CN105154479A - Microbial-conversion-based method for synthesizing 3-hydroxy beta-ionone and beta-ionone - Google Patents
Microbial-conversion-based method for synthesizing 3-hydroxy beta-ionone and beta-ionone Download PDFInfo
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- CN105154479A CN105154479A CN201510466265.5A CN201510466265A CN105154479A CN 105154479 A CN105154479 A CN 105154479A CN 201510466265 A CN201510466265 A CN 201510466265A CN 105154479 A CN105154479 A CN 105154479A
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Abstract
The invention discloses a microbial-conversion-based method for synthesizing 3-hydroxy beta-ionone and beta-ionone. The method includes the following steps: (1), culturing scattered pantoea, inoculating a triangular bottle filled with a seed culture medium with a scattered pantoea strain, and oscillating at 180-200r/min under the conditions of pH being 6.5 and temperature being 30-34 DEG C for culture for 10-18h to obtain seed liquid; (2), fermenting in a seed tank; (3), obtaining 3-hydroxy beta-ionone and beta-ionone. By searching fermentation culture media, an optimal fermentation culture medium capable of efficiently degrading lutein is obtained, and 3-hydroxy beta-ionone and beta-ionone which have aromatic substances can be generated. The 3-hydroxy beta-ionone and beta-ionone are important aromatic substances, and tobacco quality can be improved greatly by adding a small amount of the aromatic substances into tobacco. The microbial-conversion-based method has huge application prospect in utilizing biologically-degraded lutein and generating natural aromatic substances.
Description
Technical field
The invention belongs to field of bioconversion, adopt dispersion general bacterium degraded xenthophylls to generate fragrance matter 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone, relate to bioconversion medium optimization and the application thereof of general bacteria microorganism.
Background technology
Xenthophylls is extensively present in the plants such as fresh flower, fruits and vegetables, tobacco, is the precursor of many important aroma components.At flue-cured tobacco in ripe and combustion processes, xenthophylls degradable is converted into alpha, beta-lonone, Megastigmatrienone etc., and they have the strong fragrance of a flower and fruital, and fragrance threshold value is very low, has important value preparing in essence and flavoring agent.But mainly concentrate on physical chemistry degraded at present about the degraded of xenthophylls, lack specificity.The research that biological degradation method especially utilizes enzyme and microorganism to produce fragrance matter to xenthophylls of degrading is also little.Biological process degraded xenthophylls and mechanical degradation are compared with chemical degradation and are had the remarkable advantage of following two aspects: first biological process is degraded and be make use of enzymatic specificity, obtains the fragrance matter that composition is relatively single.Secondly, the biological process fragrance matter obtained of degrading is identified as " natural component ".Biological degradation xenthophylls will more and more receive everybody concern.
Disperse general bacterium to be found in the earliest in plant, seed, be the yellow flora that a class produces class yellow pigment, belong to general Pseudomonas.The general bacterium of most dispersion can L-arabinose fermentation, D-lactose, maltose, sucrose, trehalose and wood sugar.But there is not its xenthophylls of can degrading of bibliographical information to generate 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone.
Summary of the invention
The object of the invention is to utilize microbiological deterioration xenthophylls to generate fragrance matter 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone, prepare industry at essence and flavoring agent and have broad application prospects.The invention belongs to Biological preparation 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone, its product is identified as " natural component ", and application has certain advantage.Best medium when simultaneously the present invention further defines degraded xenthophylls, prepares 3-hydroxyl alpha, beta-lonone for industrialization and alpha, beta-lonone lays the foundation.
Technical scheme of the present invention is: a kind of microbial transformation synthesis 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone method, and its step is as follows:
(1) disperse general bacterium to cultivate, be inoculated into by the general bacteria strain of dispersion and be equipped with in the triangular flask of seed culture medium, under the condition of pH=6.5,30-34 DEG C, 180-200r/min shaking culture 10-18h, obtains seed liquor;
(2) seeding tank fermentation: by seed liquor by volume 1% inoculum size be seeded in the seeding tank that seeding tank liquid fermentation medium is housed and ferment, logarithmic phase is entered to thalline, described seeding tank liquid fermentation medium is: with the quality of 1L water for benchmark, following ingredients is added: xenthophylls 0.015g by following quality, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains fermentation seed liquid;
(3) by the fermentation seed liquid of step (2) seeding tank by volume the inoculum size of 5-10% be seeded in the fermentor tank that liquid fermentation medium is housed and ferment, after fermentation 48h, biomass reaches 50 ~ 80 × 108cfu/mL, go out tank, described liquid fermentation medium is: with the quality of 1L water for benchmark, adds following ingredients by following quality: xenthophylls 0.015g, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone.
Seed culture based formulas in described step (1): distilled water 1L, sucrose 10g, asparagine 4.5g, K
2hPO
41.5g, MgSO
40.5g, yeast extract paste 3.0g, FeSO
40.01g.
The invention has the beneficial effects as follows: after dichloromethane extraction obtains the fermented liquid of the general bacterium of dispersion, by the product before and after GC-MS analyses and comparison fermentation, find in the general bacteria strain fermented liquid of dispersion of the present invention containing 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone, wherein 3-hydroxyl alpha, beta-lonone content is 30%, and alpha, beta-lonone content is 20%.The fermented liquid produced has volatile violet fragrance, obtains the fragrance matter of high-quality by deep processing.
The present invention, by groping its fermention medium, obtains and the optimum fermention medium scheme of efficient degradation xenthophylls also can generate the 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone with fragrance matter.3-hydroxyl alpha, beta-lonone and alpha, beta-lonone are important fragrance matters, and adding this kind of a small amount of fragrance matter in tobacco can promote cigarette quality to a great extent.The present invention is utilizing biological degradation xenthophylls, and produces natural flavor material aspect and present huge application prospect.
Accompanying drawing explanation
Fig. 1 is the growing state of the general bacterium of dispersion on the solid medium taking xenthophylls as substrate;
Fig. 2 is the uv-absorbing light figure of dispersion general bacterium degraded xenthophylls process;
Fig. 3 is the thin-layer chromatogram of lutein degradation process;
Fig. 4 is that the product GC-MS after lutein degradation schemes.
Embodiment
A kind of microbial transformation synthesis 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone method, its step is as follows:
(1) disperse general bacterium at-80 DEG C of very low temperature Cryopreservations, protective material is 20% glycerol; Disperse general bacterium to cultivate, the general bacteria strain of dispersion is inoculated into and is equipped with in the triangular flask of seed culture medium, seed culture based formulas: distilled water 1L, sucrose 10g, asparagine 4.5g, K
2hPO
41.5g, MgSO
40.5g, yeast extract paste 3.0g, FeSO
40.01g, under the condition of pH=6.5,30-34 DEG C, 180-200r/min shaking culture 10-18h, obtains seed liquor;
(2) seeding tank fermentation: by seed liquor by volume 1% inoculum size be seeded in the seeding tank that seeding tank liquid fermentation medium is housed and ferment, logarithmic phase is entered to thalline, described seeding tank liquid fermentation medium is: with the quality of 1L water for benchmark, following ingredients is added: xenthophylls 0.015g by following quality, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains fermentation seed liquid;
(3) by the fermentation seed liquid of step (2) seeding tank by volume the inoculum size of 5-10% be seeded in the fermentor tank that liquid fermentation medium is housed and ferment, after fermentation 48h, biomass reaches 50 ~ 80 × 108cfu/mL, go out tank, described liquid fermentation medium is: with the quality of 1L water for benchmark, adds following ingredients by following quality: xenthophylls 0.015g, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains fermented liquid, fermented liquid is under lucifuge condition, the centrifugal 10min of 12000r/min, gets supernatant liquor, adds equal-volume methylene dichloride and repeatedly extracts 3 times, obtain the extract of xenthophylls fermented liquid, include reaction product 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone, add anhydrous Na 2SO4 dried overnight, and do not connect bacterium culture medium in contrast with equal-volume.
Disperse general bacterium to be preserved in American Type Culture collection warehousing (Americantypeculturecollection), preserving number is ATCC29922.Disperse general bacterium can also be that Wen bacterium (Ewingellaamericana) is liked in America.
Fig. 1 be presented at xenthophylls be substrate solid medium on, the position after microorganism line be all white, illustrates that xenthophylls obtains degraded.
Uv absorption spectra full wavelength scanner is carried out to 0h, 24h, 48h, 96h different time fermentation, extraction liquid: be in certain wavelength region, sample is measured, to reflect sample absorption value at different wavelengths.During analysis for many biological components, do not need just can out by each compound mensuration through being separated simultaneously.Condition determination: sweep limit 200-600nm, each Sample Scan 3 times, sweep velocity 600nm/min, 0.5nm interval gathers.Fig. 2 shows: three charateristic avsorption bands of xenthophylls are respectively 430nm, 448nm, 460nm, and the absorption peak of lutein degradation generating feature fragrance matter is 250-300nm.0h in Fig. 2, lutein content is the highest, does not now almost have fragrance matter to produce.Along with time variations, at 48h, the content of xenthophylls obviously declines, and fragrance matter increases gradually.When arriving 96h, most of xenthophylls is degraded, and fragrance matter content obviously increases.Whole change procedure and Changing Pattern embody clearly from scanning spectra.
Thin layer chromatography: get xenthophylls standard substance respectively, jononeionone standard substance, fermentation 48h fermented liquid, fermentation 96h fermented liquid 5uL, with this point on same thin plate.With sherwood oil: methylene dichloride: ethyl acetate=7:2:1 is developping agent, launch, taking-up is dried, and develops the color in the methanolic of 5%, and 90 ° of C baking ovens are put in taking-up dries, and contrasts, the position of colour developing spot and standard substance to determine the composition transfer in fermented liquid.In Fig. 3, from left to right sample is followed successively by xenthophylls standard specimen, jononeionone standard specimen, fermentation 48h extraction liquid, fermentation 96h extraction liquid.As can be seen from the figure, because xenthophylls self is with color, before dyeing, xenthophylls standard specimen Rf value is 0.57, fermentation 48h extraction liquid also has yellow spotting in same position, illustrate now xenthophylls some do not degrade, and in the 96h extraction liquid that ferments, almost there is no yellow spotting, illustrated that now most of xenthophylls is degraded.
Develop the color with 5% methanolic, shown in after result dyeing, the standard specimen of jononeionone is clear to be displayed, Rf value is 0.79, and the 48h extraction liquid that now ferments has had jononeionone to exist, but content is very low, and ferment in 96h extraction liquid, Rf value is that 0.79 place's spot is obvious.Therefore jononeionone is contained in degraded product.
The condition that GC-MS analyzes:
Analytical procedure: with chromatographic pure dichloromethane extractive fermentation liquid, collect organic phase in triangular flask.Because moisture remaining in fermentation, extraction liquid may impact chromatography of gases instrument, therefore add a certain amount of anhydrous sodium sulphate 4 DEG C of hold over night, take out the moisture remained in extraction liquid.Through concentration, be placed in sample injection bottle, carry out GC-MS qualitative analysis.
Gas phase condition: chromatographic column: HP-5(30m × 0.25mm; 0.25rm); Heating schedule: initial temperature 45 DEG C, keeps 2min, is raised to 120 DEG C with 4 DEG C/min, retains 2min, is raised to 160 DEG C with 1 DEG C/min, retains 5min, is raised to 200 DEG C, is raised to 280 DEG C with 5 DEG C/min with 3 DEG C/min, retains 5min; Injector temperature: 270 DEG C; Transmission line temperature: 270 DEG C; Splitting ratio: 10:1; Carrier gas: He gas; Flow velocity: 1mL/min
Mass Spectrometry Conditions: ionization mode EI, ionization voltage 70eV; Ion source temperature 230 DEG C; Mass scan range: 30-550amu.
Fig. 4 is the GC-MS analysis chart of tunning, and Fig. 4 is fermentation 48h sample, and analyzing main degradation products through GC-MS is 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone two kinds.
Claims (2)
1. microbial transformation synthesis 3-hydroxyl alpha, beta-lonone and an alpha, beta-lonone method, it is characterized in that, its step is as follows:
(1) disperse general bacterium to cultivate, be inoculated into by the general bacteria strain of dispersion and be equipped with in the triangular flask of seed culture medium, under the condition of pH=6.5,30-34 DEG C, 180-200r/min shaking culture 10-18h, obtains seed liquor;
(2) seeding tank fermentation: by seed liquor by volume 1% inoculum size be seeded in the seeding tank that seeding tank liquid fermentation medium is housed and ferment, logarithmic phase is entered to thalline, described seeding tank liquid fermentation medium is: with the quality of 1L water for benchmark, following ingredients is added: xenthophylls 0.015g by following quality, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains fermentation seed liquid;
(3) by the fermentation seed liquid of step (2) seeding tank by volume the inoculum size of 5-10% be seeded in the fermentor tank that liquid fermentation medium is housed and ferment, after fermentation 48h, biomass reaches 50 ~ 80 × 108cfu/mL, go out tank, described liquid fermentation medium is: with the quality of 1L water for benchmark, adds following ingredients by following quality: xenthophylls 0.015g, glucose 11g, sucrose 1g, yeast extract paste 1.8g, K
2hPO
40.5mg, leavening temperature is 30-34 DEG C, and ventilating ratio is 0.5 ~ 0.8:1, obtains 3-hydroxyl alpha, beta-lonone and alpha, beta-lonone.
2. microbial transformation synthesis 3-hydroxyl alpha, beta-lonone according to claim 1 and alpha, beta-lonone method, is characterized in that: seed culture based formulas in described step (1): distilled water 1L, sucrose 10g, asparagine 4.5g, K
2hPO
41.5g, MgSO
40.5g, yeast extract paste 3.0g, FeSO
40.01g.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399399A (en) * | 2016-08-30 | 2017-02-15 | 郑州轻工业学院 | Method for production of 8-methyl-alpha-ionone by biodegradation of lutein |
| CN107227314A (en) * | 2017-03-10 | 2017-10-03 | 云南中烟工业有限责任公司 | A kind of method that lutein of being degraded with aspergillus oryzae prepares tobacco aromaticss |
| CN113413377A (en) * | 2021-06-23 | 2021-09-21 | 陕西中医药大学 | Application of 3-hydroxy-beta-ionone in preparation of drugs for treating vascular related diseases |
-
2015
- 2015-07-31 CN CN201510466265.5A patent/CN105154479A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| YUE ZHAO等: "Bioconversion of lutein to form aroma compounds by Pantoea dispersa", 《BIOTECHNOL LETT》 * |
| 杨雪鹏等: "应用响应面法优化叶黄素降解发酵培养基", 《生物工程》 * |
| 杨雪鹏等: "烟叶叶黄素降解菌发酵条件研究", 《河南农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399399A (en) * | 2016-08-30 | 2017-02-15 | 郑州轻工业学院 | Method for production of 8-methyl-alpha-ionone by biodegradation of lutein |
| CN106399399B (en) * | 2016-08-30 | 2019-10-29 | 郑州轻工业学院 | A kind of biodegrade lutein generation 8- methyl-α-ionone method |
| CN107227314A (en) * | 2017-03-10 | 2017-10-03 | 云南中烟工业有限责任公司 | A kind of method that lutein of being degraded with aspergillus oryzae prepares tobacco aromaticss |
| CN113413377A (en) * | 2021-06-23 | 2021-09-21 | 陕西中医药大学 | Application of 3-hydroxy-beta-ionone in preparation of drugs for treating vascular related diseases |
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