CN103805640B - A kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid - Google Patents
A kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid Download PDFInfo
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- CN103805640B CN103805640B CN201410038032.0A CN201410038032A CN103805640B CN 103805640 B CN103805640 B CN 103805640B CN 201410038032 A CN201410038032 A CN 201410038032A CN 103805640 B CN103805640 B CN 103805640B
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- 241000018646 Pinus brutia Species 0.000 title claims abstract description 16
- 230000003647 oxidation Effects 0.000 title claims abstract description 12
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 12
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- 235000007847 Acetobacter aceti Nutrition 0.000 claims abstract description 5
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid, comprise: (1) gets in a ring bacillus aceticus access liquid seed culture medium from the agar slant culture-medium preserving bacterial classification, 12-24 hour is cultivated, with activated spawn at 20-30 DEG C; (2) by seed liquor by volume per-cent 6-10% be linked in liquid fermentation medium, cultivate or quiescent culture 12 ~ 48h in 20-30 DEG C of shaking table; (3) treat in liquid fermentation medium, have a large amount of flosss to generate, the pine uncle aldehyde solution filtered through filtration sterilization is added in substratum, to make in substratum the final concentration of pine uncle aldehyde reach 0.1-3.0mmol/L, cultivate or quiescent culture after 0.5 ~ 7 day in 20-35 DEG C of shaking table, obtain forulic acid.Conversion yield of the present invention is higher, can reach 70-98%, and reaction conditions is gentle, environmental protection, has a good application prospect.
Description
Technical field
The invention belongs to the preparation field of forulic acid, particularly a kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid.
Background technology
Ferulyolated formal name used at school is called Ferulic acid, is one of derivative of TRANSCINNAMIC ACID, and it has cis and trans two kinds.Cis is yellow oil, and trans is white extremely micro-yellow crystal thing, relative molecular mass 194.19.Wherein trans-ferulaic acid is generally acknowledged natural safe free radical quencher and antioxidant.Forulic acid has strong scavenging(action) to hydrogen peroxide, superoxide radical, hydroxy radical qiao, peroxynitrite.Japan, the U.S. and some European countries allow forulic acid to add in food as foodstuff additive as antioxidant.Early stage research shows that forulic acid also has the multiple characteristic that can be used as medicinal use: such as effectively can reduce the degree of oxidation of uv-radiation to human lymphocyte; The release of the hematoblastic gathering of obvious suppression and the material such as hydroxy-tryptamine, thromboxane, thus the formation suppressing thrombus; By suppressing hydroxyl valeric acid-5-tetra-sodium dehydrogenase activity thus suppressing liver synthesis cholesterol, reduce serum lipid concentrations, thus prevent and treat atherosclerosis; There is immunoregulation effect.Simultaneously at cosmetic industry, forulic acid is formed with very strong restraining effect to melanic, can be used for the whitening nursing of skin or does nti-freckle treatment.
Forulic acid is extensively present in some plants, is one of effective constituent of Radix Angelicae Sinensis, Ligusticum wallichii, scouring rush, rattletop, the white skin of camphor tree etc.Its content in plant is generally only about 0.03%, and few owing to measuring, therefore from plant, extraction cost is higher.Along with improving constantly of study of pharmacy and human life quality, pharmacy synthesis, healthcare products and the demand of cosmetic industry to forulic acid are increasing.In order to the restriction not by natural resource, meet the growing market requirement, in the preparation of forulic acid, except extracting directly from plant, also comprise following semi-synthesis method, biological hydrolysis process and chemical synthesis.
One, semi-synthesis method
Semi-synthesis method is as thiaminogen, the acid of peat detritus are first degraded to extract again and prepared forulic acid by the material containing ferulic acid derivative.Such as, the structural unit containing forulic acid in thiaminogen, exists with the form of ester, and is easy to hydrolysis.Therefore, can first use basic hydrolysis thiaminogen, then prepare forulic acid by the method for acidifying, its reaction formula is as follows:
This method is easy to operate, and yield is up to 85.7%.Because thiaminogen source is wide, output is large, and moderate, so this method has certain industrial production value.
Two, biological hydrolysis process
This method is the mutation utilizing several microorganism (such as Arthrobacterglobiformis), and be converted into forulic acid by extracting the eugenol cinnamate obtained in Syzygium aromaticum stem oil, reaction equation is as follows:
In addition, fungi, bacterium and yeast can secrete feruloyl esterase.Feruloyl esterase (EC3.1.1.1) refers to and by Ferulic acid methylester, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester, forulic acid can be hydrolyzed free a kind of enzyme out.Have at present and adopt microorganism to directly act on cell wall substance as the preparation such as bagasse, wheat bran forulic acid, some fruitful work have been carried out in the research of feruloyl esterase.Such as, the microorganism of a collection of energy efficient secretion feruloyl esterase has been screened; The enzymatic property of this enzyme is studied in detail; Inquire into the synergy of feruloyl esterase and some polysaccharide degrading enzymes; The influence factor of production by biological enzyme and the industrial separation method of enzyme are inquired into.
Three, chemical synthesis
(1) Wittig-Horner reaction method synthesis forulic acid
There is Wittig-Horner reaction in triethyl-phosphite acetate (TEPA) and acetyl vanillin, then obtain forulic acid with concentrated hydrochloric acid acidifying in NaOH alkali system.
(2) Knoevenagel reaction method synthesis forulic acid
In pyridine solvent, adding a small amount of organic bases as catalyzer, there is Knoevenagel reaction and generates forulic acid in vanillin food grade,1000.000000ine mesh and propanedioic acid.But this method exists open defect, because the forulic acid that it obtains is the mixture of cis and trans-ferulaic acid, and the reaction times longer (being 3 weeks), solvent load is large, and productive rate is also very low.
The reaction times of chemosynthesis forulic acid is longer, and solvent load is large, low conversion rate, and cost is high; And discharging forulic acid with feruloyl esterase cooperative polysaccharide lytic enzyme, the microorganism strains obtaining forulic acid due to screening efficient degradation raw material is more difficult, and catalytic rate is slow simultaneously, and the potentiality of therefore enzyme process practical application are also very limited.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid, and the method conversion yield is higher, can reach 70-98%, and reaction conditions is gentle, environmental protection, has a good application prospect.
A kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid of the present invention, comprising:
(1) get in a ring bacillus aceticus access liquid seed culture medium from the agar slant culture-medium preserving bacterial classification, at 20-30 DEG C, cultivate 12-24 hour, with activated spawn;
(2) by seed liquor by volume per-cent 6-10% be linked in liquid fermentation medium, cultivate or quiescent culture 12 ~ 48h in 20-30 DEG C of shaking table;
(3) treat in liquid fermentation medium, have a large amount of flosss to generate, the pine uncle aldehyde solution through sterilising filtration is added in substratum, to make in substratum the final concentration of pine uncle aldehyde reach 0.1-3.0mmol/L, cultivate or quiescent culture after 0.5 ~ 7 day in 20-35 DEG C of shaking table, obtain forulic acid.
Bacillus aceticus in described step (1) is acetic acid Pseudomonas, gluconobacter suboxydans belongs to or glyconic acid genus acetobacter.
Liquid seed culture medium in described step (1) and (2) and the component of liquid fermentation medium are: N.F,USP MANNITOL, glucose, sucrose, glycerine or fructose 25g, peptone or Tryptones 3g, yeast extract 5g, water 1L, pH3.5-7.5,121 DEG C of sterilizing 20min; Or be: N.F,USP MANNITOL, glucose, sucrose, glycerine or fructose 20-100g, yeast extract 5g, peptone or Tryptones 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH3.5-7.5,121 DEG C of sterilizing 20min.
Cultivating in liquid seed culture medium in described step (1) is that shaking table is cultivated, and speed is 160 ~ 180r/min.
The speed that shaking table in described step (2) and (3) is cultivated is 100-250r/min.
beneficial effect
Conversion yield of the present invention is higher, can reach 70-98%, and reaction conditions is gentle, environmental protection, with traditional chemical method phase specific energy consumption is low, production technique is simple, by product is few, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is the course figure of acetobacter xylinum (AcetobacterxylinumATCC23770) oxidation conversion different concns coniferyl aldehyde; A is the changing conditions of residual glucose when adding 0-2mmol/L coniferyl aldehyde in substratum; B is the changing conditions of culturing process pH value when adding 0-2mmol/L coniferyl aldehyde in substratum; C is the proliferative conditions of viable bacteria in culturing process when adding 0-2mmol/L coniferyl aldehyde in substratum; D is the change of coniferyl aldehyde concentration when adding 0-2mmol/L coniferyl aldehyde in substratum.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
(1) activated spawn
Get a ring acetobacter xylinum (AcetobacterxylinumATCC23770) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or glycerine 25g, peptone 3g, yeast extract 5g, water 1L, pH3.5,121 DEG C of sterilizing 20min) in, cultivate 12 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 6%(v/v) be linked into liquid nutrient medium (glucose, N.F,USP MANNITOL, sucrose or glycerine 25g, peptone 3g, yeast extract 5g, water 1L, pH3.5,121 DEG C of sterilizing 20min) in, in 20 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 12h under 100r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 1.5mmol/L, in 20 DEG C, shaking table is cultivated or quiescent culture after 1 day under 100r/min condition, detects the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC).
UV and DAD detector joined by HPLC(Agilent1200series, C18 post) detection method: moving phase is A phase: 2mmol/L formic acid joins in Milli-Q ultrapure water; B phase: 2mmol/L formic acid joins acetonitrile.Flow rate of mobile phase: 0.4mL/min.Column temperature: 40 DEG C.Determined wavelength: 330nm.
Result is as the situation of 1.5mM coniferyl aldehyde in Fig. 1.Found that the amount of the coniferyl aldehyde be converted reaches 98%, the content being wherein converted into forulic acid accounts for 88% of initial coniferyl aldehyde content.
Embodiment 2
(1) activated spawn
Get a ring acetobacter xylinum (AcetobacterxylinumATCC23770) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or fructose 25g, Tryptones 3g, yeast extract 5g, water 1L, pH5.5,121 DEG C of sterilizing 20min) in, cultivate 18 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 8%(v/v) be linked into liquid nutrient medium (glucose, N.F,USP MANNITOL, sucrose or fructose 25g, Tryptones 3g, yeast extract 5g, water 1L, pH5.5,121 DEG C of sterilizing 20min) in, in 30 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 18h under 250r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 3.0mmol/L, in 35 DEG C, shaking table is cultivated or quiescent culture after 4 days under 250r/min condition, detect the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC), detection method is as embodiment 1.Found that the amount of the coniferyl aldehyde be converted reaches 55%, the content being wherein converted into forulic acid accounts for 35% of initial coniferyl aldehyde content.
Embodiment 3
(1) activated spawn
Get a ring acetobacter xylinum (AcetobacterxylinumATCC23770) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or fructose 25g, Tryptones 3g, yeast extract 5g, water 1L, pH5.5,121 DEG C of sterilizing 20min) in, cultivate 24 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 10%(v/v) be linked into liquid nutrient medium (N.F,USP MANNITOL, glucose, sucrose or fructose 25g, Tryptones 3g, yeast extract 5g, water 1L, pH5.5,121 DEG C of sterilizing 20min) in, in 25 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 48h under 150r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 3.0mmol/L, in 30 DEG C, shaking table is cultivated or quiescent culture after 0.5 day under 150r/min condition, detect the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC), detection method is as embodiment 1.Found that the amount of the coniferyl aldehyde be converted reaches 100%, the content being wherein converted into forulic acid accounts for 95% of initial coniferyl aldehyde content.
Embodiment 4
(1) activated spawn
Get an epoxidation gluconobacter suboxydans (GluconobacteroxydansDSM2003 or ATCC23773) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, cultivate 12 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 6%(v/v) be linked into liquid nutrient medium (glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, in 20 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 1h under 100r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 0.1mmol/L, in 20 DEG C, shaking table is cultivated or quiescent culture after 0.5 day under 100r/min condition, detect the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC), detection method is as embodiment 1.Found that the amount of the coniferyl aldehyde be converted reaches 100%, the content being wherein converted into forulic acid accounts for 100% of initial coniferyl aldehyde content.
Embodiment 5
(1) activated spawn
Get an epoxidation gluconobacter suboxydans (GluconobacteroxydansDSM2003) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, cultivate 14 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 10%(v/v) be linked into liquid nutrient medium (N.F,USP MANNITOL, glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, in 30 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 48h under 250r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 1.5mmol/L, in 35 DEG C, shaking table is cultivated or quiescent culture after 1 day under 250r/min condition, detect the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC), detection method is as embodiment 1.Found that the amount of the coniferyl aldehyde be converted reaches 100%, the content being wherein converted into forulic acid accounts for 90% of initial coniferyl aldehyde content.
Embodiment 6
(1) activated spawn
Get a ring gluconate pyracetobacillus (GluconacetobacterxylinusATCC23770 or ATCC53524) from the agar slant culture-medium preserving bacterial classification and access liquid seed culture medium (N.F,USP MANNITOL, glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, cultivate 12 hours, with activated spawn for 160 turns at 30 DEG C;
(2) spread cultivation bacterium cell
Will containing seed liquor by 8%(v/v) be linked into liquid nutrient medium (N.F,USP MANNITOL, glucose, sucrose or fructose 100g, yeast extract 5g, peptone 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH7.5,121 DEG C of sterilizing 20min) in, in 26 DEG C, shaking table is cultivated or for subsequent use after quiescent culture 20h under 140r/min condition;
(3) add coniferyl aldehyde and prepare forulic acid
Treat in substratum, have a large amount of flosss to generate, respectively to the coniferyl aldehyde solution added in substratum through sterilising filtration, the final concentration of coniferyl aldehyde in substratum is made to reach 3.0mmol/L, in 20 DEG C, shaking table is cultivated or quiescent culture after 2 days under 100r/min condition, detect the transformation efficiency of the coniferyl aldehyde in substratum with high performance liquid chromatography (HPLC), detection method is as embodiment 1.Found that the amount of the coniferyl aldehyde be converted reaches 95%, the content being wherein converted into forulic acid accounts for 86% of initial coniferyl aldehyde content.
Claims (4)
1. utilize bacterial oxidation pine uncle aldehyde to prepare a method for forulic acid, comprising:
(1) get in a ring bacillus aceticus access liquid seed culture medium from the agar slant culture-medium preserving bacterial classification, at 20-30 DEG C, cultivate 12-24 hour, with activated spawn; Wherein, bacillus aceticus is acetobacter xylinum AcetobacterxylinumATCC23770;
(2) by seed liquor by volume per-cent 6-10% be linked in liquid fermentation medium, cultivate or quiescent culture 12 ~ 48h in 20-30 DEG C of shaking table;
(3) treat in liquid fermentation medium, have a large amount of flosss to generate, the pine uncle aldehyde solution through sterilising filtration is added in substratum, to make in substratum the final concentration of pine uncle aldehyde reach 0.1-3.0mmol/L, cultivate or quiescent culture after 0.5 ~ 7 day in 20-35 DEG C of shaking table, obtain forulic acid.
2. a kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid according to claim 1, it is characterized in that: the liquid seed culture medium in described step (1) and (2) and the component of liquid fermentation medium are: N.F,USP MANNITOL, glucose, sucrose, glycerine or fructose 25g, peptone or Tryptones 3g, yeast extract 5g, water 1L, pH3.5-7.5,121 DEG C of sterilizing 20min; Or be: N.F,USP MANNITOL, glucose, sucrose, glycerine or fructose 20-100g, yeast extract 5g, peptone or Tryptones 5g, citric acid 1.15g, Na
2hPO
42.7g, water 1L, pH3.5-7.5,121 DEG C of sterilizing 20min.
3. a kind of method utilizing bacterial oxidation pine uncle aldehyde to prepare forulic acid according to claim 1, is characterized in that: cultivating in liquid seed culture medium in described step (1) is that shaking table is cultivated, and speed is 160 ~ 180r/min.
4. a kind of bacterial oxidation pine uncle aldehyde that utilizes according to claim 1 prepares the method for forulic acid, it is characterized in that: the speed of the shaking table cultivation in described step (2) and (3) is 100-250r/min.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1325444A (en) * | 1998-10-31 | 2001-12-05 | 哈尔曼及赖默股份有限公司 | Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1325444A (en) * | 1998-10-31 | 2001-12-05 | 哈尔曼及赖默股份有限公司 | Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism |
Non-Patent Citations (3)
| Title |
|---|
| ACCESSION：CCT60361;GenPept;《GenPept》;20130809;全文 * |
| Characterization of a Novel NADPH-Dependent Oxidoreductase from Gluconobacter oxydans;Chen M.等;《Mol Biotechnol》;20101231;全文 * |
| Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene;ACHTERHOLT S.等;《JOURNAL OF BACTERIOLOGY》;19980930;第180卷(第17期);摘要、图1 * |
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