CN104372036A - Method for improving yield of fermentation produced shikimic acid with sorbitol as carbon source - Google Patents
Method for improving yield of fermentation produced shikimic acid with sorbitol as carbon source Download PDFInfo
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Abstract
The invention discloses a method for improving the yield of fermentation produced shikimic acid with sorbitol as a carbon source. The invention also discloses a fermentation medium for producing shikimic acid. The medium includes a carbon source, and the carbon source is 0.1-5% (g/ml) sorbitol. The fermentation medium for producing shikimic acid adopts sorbitol as the carbon source, so the yield of strain fermentation produced shikimic acid is significantly improved to 750% and is higher than the yield of shikimic acid produced by adopting traditional media adopting glucose as the carbon source. The method has the advantages of simple and effective process, and cheap raw material, can be applied to industrialized production of biosynthetic shikimic acid, and has a positive guiding effect on the optimization of a process for producing metabolites through Escherichia coli fermentation.
Description
Technical field
The present invention relates to a kind of method improving fermentative production shikimic acid output using sorbyl alcohol as carbon source.
Background technology
Shikimic acid (Shikimic acid), chemistry [3R-(3 α, 4 α, 5 β)]-SA by name, molecular formula is C
7h
10o
5, relative molecular weight is 174.15.
The continuous outburst of the H7N9 hypotype found at first Chinese along with influenza virus H 5 N 1 and H1N1 hypotype and 2013, the patent drugs " Tamiflu " (Ro 64-0796/002, Oseltamivir) of Roche Holding Ag becomes the first-selected anti-influenza virus medicament falling over each other to stock in countries in the world.As the crucial chiral starting materials that " Tamiflu " synthesizes, shikimic acid also causes the extensive concern of people.At present, the shikimic acid major part of synthesis needed for " Tamiflu " comes from Magnoliacea plant anise (Illicium verum), but utilize anistree extraction shikimic acid, complex manufacturing, yield is lower, raw material is subject to the factor restrictions such as seasonal climate, is difficult to meet needed for scale operation, and a large amount of felling plant also result in significant damage to environment.And utilizing microorganisms producing shikimic acid to have extremely wide application prospect, in research in recent years, utilizing Escherichia coli fermentation to produce shikimic acid also becomes focus gradually.
But this area utilizes the efficiency of intestinal bacteria production shikimic acid still not high at present, is difficult to meet the demand of market to shikimic acid, therefore, it may be necessary certain technology and improve fermenting process, to improve the output of fermentative production shikimic acid.
Summary of the invention
Technical problem to be solved by this invention is for the not high present situation of the efficiency of current fermentative production shikimic acid, and provides a kind of method improving fermentative production shikimic acid output using sorbyl alcohol as carbon source.Current this area yet there are no and utilizes sorbyl alcohol to improve the report of fermentative production shikimic acid output as carbon source, the invention belongs to pioneering, the present invention is by changing carbon source into sorbyl alcohol by common glucose, the growth performance of bacterial strain is improved, and finally to have made the output of shikimic acid have unexpected significantly improves.
One of technical scheme provided by the invention is: a kind of fermention medium producing shikimic acid, it comprises carbon source, described carbon source is 0.1 ~ 5% sorbyl alcohol, described per-cent is the grams of sorbyl alcohol contained in every 100 milliliters of fermention mediums, as in the every 100 milliliters of fermention mediums of 1% expression, the quality of sorbyl alcohol is 1 gram.
In the present invention, the component that the fermention medium that described fermention medium also comprises the production shikimic acid of this area routine comprises, the component such as nitrogenous source, inorganic salt that the fermention medium that namely described fermention medium also comprises the production shikimic acid of this area routine comprises.
Preferably, described fermention medium also comprises 1 ~ 5% nitrogenous source and 0.1 ~ 5% inorganic salt, and described nitrogenous source is selected from soy peptone, Tryptones, yeast powder, (NH
4)
2sO
4, NH
4nO
3and KNO
3in one or more, described inorganic salt are selected from MgSO
4, K
2hPO
4, KH
2pO
4, Na
2hPO
4, MnCl
2, CuSO
4with one or more in NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Wherein, preferably, the addition of described soy peptone is 0.1 ~ 2%, and the addition of described Tryptones is 0.1 ~ 2%, and the addition of described yeast powder is 0.1 ~ 2%, described (NH
4)
2sO
4addition be 0.1 ~ 3%, described NH
4nO
3addition be 0.1 ~ 3%, described KNO
3addition be 0.1 ~ 3%, described MgSO
4addition be 0.1 ~ 0.6%, described K
2hPO
4addition be 0.1 ~ 0.7%, described KH
2pO
4addition be 0.05 ~ 0.7%, described Na
2hPO
4addition be 0.05 ~ 0.6%, described MnCl
2addition be 0.05 ~ 0.6%, described CuSO
4addition be 0.1 ~ 0.6%, the addition of described NaCl is 0.05 ~ 0.6%, and described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Described nitrogenous source is soy peptone and/or NH more preferably
4nO
3, most preferably soy peptone; Described inorganic salt more preferably MgSO
4, K
2hPO
4, KH
2pO
4with the combination of NaCl.
In the present invention, described fermention medium can also comprise the organic acid in the made an addition to fermention medium of this area routine.
Preferably, described fermention medium also comprise in tyrosine, citric acid, phenylalanine and Sodium Glutamate one or more, preferably include tyrosine and citric acid, the addition of described tyrosine is 0.05 ~ 2%, the addition of described citric acid is 0.01 ~ 0.2%, the addition of described phenylalanine is 0.05 ~ 0.2%, and the addition of described Sodium Glutamate is 0.05 ~ 0.5%, and described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Preferably, described fermention medium comprises 0.1 ~ 5% sorbyl alcohol, 0.1 ~ 2% soy peptone, 0.1 ~ 3%NH in the present invention
4nO
3, 0.1 ~ 0.6%MgSO
4, 0.05 ~ 0.5% tyrosine, 0.05 ~ 0.15% citric acid, 0.1 ~ 0.7%K
2hPO
4, 0.05 ~ 0.7%KH
2pO
4with 0.05 ~ 0.6%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Further preferably, described fermention medium comprises 0.1 ~ 5% sorbyl alcohol, 0.5% soy peptone, 0.6%NH
4nO
3, 0.2%MgSO
4, 0.1% tyrosine, 0.1% citric acid, 0.3%K
2hPO
4, 0.1%KH
2pO
4and 0.1%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Best, described fermention medium comprises 2% sorbyl alcohol, 0.5% soy peptone, 0.6%NH
4nO
3, 0.2%MgSO
4, 0.1% tyrosine, 0.1% citric acid, 0.3%K
2hPO
4, 0.1%KH
2pO
4and 0.1%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Two of technical scheme provided by the invention is: a kind of method of adding sorbyl alcohol raising fermentative production shikimic acid output, and it comprises step: cultivate intestinal bacteria with aforementioned fermention medium.
In the present invention, described intestinal bacteria are for described in the routine of this area, as long as it can fermentative production shikimic acid, as E.coli BL21, E.coli JW0379-1 etc., and the preferred E. coli JW0379-1 of the present invention.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material except special instruction, all commercially.
Positive progressive effect of the present invention is:
Fermention medium for the production of shikimic acid of the present invention is using sorbyl alcohol as carbon source, and it significantly improves the output that strain fermentation produces shikimic acid, and what its output was more traditional is increased to 750% using glucose as carbon source.Technique of the present invention is simply effective, low in raw material price, during the industrialization that can be applicable to biosynthesizing shikimic acid is produced, and has positive directive function to other the process optimization producing meta-bolites with Escherichia coli fermentation.
Accompanying drawing explanation
Fig. 1 is the measurement result of shikimic acid content in comparative example 1 and embodiment 1 ~ 7 tunning, and wherein 0 is comparative example 1, and 1 ~ 7 is respectively embodiment 1 ~ 7.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Bacterial strain used in following embodiment is recombination bacillus coli JW0379-1(Baba T, Ara T, Hasegawa M, et al.Construction of Escherichia coli K-12in-frame, single-geneknockout mutants:the Keio collection.Mol Syst Biol2006.2:1-11), its genotype is F
-, Δ (araD-araB) 567, Δ lacZ4787(::rrnB-3), Δ aroL753::kan, λ
-, rph-1, Δ (rhaD-rhaB) 568, hsdR514.This recombination bacillus coli JW0379-1 bacterial strain by the present inventor in January, 2010 from E.coli Genetic Stock Center(New Haven, USA) buy obtain.
In following embodiment:
Fermention medium: specifically as described in each embodiment and comparative example 1, except fermention medium difference, each embodiment is all identical with all the other conditions of comparative example 1.
Seed culture medium: each embodiment and comparative example are all identical.It consists of: yeast extract 0.5%, Tryptones 1%, NaCl1%(Luria-Bertani substratum, with reference to " Molecular Cloning: A Laboratory guide " third edition, (J. Pehanorm Brooker D.W. Russell work)).
Culture condition: each embodiment and comparative example are all identical.By the strain inoculation of glycerine pipe preservation in the triangular flask that 20mL/250mL seed culture medium (containing 25mg/L kantlex) is housed, 37 DEG C, the cultivation of 220rpm shaker overnight, by seed liquor by (volume ratio) 3% inoculum size be seeded in 20mL/250mL fermention medium (containing 25mg/L kantlex), 37 DEG C, 220rpm shaking table cultivates 24 hours after sample 1mL, 5000rpm gets the mensuration that supernatant carries out shikimic acid content in centrifugal 5 minutes.
The measuring method of shikimic acid content: each embodiment and comparative example are all identical.The content of periodate oxidation method to shikimic acid disclosed in patent CN1351711 is adopted to measure.Get 0.25mL fermented supernatant fluid and add 0.25mL periodate reagent (0.5%H
5iO
6: 0.5%NaIO
4=1:1), add 0.5mL Na after 37 DEG C of insulation 0.5h
2sO
3with NaOH mixing solutions (0.056M Na
2sO
3: 1MNaOH=2:3) termination reaction, under 382nm wavelength, measure absorbancy after mixing.According to the output of shikimic acid contained in standard substance absorbance curve calculation sample.
Embodiment 1
Fermention medium: sorbyl alcohol 0.1%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 2
Fermention medium: sorbyl alcohol 0.5%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 3
Fermention medium: sorbyl alcohol 1%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 4
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 5
Fermention medium: sorbyl alcohol 3%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 6
Fermention medium: sorbyl alcohol 4%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 7
Fermention medium: sorbyl alcohol 5%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 8
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.05%, tyrosine 1.0%, citric acid 0.1%, K
2hPO
40.02%, NaCl0.03%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 9
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.05%, citric acid 0.01%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 10
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 2.0%, citric acid 0.2%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 11
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.05%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.05%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 12
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.6%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 13
Fermention medium: sorbyl alcohol 2%, soy peptone 0.1%, NH
4nO
33%, MgSO
40.2%, tyrosine 0.5%, citric acid 0.1%, K
2hPO
40.7%, KH
2pO
40.7%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 14
Fermention medium: sorbyl alcohol 2%, soy peptone 2%, NH
4nO
30.1%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.15%, K
2hPO
40.3%, KH
2pO
40.05%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 15
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.5%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.05%, K
2hPO
40.1%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 16
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
41%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
41%, KH
2pO
41%, NaCl2%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 17
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 18
Fermention medium: sorbyl alcohol 2%, Tryptones 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 19
Fermention medium: sorbyl alcohol 2%, Tryptones 0.1%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 20
Fermention medium: sorbyl alcohol 2%, Tryptones 2%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 21
Fermention medium: sorbyl alcohol 2%, yeast powder 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 22
Fermention medium: sorbyl alcohol 2%, yeast powder 0.1%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 23
Fermention medium: sorbyl alcohol 2%, yeast powder 2%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 24
Fermention medium: sorbyl alcohol 2%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 25
Fermention medium: sorbyl alcohol 2%, (NH
4)
2sO
40.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 26
Fermention medium: sorbyl alcohol 2%, KNO
30.1%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 27
Fermention medium: sorbyl alcohol 2%, (NH
4)
2sO
43%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 28
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 29
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 30
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 31
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, phenylalanine 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 32
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, Sodium Glutamate 0.05%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 33
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, Sodium Glutamate 0.2%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 34
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, Na
2hPO
40.3%, KH
2pO
40.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 35
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, CuSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 36
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, MnCl
20.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 37
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, CuSO
40.05%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.Embodiment 28
Embodiment 38
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, CuSO
40.6%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.Embodiment 29
Embodiment 39
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, tyrosine 0.1%, citric acid 0.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 40
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Embodiment 41
Fermention medium: sorbyl alcohol 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Comparative example 1(is except carbon source is glucose, and all the other components are all identical with embodiment 4)
Fermention medium: glucose 2%, soy peptone 0.5%, NH
4nO
30.6%, MgSO
40.2%, tyrosine 0.1%, citric acid 0.1%, K
2hPO
40.3%, KH
2pO
40.1%, NaCl0.1%.Described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
Output based on the fermentative Production shikimic acid under comparative example 1 technique is decided to be 100%, as the reference of other embodiments.
The tunning of embodiment 1 ~ 31 and comparative example 1 is carried out to the detection of shikimic acid content, result is as shown in table 1 and Fig. 1.As can be seen from Figure 1, fermention medium, using sorbyl alcohol as carbon source, compares the traditional cultural method of ratio 1(using glucose as carbon source) bacterial strain uses therefor produces the ability of shikimic acid obvious lifting.When sorbitol content is 2%, its output comparatively glucose is increased to 750% as carbon source, and when sorbyl alcohol addition is more than 2%, shikimic acid output no longer increases.If consider the Cost Problems of substratum, then the sorbyl alcohol of 2% is the optimum addition of substratum of the present invention.
The measurement result of shikimic acid relative content in each embodiment of table 1 and comparative example tunning
| Numbering | Shikimic acid relative content | Numbering | Shikimic acid relative content |
| Embodiment 1 | 148% | Embodiment 22 | 114% |
| Embodiment 2 | 232% | Embodiment 23 | 336% |
| Embodiment 3 | 397% | Embodiment 24 | 134% |
| Embodiment 4 | 750% | Embodiment 25 | 132% |
| Embodiment 5 | 747% | Embodiment 26 | 107% |
| Embodiment 6 | 745% | Embodiment 27 | 129% |
| Embodiment 7 | 746% | Embodiment 28 | 703% |
| Embodiment 8 | 740% | Embodiment 29 | 708% |
| Embodiment 9 | 733% | Embodiment 30 | 643% |
| Embodiment 10 | 645% | Embodiment 31 | 584% |
| Embodiment 11 | 725% | Embodiment 32 | 528% |
| Embodiment 12 | 746% | Embodiment 33 | 575% |
| Embodiment 13 | 258% | Embodiment 34 | 732% |
| Embodiment 14 | 374% | Embodiment 35 | 728% |
| Embodiment 15 | 637% | Embodiment 36 | 730% |
| Embodiment 16 | 677% | Embodiment 37 | 723% |
| Embodiment 17 | 702% | Embodiment 38 | 724% |
| Embodiment 18 | 573% | Embodiment 39 | 683% |
| Embodiment 19 | 232% | Embodiment 40 | 645% |
| Embodiment 20 | 315% | Embodiment 41 | 704% |
| Embodiment 21 | 732% | Comparative example 1 | 100% |
Claims (10)
1. produce a fermention medium for shikimic acid, it is characterized in that, it comprises carbon source, and described carbon source is 0.1 ~ 5% sorbyl alcohol, and described per-cent is the grams of sorbyl alcohol contained in every 100 milliliters of fermention mediums.
2. fermention medium as claimed in claim 1, it is characterized in that, described fermention medium also comprises 1 ~ 5% nitrogenous source and 0.1 ~ 5% inorganic salt, and described nitrogenous source is selected from soy peptone, Tryptones, yeast powder, (NH
4)
2sO
4, NH
4nO
3and KNO
3in one or more, described inorganic salt are selected from MgSO
4, K
2hPO
4, KH
2pO
4, Na
2hPO
4, MnCl
2, CuSO
4with one or more in NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
3. fermention medium as claimed in claim 2, it is characterized in that, the addition of described soy peptone is 0.1 ~ 2%, and the addition of described Tryptones is 0.1 ~ 2%, and the addition of described yeast powder is 0.1 ~ 2%, described (NH
4)
2sO
4addition be 0.1 ~ 3%, described NH
4nO
3addition be 0.1 ~ 3%, described KNO
3addition be 0.1 ~ 3%, described MgSO
4addition be 0.1 ~ 0.6%, described K
2hPO
4addition be 0.1 ~ 0.7%, described KH
2pO
4addition be 0.05 ~ 0.7%, described Na
2hPO
4addition be 0.05 ~ 0.6%, described MnCl
2addition be 0.05 ~ 0.6%, described CuSO
4addition be 0.1 ~ 0.6%, the addition of described NaCl is 0.05 ~ 0.6%, and described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
4. fermention medium as claimed in claim 3, it is characterized in that, described nitrogenous source is soy peptone and/or NH
4nO
3, described inorganic salt are MgSO
4, K
2hPO
4, KH
2pO
4with the combination of NaCl.
5. fermention medium as claimed in claim 3, it is characterized in that, described fermention medium also comprise in tyrosine, citric acid, phenylalanine and Sodium Glutamate one or more, the addition of described tyrosine is 0.05 ~ 2%, the addition of described citric acid is 0.01 ~ 0.2%, the addition of described phenylalanine is 0.05 ~ 2%, and the addition of described Sodium Glutamate is 0.05 ~ 0.5%, and described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
6. fermention medium as claimed in claim 5, it is characterized in that, described fermention medium comprises 0.1 ~ 5% sorbyl alcohol, 0.1 ~ 2% soy peptone, 0.1 ~ 3%NH
4nO
3, 0.1 ~ 0.6%MgSO
4, 0.05 ~ 0.5% tyrosine, 0.05 ~ 0.15% citric acid, 0.1 ~ 0.7%K
2hPO
4, 0.05 ~ 0.7%KH
2pO
4with 0.05 ~ 0.6%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
7. fermention medium as claimed in claim 6, it is characterized in that, described fermention medium comprises 0.1 ~ 5% sorbyl alcohol, 0.5% soy peptone, 0.6%NH
4nO
3, 0.2%MgSO
4, 0.1% tyrosine, 0.1% citric acid, 0.3%K
2hPO
4, 0.1%KH
2pO
4and 0.1%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
8. fermention medium as claimed in claim 7, it is characterized in that, described fermention medium comprises 2% sorbyl alcohol, 0.5% soy peptone, 0.6%NH
4nO
3, 0.2%MgSO
4, 0.1% tyrosine, 0.1% citric acid, 0.3%K
2hPO
4, 0.1%KH
2pO
4and 0.1%NaCl, described per-cent is the grams of each component contained in every 100 milliliters of fermention mediums.
9. improve a method for fermentative production shikimic acid output using sorbyl alcohol as carbon source, it comprises step: cultivate intestinal bacteria with the fermention medium as described in any one of claim 1 ~ 8.
10. the method improving fermentative production shikimic acid output using sorbyl alcohol as carbon source as claimed in claim 9, it is characterized in that, described intestinal bacteria are E.coli JW0379-1.
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