CN101200736A - Method for preparing vanillic acid methyl esters - Google Patents
Method for preparing vanillic acid methyl esters Download PDFInfo
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- CN101200736A CN101200736A CNA2006101472772A CN200610147277A CN101200736A CN 101200736 A CN101200736 A CN 101200736A CN A2006101472772 A CNA2006101472772 A CN A2006101472772A CN 200610147277 A CN200610147277 A CN 200610147277A CN 101200736 A CN101200736 A CN 101200736A
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- acid methyl
- vanillic acid
- methyl esters
- fermentation
- acetone
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Abstract
The present invention relates to a method of preparing the microorganism of vanillic acid methyl by cultivating ergot CGMCC No.3997 strain, which is that the CGMCC No.3997 strain is processed for the slant culture, the seed culture and the fermentation culture respectively at a nutrient culture medium containing usable carbon source, nitrogen source and inorganic salt; obtained fermentation liquid contains the vanillic acid methyl which is separated from culture liquid and is purified to obtain the vanillic acid methyl with high purity. The present invention creates a beginning of using the ergot or the same kind of bacterium and even using a biological method to prepare for the vanillic acid methyl, which has the advantages in a plurality of aspects.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind ofly prepare the method for vanillic acid methyl esters with ergot (Claviceps sp.) CGMCC No.3997.
Background technology
The vanilla esters of gallic acid, as vanillic acid methyl esters and ethyl vanillate, be class purposes spices very widely, not only can be used as perfume, skin or hair care, makeup, barber's tool, air freshener, the additive of Chemicals such as hidroschesis dew and house reodorant, also can be used as the numerous food product seasonings, be used for food, tobacco, seasonings and daily chemical essence, is good fixative.
Vanillic acid methyl esters is the raw material of synthetic a lot of medicines still, the raw material and the precursor substance of synthetic many Chemicals.
At present, most vanillic acid methyl esters derive from chemosynthesis, take from plant on a small quantity, and vanillic acid methyl esters also is many main volatile flavour ingredients vinous, its source be yeast saccharomyces cerevisiae or other bacterium with the vanillic acid methyl esters glucose glycoside material hydrolysis that contains in the grape, and discharge.
Vanillin is the important as precursors of vanillic acid methyl esters chemosynthesis.The Vanillin of supplying on the market has two kinds: the Vanillin of (1) chemosynthesis, and world's year sales volume surpasses 10,000 tons, is that raw material is synthetic with methyl catechol and xylogen mainly, and its market value is lower.(2) natural herb aldehyde is meant to be obtained through physics (comprise distillation, extraction) method, enzyme process or microbial process by the animals and plants material.Mainly from the alcohol extract of natural vanilla pod, the natural herb aldehyde price in this source is higher than the price (once reported and reach 300 times more than) of sintetics far away to the natural herb aldehyde that extracts from plant.Costing an arm and a leg of natural herb aldehyde is because the source of vanilla pod is very limited on the one hand, is subjected to the influence of the place of production, natural factor, cultivation, results and processing labour intensity are big, with high costs, particularly need in the vanilla planting process flower is carried out artificial pollination, be difficult to implant mass.To be people to the favor of natural flavor material have another reason adds.
The market requirement of natural herb aldehyde is growing, and as a kind of safe and reliable spices and bulk drug intermediate, it more and more comes into one's own.
The situation of vanillic acid methyl esters that uses and Vanillin is quite similar at present, and great majority derive from chemosynthesis; And come from the natural herb acid methyl esters of plant roots and stems because the source is difficult, extraction expends the cost height, it is big to consume, thereby cost is very expensive.If can come preparation of industrialization,, will become a quantum jump bright spot owing to demonstrate very big advantage in all many-sides such as starting material source, cost, production capacity, ecology, environmental benefits by microbial fermentation.The vanillic acid methyl esters that comes from microorganism preparation is expected to substitute expensive plant origin product or chemosynthesis product and becomes safe natural green product.
Summary of the invention
Technical problem to be solved by this invention is exactly the method with microorganism culturing, and promptly vanillic acid methyl esters can extract and get through cultivation, fermentation, purifying from ergot (Claviceps sp.) CGMCC No.3997.The present invention carries out slant culture, seed culture and fermentation culture respectively with bacterial classification on substratum, separation and purification obtains the product vanillic acid methyl esters again.Method of the present invention has been started with ergot or similar bacterial classification even has been prepared the beginning of vanillic acid methyl esters with biological method.A kind of preparation method is characterized in that being
Substratum:
1. slant medium (PDA) (%): potato extract 20, glucose 2, agar 2, pH6.0.
2. seed culture medium (%): glucose 10, citric acid 1, yeast extract paste 0.01, KH
2PO
40.05, MgSO
47H
2O 0.03, and KCl 0.03, FeSO
47H
2O 0.0007, ZnSO
47H
2O 0.0006, and ammoniacal liquor is transferred pH to 5.0.
3. the fermention medium of Cai Yonging contains: sucrose 1%-30%, and citric acid 0.1%-3.0%, yeast powder 0-0.5% or peptone 0-0.1%, inorganic salt comprise KH
2PO
4, MgSO
4, KCl, FeSO
4, ZnSO
4Deng a small amount of.
Contain through the more excellent fermention medium of test: sucrose 1%-10%, citric acid 0.1%-0.5%, yeast powder 0.01%-0.1% or peptone 0.01%-0.1%, KH
2PO
40.05%, MgSO
47H
2O 0.05%, and KCl 0.012%, FeSO
47H2O 0.0007%, ZnSO
47H
2O 0.0006%.
Culture condition:
Adopt second order fermentation, the bacterial classification inoculation of freeze pipe preservation is to slant medium, and in 25 ℃ of cultivations, after the slant pore maturation, the preparation spore suspension with spore suspension inoculation seed culture medium, is transferred to fermention medium in 25 ℃ of cultivations on the shaking table after 4-5 days.Fermentation culture conditions is: pH5-7, temperature 22-28 ℃, fermentation period 5-12 days.More excellent fermentation culture conditions is: pH5.0-6.0, temperature 23-26 ℃, fermentation period 8-10 days.
The separation and purification of product:
It is centrifugal to get fermented liquid, takes out mycelium and soaks with acetone.Soak solution is dissolved in acetone behind concentrating under reduced pressure: in the sherwood oil (10: 1), admixing proper silica gel (200～300 order) mixes, suitably after the drying silica gel packed into and use sherwood oil: in the good silicagel column of acetone (100: 1) pre-balance, use sherwood oil: the constant wash-out of acetone (10: 1) system, fraction collection, TLC are followed the tracks of to detect and are respectively flowed out part, collect to merge the part that contains target product, the concentrating under reduced pressure evaporate to dryness obtains vanillic acid methyl esters crude product I.
The centrifugal fermented supernatant fluid ethyl acetate extraction that obtains is told the extraction phase concentrating under reduced pressure.Concentrated solution is directly gone up silica gel column chromatography, with sherwood oil: acetone (50: 1), sherwood oil: acetone (10: 1) and sherwood oil: acetone (9: 1) carries out gradient elution, fraction collection, TLC follows the tracks of to detect and respectively flows out part, collection merges the part that contains target product, and the concentrating under reduced pressure evaporate to dryness obtains vanillic acid methyl esters crude product II.
Crude product I and II are merged, obtain crude product III.Sherwood oil has been used in crude product III adding: the silicagel column that acetone (100: 1) pre-balance is good (carries out the gradient elution second time in 40 * 1.5cm), elution system is a sherwood oil: acetone (100: 1), sherwood oil: acetone (50: 1), sherwood oil: acetone (40: 1); Fraction collection, TLC are followed the tracks of to detect and are respectively flowed out part, merge the part that contains target product, obtain the yellow oily material after concentrating, and analyze through HPLC, and its purity is more than 95%.
Above-mentioned prepared product is dissolved in small amount of acetone, and crystallization in methyl alcohol gets the white powder crystal, analyzes through HPLC, and its purity reaches more than 98%.
Analysing and detecting method:
HPLC (qualitative and quantitative):
Chromatographic column ZORBA * 80A Extend-C18 (4.6 * 250mm, 5 μ m), moving phase, methyl alcohol: water=60: 40, flow velocity 1.0ml/min, sample size 5 μ l detect wavelength 254nm.
TLC (qualitative):
Adopt Merck silica gel column chromatography plate GF254, the expansion system is as follows:
Sherwood oil: acetone=5: 2 Rf=0.5
Chloroform: methyl alcohol=9: 1 Rf=0.46
The physico-chemical property of product:
The conclusive evidence of product chemical structure:
According to the analysis-by-synthesis of physico-chemical property, ultraviolet, mass spectrum, hydrogen spectrum and carbon spectrum and hydrocarbon relevant spectrum, and reference literature report data compare, and determine that resulting material is a vanillic acid methyl esters.Structure is as follows:
Description of drawings
Fig. 1. be the HPLC collection of illustrative plates of refining back sample.
Embodiment
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment 1
Shake flask fermentation
The ergot CGMCC No.3997 bacterial classification inoculation of getting the freeze pipe preservation in 25 ℃ of cultivations, after the slant pore maturation, prepares spore suspension to slant medium.With spore suspension inoculation seed culture medium, on shaking table, cultivated 4 days for 25 ℃, seed culture fluid is arrived the shake flask fermentation substratum with 10% inoculum size transferred species.Fermentative medium formula is (%): sucrose 10, citric acid 0.1, peptone 0.02, KH
2PO
40.05, MgSO
47H
2O0.05, KCl0.012, FeSO
47H
2O 0.0001, ZnSO
47H
2O 0.0006; Ammoniacal liquor is transferred pH to 5.8; 26 ℃ of leavening temperatures, fermentation period 9 days.
Shake flask fermentation
After obtaining seed culture fluid with embodiment 1, with 10% inoculum size transferred species to the shake flask fermentation substratum.Fermentative medium formula is (%): sucrose 3, citric acid 0.3, peptone 0.08, KH
2PO
40.05, MgSO
47H
2O 0.05, and XCl 0.012, FeSO
47H
2O 0.0001, ZnSO
47H
2O 0.0006; Ammoniacal liquor is transferred pH to 5.2; 24 ℃ of leavening temperatures, fermentation period 8 days.
Embodiment 3
Shake flask fermentation
After obtaining seed culture fluid with embodiment 1, with 10% inoculum size transferred species to the shake flask fermentation substratum.Fermentative medium formula is (%): sucrose 2, citric acid 0.4, yeast powder 0.01, KH
2PO
40.05, MgSO
47H
2O 0.05, and KCl 0.012, FeSO
47H
2O 0.0001, ZnSO
47H
2O 0.0006; Ammoniacal liquor is transferred pH to 6.0; 23 ℃ of leavening temperatures, fermentation period 10 days.
Embodiment 4
The glass ferment tank
After obtaining seed culture fluid with above embodiment, with 10% inoculum size transferred species in the 10L glass fermentor tank that the 4L fermention medium is housed.Fermentative medium formula is (%): sucrose 5, citric acid 0.5, yeast powder 0.06, KH
2PO
40.05, MgSO
47H
2O 0.05, and KCl 0.012, FeSO
47H
2O 0.0001, ZnSO
47H
2O0.0006; Ammoniacal liquor is transferred pH to 5.5; 25 ℃ of leavening temperatures, fermentation period 8 days.
Embodiment 5
Product rough
Fermented liquid is collected in fermentation as embodiment 4 methods.Two jars merge the about 7L of fermented liquid, the about 600g of mycelium is taken out in centrifugal back, soaks with 600ml acetone.Soak solution is dissolved in 20ml acetone behind concentrating under reduced pressure: in the sherwood oil (10: 1), admixing 25g silica gel (200～300 order) mixes, suitably after the drying silica gel packed into and use sherwood oil: in the good silicagel column of acetone (100: 1) pre-balance, use sherwood oil: the constant wash-out of acetone (10: 1) system, fraction collection, TLC are followed the tracks of to detect and are respectively flowed out part, collect to merge the part that contains target product, the concentrating under reduced pressure evaporate to dryness obtains vanillic acid methyl esters crude product I.
The centrifugal fermented supernatant fluid that obtains equivalent ethyl acetate extraction is told the extraction phase concentrating under reduced pressure.Concentrated solution is directly gone up silica gel column chromatography, with sherwood oil: acetone (50: 1), sherwood oil: acetone (10: 1) and sherwood oil: acetone (9: 1) carries out gradient elution, fraction collection, TLC follows the tracks of to detect and respectively flows out part, collection merges the part that contains target product, and the concentrating under reduced pressure evaporate to dryness obtains vanillic acid methyl esters crude product II.
Crude product I and II are merged, obtain crude product III.Sherwood oil has been used in crude product III adding: the silicagel column that acetone (100: 1) pre-balance is good (carries out the gradient elution second time in 40 * 1.5cm), elution system is followed successively by sherwood oil: acetone (100: 1), sherwood oil: acetone (50: 1), sherwood oil: acetone (40: 1); Fraction collection, TLC are followed the tracks of to detect and are respectively flowed out part, merge the part that contains target product, obtain the yellow oily material after concentrating, and analyze through HPLC, and its purity is more than 95%.
Embodiment 6
Making with extra care of product
Embodiment 5 described prepared products are dissolved in small amount of acetone, and crystallization in methyl alcohol gets the about 230mg of white powder crystal after the drying, analyze through HPLC, and its purity reaches more than 98%.
Claims (3)
1. the preparation method of a vanillic acid methyl esters is characterized in that it being to extract and get through cultivation, fermentation, purifying from ergot (Claviceps sp.) CGMCCNo.3997.
2. method according to claim 1 is characterized in that the fermention medium that adopts contains: sucrose 1%-30%, and citric acid 0.1%-3.0%, yeast powder 0-0.5% or peptone 0-0.1%, inorganic salt comprise a small amount of KH
2PO
4, MgSO
4, KCl, FeSO
4, ZnSO
4Deng, fermentation culture conditions is: pH5-7, temperature 22-28, fermentation period 6-14 days.
3. method according to claim 1 and 2 is characterized in that fermention medium contains: sucrose 1%-10%, citric acid 0.1%-0.5%, yeast powder 0.01%-0.1% or peptone 0.01%-0.1%, KH
2PO
40.05%, MgSO
47H
2O 0.05%, KCl0.012%, FeSO
47H
2O0.0007%, ZnSO
47H
2O0.0006%, fermentation culture conditions is: pH5.0-6.0, temperature 23-26, fermentation period 8-10 days.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101472772A CN101200736A (en) | 2006-12-14 | 2006-12-14 | Method for preparing vanillic acid methyl esters |
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|---|---|---|---|
| CNA2006101472772A CN101200736A (en) | 2006-12-14 | 2006-12-14 | Method for preparing vanillic acid methyl esters |
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| Publication Number | Publication Date |
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| CN101200736A true CN101200736A (en) | 2008-06-18 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11219656B2 (en) | 2011-03-31 | 2022-01-11 | Industry-Academic Cooperation Foundation, Yonsei University | Method of using composition containing Hovenia dulcis Thunb. extract as active ingredient for prevention and treatment of bone diseases |
-
2006
- 2006-12-14 CN CNA2006101472772A patent/CN101200736A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11219656B2 (en) | 2011-03-31 | 2022-01-11 | Industry-Academic Cooperation Foundation, Yonsei University | Method of using composition containing Hovenia dulcis Thunb. extract as active ingredient for prevention and treatment of bone diseases |
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| TA01 | Transfer of patent application right |
Effective date of registration: 20080808 Address after: No. 1, Newton Road, Zhangjiang hi tech park, Shanghai, No. 200, 2B Applicant after: Shanghai Laiyi Biomedical Research and Development Center LLC Address before: Room 11, No. 258, Lane 3001, Zhuzhou Road, Shanghai, Hongkou District Applicant before: Song Zhengyu |
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Open date: 20080618 |