CN106011178B - A method of β-farnesene is prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues - Google Patents
A method of β-farnesene is prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues Download PDFInfo
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Abstract
Prepare β-farnesene method using sugared sorghum stalk and sugarcane produced by fermenting waste residues the present invention relates to a kind of, it is characterised in that use following steps: A: sugared sorghum juice is made in the mature fresh sugared sorghum squeezing of harvesting；B: sugared sorghum juice is concentrated；C: sugared sorghum juice concentrate is uniformly mixed with the by-product sugarcane waste residue that sugar refinery produces, obtains sugared sorghum and sugarcane waste residue mixed liquor；D: shaking flask strain is prepared using the saccharomyces cerevisiae that deposit number is CICC No.32475 as strain using sugared sorghum and sugarcane waste residue mixed liquor as the carbon source of fermentation；E: shaking flask strain is inoculated into seed culture medium and is cultivated；G: seed culture fluid is accessed into the 30-50 h that ferments in fermentation medium, flocculant is added into fermentation liquid, siphon obtains supernatant after precipitating.The raw materials used in the present invention is at low cost, and β-farnesene content is high in gained supernatant, conducive to the preparation cost for reducing plant origin saualane.
Description
Technical field
It is the present invention relates to technical field of microbial fermentation, in particular to a kind of to utilize sugared sorghum stalk and sugarcane produced by fermenting waste residues system
The method of standby β-farnesene.
Background technique
β-farnesene also known as farnesene are the linear alkenes with 15 carbon.β-farnesene can be used as diesel oil and spy
The precursor and important fragrance component precursor of different chemicals, further the product after synthesis, it is fragrant to can be applied to cosmetics, essence
The fields such as material, macromolecule, lubricating oil, diesel oil, jet fuel.Currently, β-farnesene mainly passes through nerolidol in various acidity
Synthetic method preparation under catalyst action through being dehydrated causes separating technology complicated there are isomers is more, and production cost is high.
The development of biotechnology provides inexpensive, renewable, sustainable development for beam system for β-farnesene of high-purity
The approach of exhibition.U.S. Amyris takes the lead in preparing β-farnesene using bioanalysis, becomes the pioneer for representing American industry biotechnology,
Amyris factory, the U.S. is located at Brazil, as substrate after using the local sugar-cane juice abounded with to be concentrated.Compared to Brazil, Chinese sugarcane
Higher cost, it is predominantly edible or refine sugar, it is too high for producing β-farnesene cost.So if using the useless of industrial crops
Production cost can be substantially reduced, meet China's actual conditions for producing β-farnesene by abandoning part.
Sugared sorghum, popular name " sweet tea sorgo " belong to grass family sorghum, are one kind of grain sorghum.The sugared same sorghum of sorghum shape
Similar, leaf cinerous has liquid containing syrup in stem.Sugared sorghum is not stringent to soil and climate requirements, widely distributed, is that sugar material is made
One of object, it is also possible to make the purposes such as feed, wine brewing.It produces all over China.It is the same with common sorghum, can bear per acre
The grain of 150-500 kg.The stalk of sugared sorghum contains sugar, and per unit area yield is up to 5000-10000 kg per acre.Sugared sorghum because
Stalk short sweetness, by as fuel or as animal feed processing after usually drying.
Sugarcane is planted in the southern torrid areas such as China Fujian, Guangdong, Hainan, Guangxi, Sichuan, Yunnan, is mainly used for straight
Connect edible and manufacture sucrose raw material.Sugarcane genotypes are rich in sugar, and per unit area yield is up to 4000-6000 kg per acre.It is sweet after squeezing sugar
Sugarcane waste residue is used for fuel after generally drying.
Summary of the invention
The technical problem to be solved by the present invention is to provide and a kind of utilize sugared sorghum stalk and sugarcane produced by fermenting waste residues preparation β-method
The method of alkene, this method is at low cost, raw material sources are wide, is conducive to environmental protection.
In order to solve the above technical problems, the invention adopts the following technical scheme: a kind of utilize sugared sorghum stalk and sugarcane waste residue
Fermentation prepares β-farnesene method, it is characterised in that uses following steps:
A: the mature fresh sugared sorghum of harvesting, removal sorghum fringe, root and leaf, then clean remove dust on sugared sorghum stalk and
Soil enters squeezer and carries out squeezing obtained sugared sorghum juice；
B: sugared sorghum juice obtained by step A is filtered, evaporate, is concentrated, the sugared sorghum juice that water content is 20%-25% is obtained
Concentrate；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the quality of 1 ~ 3:1
Than being uniformly mixed, sugared sorghum and sugarcane waste residue mixed liquor are obtained；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The Saccharomyces Cerevisiae in S accharomyces cerevisiae of CICC No. 32475 be strain, shaking speed 50-200 r/min,
Culture 12-48 h obtains shaking flask strain；
E: seed culture medium: 5-10% glucose, 0.1-0.3%NaNO is prepared by following mass ratio3, 0.05-0.15%
K2HPO4, 0.05-0.15%KH2PO4, 0.01-0.1%MgSO4, 0.01-0.1%FeSO4, 0.01-0.1%ZnSO4, 0.01-0.05%
MnSO4, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by the Volume fraction of 2-10%, the range of 100-300 r/min
Interior shaken cultivation 12-24 h, obtains seed culture fluid；
G: fermentation medium: 10-20% sugar sorghum and sugarcane waste residue mixed liquor, 0.1-0.3% is prepared by following mass ratio
NaNO3, 0.05-0.15%K2HPO4, 0.05-0.15%KH2PO4, 0.01-0.1%MgSO4, 0.01-0.1%FeSO4, 0.01-0.1%
ZnSO4, 0.01-0.05%MnSO4, surplus is water；
H: the fermented and cultured for preparing the access of seed culture fluid obtained by step F step G by the inoculum concentration of volume fraction 5-20%
In base, be passed through air maintain micro-oxygen conditions, ventilatory capacity 100-400 ml/min, 37 DEG C of temperature, speed of agitator 200-400 r/
Min adjusts pH to 5 ~ 7 with 3 ~ 5 mol/L NaOH solutions, stops fermentation after the 30-50 h that ferments, 0.75 is added into fermentation liquid
15 min are stirred in g/L flocculant chitosan and 0.125 g/L flocculant aid sodium alginate, regulation system pH=5, stand 1 h
Afterwards, siphon obtains supernatant.
The present invention is using sugared sorghum stalk and sugarcane waste residue cheap and easy to get as raw material, after fermentation using this method, gained
β-farnesene content is up to 516 mg/L-750 mg/L in supernatant.In addition, a kind of fermentation of the present invention prepares β-farnesene
Method and process condition it is simple, it is mild, remained without toxic solvent, it can be achieved that industrializing and clean manufacturing.
Using in the supernatant of this method after fermentation, β-farnesene content is up to 516 mg/L-750 mg/L, by supernatant
In liquid after β-farnesene purification, gained β-farnesene can especially be made directly as diesel oil, speciality chemical, fragrance precursor
Standby plant origin saualane, convenient for the production and manufacture of downstream product.
The present invention has abundance, cost using sugared sorghum stem and sugarcane waste residue as production β-farnesene raw material
Cheap, raw material is easy to get, reproducible advantage；β-farnesene content is high in gained supernatant, is conducive to reduce plant origin saualane
Preparation cost.
Specific embodiment
Technical solution of the present invention is described further by the following examples, but protection scope of the present invention and unlimited
In this.
Embodiment 1:
A method of β-farnesene being prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues, is prepared using following steps:
A: mature sugared sorghum, removal sorghum fringe, root and leaf are gathered in from sugared sorghum planting site, then cleans the sugared sorghum stalk of removal
On dust and soil, enter squeezer and squeeze sugared sorghum juice is made；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and obtaining water content is 20% sugared sorghum juice concentration
Liquid；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the mass ratio of 1:1
It is uniformly mixed；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The Saccharomyces Cerevisiae in S accharomyces cerevisiae of CICC No. 32475 is strain, 80 r/min of shaking speed, culture
18 h obtain shaking flask strain；
E: seed culture medium group becomes glucose 3%, NaNO3 0.1%；K2HPO4 0.05%；KH2PO4 0.1%；MgSO4
0.01%；FeSO40.01%；ZnSO40.01%；MnSO40.01%, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by 4% Volume fraction, is vibrated in the range of 100 r/min
15 h are cultivated, seed culture fluid is obtained；
G: fermentation medium group becomes 10% sugared sorghum and sugarcane waste residue mixed liquor, 0.1%NaNO3, 0.05%K2HPO4, 0.1%
KH2PO4, 0.01%MgSO4, 0.01%FeSO4, 0.01%ZnSO4, 0.01%MnSO4, surplus is water；
H: it by the inoculum concentration of volume fraction 6% prepared by seed culture fluid cultured in step (F) access step (G)
In culture medium, be passed through air maintain micro-oxygen conditions, 100 ml/min of ventilatory capacity, 37 DEG C of temperature, 200 r/min of speed of agitator, with 3
~ 5 mol/L NaOH solutions adjust pH to 5, stop fermentation after 20 h that ferment.0.75 g/L flocculant shell is added into fermentation liquid
Glycan and 0.125 g/L flocculant aid sodium alginate, regulation system pH 5 stir 15 min, after standing 1 h, are siphoned off supernatant
Liquid, β-farnesene content is up to 556 mg/L in supernatant.
Embodiment 2:
A method of β-farnesene being prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues, is prepared using following steps:
A: mature sugared sorghum, removal sorghum fringe, root and leaf are gathered in from sugared sorghum planting site, then cleans the sugared sorghum stalk of removal
On dust and soil, enter squeezer and squeeze sugared sorghum juice is made；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and obtaining water content is 22% sugared sorghum juice concentration
Liquid；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the mass ratio of 2:1
It is uniformly mixed；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The saccharomyces cerevisiae (Saccharomyces cerevisiae) of CICC No. 32475 is strain, 100 r/min of shaking speed, training
It supports 20 h and obtains shaking flask strain；
E: seed culture medium group becomes glucose 5%, NaNO3 0.15%；K2HPO4 0.1%；KH2PO4 0.15%；MgSO4
0.03%；FeSO40.03%；ZnSO40.03%；MnSO40.03%, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by 6% Volume fraction, is vibrated in the range of 200 r/min
18 h are cultivated, seed culture fluid is obtained；
G: fermentation medium group becomes 12% sugared sorghum and sugarcane waste residue mixed liquor, 0.15%NaNO3, 0.1%K2HPO4,
0.15%KH2PO4, 0.03%MgSO4, 0.03%FeSO4, 0.03%ZnSO4, 0.03%MnSO4, surplus is water；
H: it by the inoculum concentration of volume fraction 8% prepared by seed culture fluid cultured in step (F) access step (G)
In culture medium, be passed through air maintain micro-oxygen conditions, 200 ml/min of ventilatory capacity, 37 DEG C of temperature, 300 r/min of speed of agitator, with 3
~ 5 mol/L NaOH solutions adjust pH to 6, stop fermentation after 30 h that ferment.0.75 g/L flocculant shell is added into fermentation liquid
Glycan and 0.125 g/L flocculant aid sodium alginate, regulation system pH 5 stir 15 min, after standing 1 h, are siphoned off supernatant
Liquid, β-farnesene content is up to 614 mg/L in supernatant.
Embodiment 3:
A method of β-farnesene being prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues, is prepared using following steps:
A: mature sugared sorghum, removal sorghum fringe, root and leaf are gathered in from sugared sorghum planting site, then cleans the sugared sorghum stalk of removal
On dust and soil, enter squeezer and squeeze sugared sorghum juice is made；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and obtaining water content is 23% sugared sorghum juice concentration
Liquid；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the mass ratio of 3:1
It is uniformly mixed；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The Saccharomyces Cerevisiae in S accharomyces cerevisiae of CICC No. 32475 is strain, 150 r/min of shaking speed, culture
30 h obtain shaking flask strain；
E: seed culture medium group becomes glucose 8%, NaNO3 0.2%；K2HPO4 0.1%；KH2PO4 0.15%；MgSO4
0.05%；FeSO40.05%；ZnSO40.05%；MnSO40.05%, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by 8% Volume fraction, is vibrated in the range of 300 r/min
24 h are cultivated, seed culture fluid is obtained；
G: fermentation medium group becomes 15% sugared sorghum and sugarcane waste residue mixed liquor, 0.2%NaNO3,0.1%K2HPO4, 0.15%
KH2PO4, 0.05%MgSO4, 0.05%FeSO4, 0.05%ZnSO4, 0.05%MnSO4, surplus is water；
H: it by the inoculum concentration of volume fraction 15% prepared by seed culture fluid cultured in step (F) access step (G)
In culture medium, it is passed through air and maintains micro-oxygen conditions, 300 ml/min of ventilatory capacity, is used by 37 DEG C of temperature, 400 r/min of speed of agitator
NaOH solution controls pH 6, stops fermentation after 40 h that ferment.0.75 g/L flocculant chitosan and 0.125 are added into fermentation liquid
G/L flocculant aid sodium alginate, regulation system pH 5 stir 15 min, after standing 1 h, are siphoned off supernatant, β-in supernatant
Farnesene content is up to 698 mg/L.
Embodiment 4:
A method of β-farnesene being prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues, is prepared using following steps:
A: mature sugared sorghum, removal sorghum fringe, root and leaf are gathered in from sugared sorghum planting site, then cleans the sugared sorghum stalk of removal
On dust and soil, enter squeezer and squeeze sugared sorghum juice is made；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and obtaining water content is 21% sugared sorghum juice concentration
Liquid；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the mass ratio of 3:1
It is uniformly mixed；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The Saccharomyces Cerevisiae in S accharomyces cerevisiae of CICC No. 32475 is strain, 200 r/min of shaking speed, culture
36 h obtain shaking flask strain；
E: seed culture medium group becomes glucose 10%, NaNO3 0.3%；K2HPO4 0.1%；KH2PO4 0.15%；MgSO4
0.08%；FeSO40.08%；ZnSO40.08%；MnSO40.05%, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by 10% Volume fraction, is vibrated in the range of 300 r/min
24 h are cultivated, seed culture fluid is obtained；
G: fermentation medium group becomes 20% sugared sorghum and sugarcane waste residue mixed liquor, 0.3%NaNO3,0.1%K2HPO4, 0.15%
KH2PO4, 0.08%MgSO4, 0.08%FeSO4, 0.08%ZnSO4, 0.08%MnSO4, surplus is water；
H: it by the inoculum concentration of volume fraction 20% prepared by seed culture fluid cultured in step (F) access step (G)
In culture medium, be passed through air maintain micro-oxygen conditions, 400 ml/min of ventilatory capacity, 37 DEG C of temperature, 400 r/min of speed of agitator, with 3
~ 5 mol/L NaOH solutions adjust pH to 5, stop fermentation after 50 h that ferment.0.75 g/L flocculant shell is added into fermentation liquid
Glycan and 0.125 g/L flocculant aid sodium alginate, regulation system pH 5 stir 15 min, after standing 1 h, are siphoned off supernatant
Liquid, β-farnesene content is up to 730 mg/L in supernatant.
Embodiment 5:
A method of β-farnesene being prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues, is prepared using following steps:
A: mature sugared sorghum, removal sorghum fringe, root and leaf are gathered in from sugared sorghum planting site, then cleans the sugared sorghum stalk of removal
On dust and soil, enter squeezer and squeeze sugared sorghum juice is made；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and obtaining water content is 25% sugared sorghum juice concentration
Liquid；
C: by sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production with the mass ratio of 1:1
It is uniformly mixed；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, is with deposit number
The Saccharomyces Cerevisiae in S accharomyces cerevisiae of CICC No. 32475 is strain, 200 r/min of shaking speed, culture
40 h obtain shaking flask strain；
E: seed culture medium group becomes glucose 7%, NaNO3 0.1%；K2HPO4 0.05%；KH2PO4 0.1%；MgSO4
0.06%；FeSO40.06%；ZnSO40.06%；MnSO40.03%, surplus is water；
F: shaking flask strain is inoculated into seed culture medium by 6% Volume fraction, is vibrated in the range of 200 r/min
24 h are cultivated, seed culture fluid is obtained；
G: fermentation medium group becomes 18% sugared sorghum and sugarcane waste residue mixed liquor, 0.1%NaNO3,0.05%K2HPO4, 0.1%
KH2PO4, 0.06%MgSO4, 0.06%FeSO4, 0.06%ZnSO4, 0.03%MnSO4, surplus is water；
H: it by the inoculum concentration of volume fraction 20% prepared by seed culture fluid cultured in step (F) access step (G)
In culture medium, be passed through air maintain micro-oxygen conditions, 300 ml/min of ventilatory capacity, 37 DEG C of temperature, 350 r/min of speed of agitator, with 3
~ 5 mol/L NaOH solutions adjust pH to 5.5, stop fermentation after 45 h that ferment.0.75 g/L flocculant is added into fermentation liquid
Chitosan and 0.125 g/L flocculant aid sodium alginate, regulation system pH 5 stir 15 min, after standing 1 h, are siphoned off
Clear liquid, β-farnesene content is up to 640 mg/L in supernatant.
Claims (1)
1. a kind of prepare β-farnesene method using sugared sorghum stalk and sugarcane produced by fermenting waste residues, it is characterised in that use following step
It is rapid:
A: the mature fresh sugared sorghum of harvesting, removal sorghum fringe, root and leaf, then clean the dust and mud removed on sugared sorghum stalk
Soil enters squeezer and carries out squeezing obtained sugared sorghum juice；
B: sugared sorghum juice obtained by step A being filtered, evaporate, is concentrated, and is obtained the sugared sorghum juice that water content is 20%-25% and is concentrated
Liquid；
C: sugared sorghum juice concentrate obtained by step B and the by-product sugarcane waste residue of sugar refinery's production is mixed with the mass ratio of 1 ~ 3:1
It closes uniformly, obtains sugared sorghum and sugarcane waste residue mixed liquor；
D: the carbon source of sugared sorghum and sugarcane waste residue mixed liquor as fermentation obtained by appropriate step C is weighed, with saccharomyces cerevisiae
(Saccharomyces cerevisiae) CICC No. 32475 is strain, and shaking speed 50-200 r/min cultivates 12-48
H obtains shaking flask strain；
E: seed culture medium: 5-10% glucose, 0.1-0.3%NaNO is prepared by following mass ratio3, 0.05-0.15%K2HPO4,
0.05-0.15%KH2PO4, 0.01-0.1%MgSO4, 0.01-0.1%FeSO4, 0.01-0.1%ZnSO4, 0.01-0.05%MnSO4,
Surplus is water；
F: shaking flask strain is inoculated into seed culture medium by the Volume fraction of 2-10%, is shaken in the range of 100-300 r/min
Culture 12-24 h is swung, seed culture fluid is obtained；
G: fermentation medium: 10-20% sugar sorghum and sugarcane waste residue mixed liquor, 0.1-0.3%NaNO is prepared by following mass ratio3,
0.05-0.15%K2HPO4, 0.05-0.15%KH2PO4, 0.01-0.1%MgSO4, 0.01-0.1%FeSO4, 0.01-0.1%ZnSO4,
0.01-0.05%MnSO4, surplus is water；
H: the fermentation medium for preparing the access of seed culture fluid obtained by step F step G by the inoculum concentration of volume fraction 5-20%
In, be passed through air maintain micro-oxygen conditions, ventilatory capacity 100-400 ml/min, 37 DEG C of temperature, speed of agitator 200-400 r/min,
PH to 5 ~ 7 is adjusted with 3 ~ 5 mol/L NaOH solutions, stops fermentation after the 30-50 h that ferments, 0.75 g/L is added into fermentation liquid
15 min are stirred in flocculant chitosan and 0.125 g/L flocculant aid sodium alginate, regulation system pH=5, after standing 1 h, rainbow
Inhale to obtain supernatant.
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|---|---|---|---|---|
| CN101868532A (en) * | 2007-09-20 | 2010-10-20 | 阿迈瑞斯生物技术公司 | The preparation of isoprenoid |
| WO2012106405A1 (en) * | 2011-02-02 | 2012-08-09 | Amyris, Inc. | Methods of developing terpene synthase variants |
| CN103571763A (en) * | 2013-11-20 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae capable of auto-synthesizing terpenoid substances and applications thereof |
-
2016
- 2016-07-14 CN CN201610551806.9A patent/CN106011178B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101868532A (en) * | 2007-09-20 | 2010-10-20 | 阿迈瑞斯生物技术公司 | The preparation of isoprenoid |
| WO2012106405A1 (en) * | 2011-02-02 | 2012-08-09 | Amyris, Inc. | Methods of developing terpene synthase variants |
| CN103571763A (en) * | 2013-11-20 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae capable of auto-synthesizing terpenoid substances and applications thereof |
Non-Patent Citations (1)
| Title |
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| Advanced biofuel production by the yeast Saccharomyces;Nicolaas A Buijs等;《Chemical Biology》;20131231;第480-488页 |
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