinc chloride 0.01% ~ 0.02%.In fermention medium, add seed culture fluid, and in 37 DEG C, pH7.5,180r/min shaking table shaking culture adds vanillin food grade,1000.000000ine mesh to make the ultimate density of vanillin food grade,1000.000000ine mesh after 10 hours be 1mmol/L, continues to cultivate 8 hours as inductor, finally obtains fermentation culture.
Transform and cultivate: in bio-transformation substrate, add the wet thallus obtaining from fermentation culture, wherein bio-transformation substrate is according to the mixture of weighing scale 80% isoeugenol and 20% water, then in bio-transformation substrate, add the promotor of 0.03% ~ 0.20%w/v and the wet thallus of 8 ~ 80g/L, controlled fermentation temperature is 37 DEG C, pH5 ~ 8, transform 24 ~ 240 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Embodiment 1
Get bacterial classification and after slant culture and seed culture, carry out fermentation culture, wherein slant medium is glucose agar medium, seed culture medium is for comprising corn steep liquor 1%, potassium hydrogen phosphate 0.01%, potassium primary phosphate 0.01%, magnesium sulfate 0.01%, glucose 0.1%, ammonium sulfate 0.1% and yeast powder 0.2%, all the other are culture medium carrier, can be water; Fermention medium comprises corn steep liquor 1%, potassium hydrogen phosphate 0.1%, and magnesium sulfate 0.1%, peptone 0.2%, zinc chloride 0.01%, all the other are culture medium carrier.
Transform and cultivate: in bio-transformation substrate, add the promotor of wet thallus and the 0.03%w/v of 8g/L, wherein promotor is the solution of ferrous chloride of the 1mmol/L of 40% sodium lauryl sulphate by weight and 60%.Then controlled fermentation temperature is 37 DEG C, and pH7 transforms 72 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Embodiment 2
Get bacterial classification and after slant culture and seed culture, carry out fermentation culture, wherein slant medium is glucose agar medium, seed culture medium is for comprising corn steep liquor 1%, potassium hydrogen phosphate 0.01%, potassium primary phosphate 0.01%, magnesium sulfate 0.01%, glucose 0.1%, ammonium sulfate 0.1% and yeast powder 0.2%, all the other are culture medium carrier, can be water; Fermention medium comprises corn steep liquor 1%, potassium hydrogen phosphate 0.1%, and magnesium sulfate 0.1%, peptone 0.2%, zinc chloride 0.01%, all the other are culture medium carrier.
Transform and cultivate: in bio-transformation substrate, add the promotor of wet thallus and the 0.03%w/v of 80g/L, wherein promotor is the solution of ferrous chloride of the 1mmol/L of 40% sodium lauryl sulphate by weight and 60%.Then controlled fermentation temperature is 37 DEG C, and pH7 transforms 72 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Embodiment 3
Get bacterial classification and after slant culture and seed culture, carry out fermentation culture, wherein slant medium is glucose agar medium, seed culture medium is for comprising corn steep liquor 10%, potassium hydrogen phosphate 0.05%, potassium primary phosphate 0.05%, magnesium sulfate 0.05%, glucose 1%, ammonium sulfate 0.2% and yeast powder 0.5%, all the other are culture medium carrier, can be water; Fermention medium comprises corn steep liquor 10%, potassium hydrogen phosphate 0.5%, and magnesium sulfate 0.5%, peptone 0.5%, zinc chloride 0.02%, all the other are culture medium carrier.
Transform and cultivate: in bio-transformation substrate, add the promotor of wet thallus and the 0.2%w/v of 50g/L, wherein promotor is the solution of ferrous chloride of 1mmol/L of 40% sodium lauryl sulphate by weight and 35% and 35% butyrolactam.Then controlled fermentation temperature is 37 DEG C, and pH8 transforms 72 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Embodiment 4
Get bacterial classification and after slant culture and seed culture, carry out fermentation culture, wherein slant medium is glucose agar medium, seed culture medium is for comprising corn steep liquor 10%, potassium hydrogen phosphate 0.05%, potassium primary phosphate 0.05%, magnesium sulfate 0.05%, glucose 1%, ammonium sulfate 0.2% and yeast powder 0.5%, all the other are culture medium carrier, can be water; Fermention medium comprises corn steep liquor 10%, potassium hydrogen phosphate 0.5%, and magnesium sulfate 0.5%, peptone 0.5%, zinc chloride 0.02%, all the other are culture medium carrier.
Transform and cultivate: in bio-transformation substrate, add the promotor of wet thallus and the 0.2%w/v of 40g/L, wherein promotor is the solution of ferrous chloride of 1mmol/L of 65% sodium lauryl sulphate by weight and 35% and 10% butyrolactam.Then controlled fermentation temperature is 37 DEG C, and pH8 transforms 72 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Embodiment 5
Get bacterial classification and after slant culture and seed culture, carry out fermentation culture, wherein slant medium is glucose agar medium, seed culture medium is for comprising corn steep liquor 10%, potassium hydrogen phosphate 0.05%, potassium primary phosphate 0.05%, magnesium sulfate 0.05%, glucose 1%, ammonium sulfate 0.2% and yeast powder 0.5%, all the other are culture medium carrier, can be water; Fermention medium comprises corn steep liquor 10%, potassium hydrogen phosphate 0.5%, and magnesium sulfate 0.5%, peptone 0.5%, zinc chloride 0.02%, all the other are culture medium carrier.
Transform and cultivate: the wet thallus that adds 50g/L in bio-transformation substrate.Then controlled fermentation temperature is 37 DEG C, and pH8 transforms 72 hours, simultaneously the concentration of isoeugenol and the concentration of product vanillin food grade,1000.000000ine mesh in 12 hours detection of biological conversion of substrate.
Experimental result
Take high performance liquid chromatography or additive method, the vanillin food grade,1000.000000ine mesh content in embodiment 1 ~ embodiment 5 is detected, its result is as follows.
In embodiment 1, be respectively every the content of 12 hours vanillin food grade,1000.000000ine meshs: 9.6g/L; 18.5 g/L; 24.9 g/L; 31.8 g/L; 28.9 g/L.
In embodiment 2, be respectively every the content of 12 hours vanillin food grade,1000.000000ine meshs: 12.8g/L; 19.6 g/L; 23.4 g/L; 31.9g/L; 27.9 g/L.
In embodiment 3, be respectively every the content of 12 hours vanillin food grade,1000.000000ine meshs: 16.8g/L; 23.2 g/L; 29.6 g/L; 36.8 g/L; 30.2 g/L.
In embodiment 4, be respectively every the content of 12 hours vanillin food grade,1000.000000ine meshs: 15.5g/L; 22.5 g/L; 29.9 g/L; 35.8 g/L; 29.8 g/L.
In embodiment 5, be respectively every the content of 12 hours vanillin food grade,1000.000000ine meshs: 7.2g/L; 12.5 g/L; 18.5 g/L; 22.5 g/L; 24.6 g/L.
Interpretation of result:
Embodiment 1 to embodiment 4 carries out bio-transformation cultivation for adding promotor.As can be known from the results, add
The amount of promotor is more, and the efficiency of bio-transformation is more, and the target product vanillin food grade,1000.000000ine mesh content in conversion fluid is higher.Meanwhile, from fermentation time, between 48 hours to 72 hours after the tangible fermentation of the high-content of vanillin food grade,1000.000000ine mesh, greatly lowered fermentation time.Therefore, the Data Comparison from embodiment 4 and embodiment 5 is known, adds the embodiment 4 of promotor than the embodiment 5 that does not add promotor, and the fermentation time of vanillin food grade,1000.000000ine mesh shortens, and content obviously increases.
Claims (6)
1. microbial transformation isoeugenol is produced a method for vanillin food grade,1000.000000ine mesh, chooses the microbial strains SW-B9 that screening is obtained from soil, and it can tolerate high concentration of substrate, isoeugenol can be converted into vanillin food grade,1000.000000ine mesh, and preserving number is CGMCC1347; Bacterial classification proceeds in seed culture medium and cultivates after slant medium is cultivated, join again and in fermentation culture, carry out fermentation culture, then add in the bio-transformation substrate that contains isoeugenol and carry out conversion reaction, it is characterized in that: the promotor that adds 0.03% ~ 0.20%w/v in bio-transformation substrate; Described promotor comprises 40% ~ 65% sodium lauryl sulphate and the iron protochloride of surplus, and wherein the concentration of iron protochloride is 1mmol/L.
2. method according to claim 1, is characterized in that: described slant medium is glucose agar medium.
3. method according to claim 1, is characterized in that: described seed culture medium comprises corn steep liquor 1% ~ 10%, potassium hydrogen phosphate 0.01% ~ 0.05%, potassium primary phosphate 0.01% ~ 0.5%, magnesium sulfate 0.01% ~ 0.5%, glucose 0.1% ~ 1%, ammonium sulfate 0.1 ~ 0.2% and yeast powder 0.2% ~ 0.5%.
4. method according to claim 1, is characterized in that: described fermention medium comprises corn steep liquor 1% ~ 10%, potassium hydrogen phosphate 0.1% ~ 0.5%, magnesium sulfate 0.1% ~ 0.5%, peptone 0.2% ~ 0.5%, zinc chloride 0.01% ~ 0.02%.
5. method according to claim 1, is characterized in that: described promotor comprises 40% ~ 65% sodium lauryl sulphate and 35% iron protochloride, and surplus is butyrolactam.
6. method according to claim 1 or 5, is characterized in that: described promotor comprises 40% sodium lauryl sulphate, 35% iron protochloride and 35% butyrolactam.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849590A (en) * | 2015-08-07 | 2018-03-27 | 罗地亚经营管理公司 | Pass through the production for the improved vanillic aldehyde that ferments |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600853A (en) * | 2003-09-25 | 2005-03-30 | 江南大学 | Coarse enzyme preparation and method for preparing vanillin through biotransformation |
| CN1712518A (en) * | 2005-04-19 | 2005-12-28 | 江南大学 | Fungus and method for preparing vanillin from isoeugenol converted by microorgan |
| CN101078005A (en) * | 2006-05-26 | 2007-11-28 | 上海凯信生物科技有限公司 | Bacillus pumilus and application of the same in producing natural vanillin by biologically converting iso-eugenol |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600853A (en) * | 2003-09-25 | 2005-03-30 | 江南大学 | Coarse enzyme preparation and method for preparing vanillin through biotransformation |
| CN1712518A (en) * | 2005-04-19 | 2005-12-28 | 江南大学 | Fungus and method for preparing vanillin from isoeugenol converted by microorgan |
| CN101078005A (en) * | 2006-05-26 | 2007-11-28 | 上海凯信生物科技有限公司 | Bacillus pumilus and application of the same in producing natural vanillin by biologically converting iso-eugenol |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849590A (en) * | 2015-08-07 | 2018-03-27 | 罗地亚经营管理公司 | Pass through the production for the improved vanillic aldehyde that ferments |
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