CN105713933B - A kind of biological preparation method of 2 phenylethyl alcohol - Google Patents
A kind of biological preparation method of 2 phenylethyl alcohol Download PDFInfo
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- CN105713933B CN105713933B CN201610256845.6A CN201610256845A CN105713933B CN 105713933 B CN105713933 B CN 105713933B CN 201610256845 A CN201610256845 A CN 201610256845A CN 105713933 B CN105713933 B CN 105713933B
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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Abstract
The invention discloses a kind of biological preparation methods of 2 phenylethyl alcohol.Choose Candida glycerolgenesis resistant to high osmotic pressure (Candida glycerinogenes CCTCC M 93018) conversion L-phenylalanine fermenting and producing 2 phenylethyl alcohol.The yeast, up to 4g/L, effectively overcomes the growth inhibition that 2 phenylethyl alcohol produces bacterium to host, the yield of 2 phenylethyl alcohol reaches 5g/L, conversion ratio reaches 0.71 (g/g) to the tolerance of 2 phenylethyl alcohol.Preparation method of the invention has the characteristics that easy to operate, high conversion efficiency, production cost are low, industrial applications prospect is wide.
Description
Technical field
The present invention relates to a kind of biological preparation methods of 2 phenylethyl alcohol, belong to microorganisms technical field.
Background technique
2 phenylethyl alcohol, also known as bata-phenethyl alcohol, diphenyl ethyl alcohol are a kind of aromatic alcohols with Rose Essentielle, lightly seasoned refined
Exquisiteness is present in the plants essential oils such as rose, narcissus, lily, jasmine more, and 2 phenylethyl alcohol is also bread, cheese, biscuit, grape
Flavor substance in the food such as wine, the substance derive Ester in alkaline environment and to the oxidatively stable of air,
Such as phenethyl acetate is also important aromatic props.It is a kind of aromatic alcohol with Rose Essentielle, lightly seasoned refined exquisiteness is deposited more
It is in the plants essential oils such as rose, narcissus, lily, jasmine, 2 phenylethyl alcohol is also in the food such as bread, cheese, biscuit, grape wine
Flavor substance, which derives Ester, such as acetic acid benzene second in alkaline environment and to the oxidatively stable of air
Ester etc. is also important aromatic props.
The bacterial strain of the 2 phenylethyl alcohol of bioconversion production at present is concentrated mainly on yeast.Common 2 phenylethyl alcohol yeast production bacterium
Strain includes: saccharomyces cerevisiae (Saccharomyces cerevisiae), kluyveromyces marxianus (Kluyveromyces
Marxianus), Yarrowia lipolytica (Yarrowia lipolytica) etc..2 phenylethyl alcohol (the Log Pow=of high concentration
1.36) there is certain toxicity to microbial cell.Research shows that the 2 phenylethyl alcohol that concentration is 2g/L can completely inhibit Marx
Growth (Fabre CE, Blanc PJ, the Goma G.Production of 2-phenylethyl alcohol of kluyveromyces
by Kluyveromyces marxianus[J].Biotechnol Prog,1998,14:270–274.)；2.5g/L 2- benzene second
Alcohol, which can make saccharomyces cerevisiae biomass reduce by 75%, 3g/L 2 phenylethyl alcohol, can almost completely inhibit Wine brewing yeast strain growth
(Seward RJ,Willets MG,Dinsdale,et al.The effects of ethanol,hexan-1-ol,and 2-
phenylethanol on cider yeast growth,viability,and energy status；Synergistic
inhibition[J].J I Brewing.1996,102(6):439-443.).The yield for eventually leading to 2 phenylethyl alcohol is lower, hair
The concentration of 2 phenylethyl alcohol is below greatly 4g/L in zymotic fluid.
Candida glycerolgenesis (Candida glycerinogenes CCTCC M 93018) is that China possesses and independently knows
One plant of industrial strain with excellent fermenting property for knowing property right, can be in the hyperosmosis culture of 55% glucose or 15%NaCl
Normal growth is bred on base, has the characteristics that resistance to hypertonic and high resistance to cold and diseases.
Summary of the invention
The present invention in order to solve the above technical problems, is provided one kind and is made a living with Candida glycerolgenesis resistant to high osmotic pressure
The method for producing bacterial strain production 2 phenylethyl alcohol.
A kind of biological preparation method of 2 phenylethyl alcohol, steps are as follows:
(1) in 1 ring Candida glycerolgenesis of picking access seed culture medium, at 30 DEG C, under the conditions of 200r/min, oscillation training
18h is supported, liquid seed is obtained.
(2) the liquid seed for obtaining step (1) by the inoculum concentration access fermentation medium of 4% (v/v), ferment by control
Temperature is 30 DEG C, revolving speed 500r/min, time 50h, fermentation ends.
Preferred according to the present invention, seed culture medium is yeast powder 10g/L, peptone 20g/L, Portugal in the step (1)
Grape sugar 20g/L, surplus are water；Preferred according to the present invention, fermentation medium is L-phenylalanine 7g/L in the step (2),
Glucose 90g/L, KH2PO45g/L, yeast powder 4g/L, MgSO4·7H2O 0.5g/L, surplus are water.
Beneficial effects of the present invention:
1. Candida glycerolgenesis bacterial strain 2 phenylethyl alcohol tolerance selected by the present invention is high, up to 4g/L.Further overcome
2 phenylethyl alcohol is conducive to the bacterial strain answering in 2 phenylethyl alcohol industrial production during the fermentation to the toxic action of bacterial strain
With.
The characteristics of 2 phenylethyl alcohol yield is high 2. Candida glycerolgenesis bacterial strain selected by the present invention has, high conversion rate, warp
Detection, the bacterial strain can generate the benzyl carbinol of 5g/L using the L-phenylalanine of 7g/L, and conversion ratio is 0.71 (g/g).
Detailed description of the invention
Fig. 1 is growing state of the Candida glycerolgenesis in the culture medium of 2 phenylethyl alcohol containing various concentration.
Fig. 2 is the gas chromatography-mass spectrography map that Candida glycerolgenesis bacterial strain produces 2 phenylethyl alcohol sample.A is that production is sweet
Oily candida bacterial strain produces the gas chromatogram of 2 phenylethyl alcohol sample；B is that Candida glycerolgenesis bacterial strain produces 2 phenylethyl alcohol sample
Mass spectrogram.
Fig. 3 is the conditional curve of Candida glycerolgenesis fermentation synthesis 2 phenylethyl alcohol.
Specific embodiment
The present invention is described in further detail below by embodiment.
Example 1
1 ring Candida glycerolgenesis of picking accesses seed culture medium (yeast powder 10g/L, peptone 20g/L, glucose
20g/L, surplus are water) in, at 30 DEG C, under the conditions of 200r/min, shaken cultivation 18h obtains liquid seed.The liquid that will be obtained
Seed accesses 2 phenylethyl alcohol culture medium (yeast powder 10g/L, peptone 20g/ containing various concentration by the inoculum concentration of 4% (v/v)
L, glucose 20g/L, surplus are water) in, the concentration of 2 phenylethyl alcohol is respectively 0g/L, 1.0g/L, 2.0g/L, 3.0g/L, 3.5g/
L, 4.0g/L.Liquid amount is 50mL/250mL, and control cultivation temperature is 30 DEG C, revolving speed 200r/min, is sampled in different time
Measure the increment OD of yeast600(attached drawing 1).
Example 2
1 ring Candida glycerolgenesis of picking accesses seed culture medium (yeast powder 10g/L, peptone 20g/L, glucose
20g/L, surplus are water) in, at 30 DEG C, under the conditions of 200r/min, shaken cultivation 18h obtains liquid seed.The liquid that will be obtained
Seed accesses fermentation medium (L-phenylalanine 7g/L, glucose 90g/L, KH by the inoculum concentration access of 4% (v/v)2PO4 5g/
L, no amino acid yeast nitrogen YNB 0.17g/L, MgSO4·7H2O 0.5g/L, surplus are water) in, liquid amount 30mL/
250mL, control fermentation temperature are 30 DEG C, revolving speed 200r/min, time 50h, fermentation ends.
Product confirmation is detected using gas chromatography-mass spectrography, by 4 DEG C of fermentation liquid made from embodiment 2,10,000r/m
It is centrifuged 10min, takes supernatant in separatory funnel, the pentane of same volume and the mixed solution (2:1, v:v) of hexamethylene is added
It mixes, extracted overnight, by organic phase addition anhydrous sodium sulfate dehydration, supernatant is taken to detect for compounds GC-MS.
Gas chromatography-mass spectrum (GC-MS) instrument (Broker SCION SQ, USA), chromatographic column DB-wax, (30m ×
0.25mm, 0.25 μm), sample volume 0.2 μ L, carrier gas He, flow velocity 0.8mL/min, injector temperature are 260 DEG C, and column oven rises
Beginning temperature is 50 DEG C, retains 1min, with 10 DEG C/min temperature programming to 230 DEG C, retains 5min to terminal.Connecting rod temperature 250
DEG C, mass scan range 33-400m/z, ionization voltage 70eV.Library, which is composed, using Nist 98 carries out map retrieval.Mass ions figure
Shown in parsing result attached drawing 2.
Example 3
1 ring Candida glycerolgenesis of picking accesses seed culture medium (yeast powder 10g/L, peptone 20g/L, glucose
20g/L, surplus are water) in, at 30 DEG C, under the conditions of 200r/min, shaken cultivation 18h obtains liquid seed.The liquid that will be obtained
Seed by 4% (v/v) inoculum concentration access access containing 2.5L fermentation medium (L-phenylalanine 7g/L, glucose 90g/L,
KH2PO45g/L, yeast powder 4g/L, MgSO4·7H2O 0.5g/L, surplus are water) 5L fermentor in, control fermentation temperature be
30 DEG C, revolving speed 500r/min, time 50h, fermentation ends.
The measuring method of 2 phenylethyl alcohol in fermentation liquid is analyzed using high performance liquid chromatography (HPLC), specific as follows: will to send out
Zymotic fluid 10,000r/min centrifugation, is handled with 0.45 μm of filtering with microporous membrane again after centrifugation, high performance liquid chromatograph (Agilent,
USA), chromatographic column be C18 column (250mm × 4.6mm, 10 μm；Ailite, China), mobile phase is methanol: water=50:50 (v/
V), flow rate of mobile phase 0.7mL/min, 30 DEG C of column temperature, Detection wavelength 260nm, 10 μ L of sample introduction detection.2 phenylethyl alcohol after fermentation
Concentration be 5g/L (attached drawing 3), conversion ratio be 0.71 (g/g).
Above said content is only the basic explanation under present inventive concept, and is appointed made by technical solution according to the present invention
What equivalent transformation is within the scope of protection of the invention.
Claims (4)
1. a kind of biological preparation method of 2 phenylethyl alcohol, which is characterized in that by choosing osmophilic strain and the 2- of resistance to 4g/L benzene second
Candida glycerolgenesis (Candida glycerinogenes) CCTCC M 93018 of alcohol, conversion L-phenylalanine fermentation life
Produce 2 phenylethyl alcohol.
2. the preparation method of 2 phenylethyl alcohol as described in claim 1, steps are as follows:
(1) in 1 ring Candida glycerolgenesis of picking access seed culture medium, in 30 DEG C, under the conditions of 200r/min, shaken cultivation
18h obtains liquid seed；
(2) the liquid seed for obtaining step (1) controls fermentation temperature by the inoculum concentration access fermentation medium of 4% (v/v)
It is 30 DEG C, revolving speed 500r/min, time 50h, fermentation ends.
3. method as claimed in claim 2, which is characterized in that seed culture medium is yeast powder 10g/L, albumen in the step (1)
Peptone 20g/L, glucose 20g/L, surplus are water.
4. method as claimed in claim 2, which is characterized in that fermentation medium is L-phenylalanine 7g/L, Portugal in the step (2)
Grape sugar 90g/L, KH2PO45g/L, yeast powder 4g/L, MgSO4·7H2O 0.5g/L, surplus are water.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110079468A (en) * | 2019-05-09 | 2019-08-02 | 江南大学 | A method of enhancing Candida glycerolgenesis 2 phenylethyl alcohol tolerance |
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| CN107858361B (en) * | 2017-12-12 | 2020-02-18 | 江南大学 | Candida glycerinogenes heat shock protein gene CgHsp10 and application thereof |
| CN109679983A (en) * | 2019-02-01 | 2019-04-26 | 福建师范大学 | A kind of Yeast engineering bacteria and the preparation method and application thereof of high yield 2 phenylethyl alcohol |
| CN110656056B (en) * | 2019-10-31 | 2021-07-27 | 江南大学 | Construction method of pinene-producing engineering bacteria with high-concentration pinene tolerance |
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| CN1244586A (en) * | 1999-08-04 | 2000-02-16 | 无锡轻工大学 | Aerobic fermentation process of producing glycerine by utilizing glycerine producing candida mutant strain |
| CN101608190A (en) * | 2009-07-17 | 2009-12-23 | 上海应用技术学院 | The preparation method of natural spice 2-phenethyl alcohol |
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