CN107022585A - A kind of method that Aspergillus niger strain produces furfural - Google Patents
A kind of method that Aspergillus niger strain produces furfural Download PDFInfo
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Abstract
The invention discloses a kind of method that Aspergillus niger strain produces furfural, including by aspergillus niger xj inoculations in PDA solid slope culture mediums, 27 DEG C are cultivated 35 days, activate 23 times, then access in the 10L fermentation tanks equipped with PDA liquid seed culture mediums, 26 28 DEG C, 100 120rpm, throughput is controlled in 500 700L/h, and canister culture 23 days obtains seed liquor；Take seed liquor 10 15%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 26 28 °C, mixing speed 80rpm, throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation for 3 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, collect mycelium, produce.Culture matrix of the present invention is readily available, and fermentation condition is stable, technique is simple and convenient.
Description
Technical field
The present invention relates to biological technical field, a kind of method that Aspergillus niger strain produces furfural is related in particular to.
Background technology
Aspergillus niger（Aspergillus niger）The Common Species belonged in fungi, are distributed widely in all over the world
In grain, plant product and soil, its producing enzyme class is enriched, and is one of most common strain in commercial Application.Because having very
High β-Isosorbide-5-Nitrae glucosidase activity, more full cellulose components and security, as study hotspot in recent years.Furfural
Also known as furtural, is important heterocyclic organic compound, has been widely used in the industrial production.Furfural chemical property ten
Point active, the chemical products directly or indirectly derived using furfural as raw material are up to kind more than 1600, such as furfuryl alcohol, furancarboxylic acid, furans, tetrahydrochysene
Furfuryl alcohol etc..In addition, furfural can also pass through hydrogenation by generating 5 hydroxymethyl furfural with the hydroxymethylation of formaldehyde by furfural
Act on generation methyltetrahydrofuran can directly as gasoline base-material.Industrially furfural is by agricultural byproducts bagasse, flower
Heat is produced altogether with dilute sulfuric acid for raw shell, sorghum rod, oat shell and corncob etc., can also be generated by mailland reaction.But existing furfural
Energy consumption in production process is huge, and environmental pollution is extremely serious, and production cost is higher and the rate of recovery is low.The furfural production of current China
Primary raw material is the agricultural byproducts such as corncob, is not directed to study in terms of microbial fermentation solution production furfural.Microorganism has suitable
Ying Xingqiang, growth and breeding are rapid, growth cycle is short, easy culture, the features such as do not limited by the places of origin of raw materials.Aspergillus niger is generally recognized as safe
Microorganism, with the powerful enzyme system of various active, such as very high β-Isosorbide-5-Nitrae glucosidase activity, more full cellulase
Component.Prepared by current furfural is mainly aspergillus niger united hydrolysis peanut shell, corncob etc to prepare, and wherein whether furfural has portion
Divide even unknown from fermentation of Aspergillus niger thing.Using aspergillus niger superior strain as starting strain, fermentation is moved into after seed culture
Tank carries out the fermenting and producing of furfural, during fermentation according to the OUR of on-line determination（OUR）It is automatically adjusted fermentation tank
Intake and speed of agitator, it is ensured that enough dissolved oxygen concentrations, are realized to the direct, real-time, accurate of furfural fermentation process
Control.Therefore, it is a new way to produce furfural using microorganism, seeks superior strain and research zymotechnique, and new carbon source
Selection and low-cost resource application just turn into research direction.
The content of the invention
It is an object of the invention to be found that a kind of culture matrix is readily available, fermentation condition is stable, technique is simple and environmentally-friendly
Aspergillus niger strain produce furfural method.
The method that a kind of Aspergillus niger strain of the present invention produces furfural, comprises the following steps：
（1）Actication of culture
By aspergillus niger xj（Aspergillus nigerxj）Bacterial strain is connected in PDA solid slope culture mediums, 27 DEG C of quiescent cultures
3-5 days, activate 2-3 times, saved backup in 4 DEG C；
（2）The preparation of spore suspension
By the bacterial strain activated, with containing spore under the sterile washing of 0.05% Tween 80,100 mL with bead are transferred to sterile
In water, shaken again on vortex oscillator after stirring, fully break up spore, be filtered to remove mycelia with 4 layers of sterile lens paper, be made
Homogeneous spore suspension；
（3）The preparation of seed liquor
By spore suspension, with 8-12%（v/v）Inoculum concentration is inoculated in the 10L fermentation tanks equipped with PDA liquid seed culture mediums, 26-
28 DEG C, 100-120rpm, throughput is controlled in 500-700L/h, and canister culture 2-3 days obtains seed liquor；
（4）Fermented and cultured
Take seed liquor 10-15%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 26-28 °C, mixing speed
80rpm, throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation in 3-5 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, receives
Collect mycelium, produce.
The method that a kind of above-mentioned Aspergillus niger strain produces furfural, the wherein preparation method of PDA solid slope culture mediums are such as
Under：Potato 200g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 20g, and agar 20g, water supplies 1000mL, pH
It is natural.Sterilising conditions：121 DEG C, 30min.
The method that a kind of above-mentioned Aspergillus niger strain produces furfural, the wherein preparation method of PDA liquid seed culture mediums are such as
Under：Potato 200g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 20g, and water supplies 1000mL, and pH is natural.Sterilizing
Condition：121 DEG C, 30min.
The method that a kind of above-mentioned Aspergillus niger strain produces furfural, the preparation method of wherein PDA fermentation mediums is as follows：Horse
Bell potato 2600g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 260g, and water supplies 13000mL, pH naturally, 121 DEG C,
Sterilize 30min.
The strain is deposited in China typical culture collection center on March 7th, 2006（Address：Wuhan, China is military
Chinese university）, preserving number：CCTCC NO：M 206021, it is entitled：Aspergillus niger xj（Aspergillus nigerxj）.
The present invention compared with prior art, with obvious beneficial effect, as can be known from the above technical solutions：The present invention is utilized
Aspergillus niger xj ferments in more single PDA culture medium matter produces furfural, can provide a new way to solve furfural raw material sources
Footpath.And aspergillus niger（Aspergillus nigerxj）Bacterium source is reliable, to the adaptation model of the natural environmental conditions such as temperature, pH
Enclose wide, easily culture and preservation, the bacterial strain for extracting furfural can be used as.The technology path is short, and production cost is greatly lowered, without useless
Water waste gas is produced, and greatly reduces environmental pollution.
Embodiment
Embodiment 1
A kind of method that Aspergillus niger strain produces furfural, comprises the following steps：
（1）Actication of culture
By aspergillus niger xj（Aspergillus nigerxj）Bacterial strain is connected to PDA solid slope culture mediums（Potato 200g strippings and slicings,
30min is boiled with water, juice, plus glucose 20g, agar 20g is filtered to take, water supplies 1000mL, pH naturally, 121 DEG C, sterilizing
30min）On, 27 DEG C of quiescent cultures 3 days activate 3 times, are stored in 4 DEG C of refrigerators standby；
（2）The preparation of spore suspension
By the bacterial strain activated, with containing 0.05% Tween 80（Toween-80）Spore under sterile washing, is transferred to bead
In the 250 mL triangular flasks equipped with 100 mL sterilized waters, shaken again on vortex oscillator after being stirred with glass rod, fully break up spore
Son, is filtered to remove mycelia with 4 layers of sterile lens paper, homogeneous spore suspension is made；
（3）The preparation of seed liquor
By spore suspension, with 8%（v/v）Inoculum concentration is inoculated in the 10L fermentation tanks equipped with PDA liquid seed culture mediums, 28 DEG C,
120rpm, throughput is controlled in 700L/h, and canister culture 3 days obtains seed liquor；
（4）Fermented and cultured
Take seed liquor 10%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 28 °C, mixing speed 80rpm,
Throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation for 5 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, collect mycelium,
As furfural.
Experimental example：Aspergillus niger xj (Aspergillus nigerXj) in mycelium furfural identification：
（1）Sample pretreatment
Take 200g obtained above to dry mycelium, be ground into powder and be well mixed, take 50g as equipment and clean it
With remaining 150g is extracted using ether, and point 2 extractions are completed, and are analyzed for GC-MS.Extracting pressure is 5MPa, temperature 50
DEG C, flow 160-200L/h obtains 15g supercritical extracts section sample.
（2）GC-MS analysis conditions
GC conditions：HP-5MS (0.25mm × 30m, 0.25 μm)；50 DEG C of column temperature（Retain 2min）, it is warming up to 280 DEG C（5
℃/min）, keep 3min；Temperature of vaporization chamber is 250 DEG C；Using high-pure helium as carrier gas；7.62psi, carrier gas flux are pressed before post
1.0mL/min；The μ L of sample size 1；Split ratio 50：1.
Mass Spectrometry Conditions：Ion gun is EI sources；280 DEG C of interface temperature；230 DEG C of ion source temperature；Multiplier voltage 1785V；
150 DEG C of quadrupole rod temperature；Electron energy 70eV；The μ A of emission current 34.6；10~550amu of mass range；Solvent delay 3min.
（3）Conclusion：
Detected by GC-MS, aspergillus niger(Aspergillus nigerXj) content of furfural is in hypha extract
1.03%。
Embodiment 2
A kind of method for producing furfural, comprises the following steps：
（1）Actication of culture
By aspergillus niger xj（Aspergillus nigerxj）Bacterial strain is connected to PDA solid slope culture mediums（Potato 200g strippings and slicings,
30min is boiled with water, juice, plus glucose 20g, agar 20g is filtered to take, water supplies 1000mL, pH naturally, 121 DEG C, sterilizing
30min）On, 27 DEG C of quiescent cultures 4 days activate 2 times, are stored in 4 DEG C of refrigerators standby；
（2）The preparation of spore suspension
By the bacterial strain activated, with containing 0.05% Tween 80（Toween-80）Spore under sterile washing, is transferred to bead
In the 250 mL triangular flasks equipped with 100 mL sterilized waters, shaken again on vortex oscillator after being stirred with glass rod, fully break up spore
Son, is filtered to remove mycelia with 4 layers of sterile lens paper, homogeneous spore suspension is made；
（3）The preparation of seed liquor
By spore suspension, with 10%（v/v）Inoculum concentration is inoculated in the 10L fermentation tanks equipped with PDA liquid seed culture mediums, 27 DEG C,
110rpm, throughput is controlled in 600L/h, and canister culture 2 days obtains seed liquor；
（4）Fermented and cultured
Take seed liquor 12%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 27 °C, mixing speed 80rpm,
Throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation for 4 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, collect mycelium,
As furfural.
Aspergillus niger xj (Aspergillus nigerXj) in mycelium furfural identification：
（1）Sample pretreatment
Take 200g obtained above to dry mycelium, be ground into powder and be well mixed, take 50g as equipment and clean it
With remaining 150g is extracted using ether, and point 2 extractions are completed, and are analyzed for GC-MS.Extracting pressure is 5MPa, temperature 50
DEG C, flow 160-200L/h obtains 15g supercritical extracts section sample.
（2）GC-MS analysis conditions
GC conditions：HP-5MS (0.25mm × 30m, 0.25 μm)；50 DEG C of column temperature（Retain 2min）, it is warming up to 280 DEG C（5
℃/min）, keep 3min；Temperature of vaporization chamber is 250 DEG C；Using high-pure helium as carrier gas；7.62psi, carrier gas flux are pressed before post
1.0mL/min；The μ L of sample size 1；Split ratio 50：1.
Mass Spectrometry Conditions：Ion gun is EI sources；280 DEG C of interface temperature；230 DEG C of ion source temperature；Multiplier voltage 1785V；
150 DEG C of quadrupole rod temperature；Electron energy 70eV；The μ A of emission current 34.6；10~550amu of mass range；Solvent delay 3min.
（3）Conclusion：
Detected by GC-MS, aspergillus niger(Aspergillus nigerXj) content of furfural is in hypha extract
1.041%。
Embodiment 3
A kind of method for producing furfural, comprises the following steps：
（1）Actication of culture
By aspergillus niger xj（Aspergillus nigerxj）Bacterial strain is connected to PDA solid slope culture mediums（Potato 200g strippings and slicings,
30min is boiled with water, juice, plus glucose 20g, agar 20g is filtered to take, water supplies 1000mL, pH naturally, 121 DEG C, sterilizing
30min）On, 27 DEG C of quiescent cultures 5 days activate 2 times, are stored in 4 DEG C of refrigerators standby；
（2）The preparation of spore suspension
By the bacterial strain activated, with containing 0.05% Tween 80（Toween-80）Spore under sterile washing, is transferred to bead
In the 250 mL triangular flasks equipped with 100 mL sterilized waters, shaken again on vortex oscillator after being stirred with glass rod, fully break up spore
Son, is filtered to remove mycelia with 4 layers of sterile lens paper, homogeneous spore suspension is made；
（3）The preparation of seed liquor
By spore suspension, with 12%（v/v）Inoculum concentration is inoculated in the 10L fermentation tanks equipped with PDA liquid seed culture mediums, 26 DEG C,
100rpm, throughput is controlled in 500L/h, and canister culture 2 days obtains seed liquor；
（4）Fermented and cultured
Take seed liquor 15%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 26 °C, mixing speed 80rpm,
Throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation for 3 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, collect mycelium,
As furfural.
Aspergillus niger xj (Aspergillus nigerXj) in mycelium furfural identification：
（1）Sample pretreatment
Take 200g obtained above to dry mycelium, be ground into powder and be well mixed, take 50g as equipment and clean it
With remaining 150g is extracted using ether, and point 2 extractions are completed, and are analyzed for GC-MS.Extracting pressure is 5MPa, temperature 50
DEG C, flow 160-200L/h obtains 15g supercritical extracts section sample.
（2）GC-MS analysis conditions
GC conditions：HP-5MS (0.25mm × 30m, 0.25 μm)；50 DEG C of column temperature（Retain 2min）, it is warming up to 280 DEG C（5
℃/min）, keep 3min；Temperature of vaporization chamber is 250 DEG C；Using high-pure helium as carrier gas；7.62psi, carrier gas flux are pressed before post
1.0mL/min；The μ L of sample size 1；Split ratio 50：1.
Mass Spectrometry Conditions：Ion gun is EI sources；280 DEG C of interface temperature；230 DEG C of ion source temperature；Multiplier voltage 1785V；
150 DEG C of quadrupole rod temperature；Electron energy 70eV；The μ A of emission current 34.6；10~550amu of mass range；Solvent delay 3min.
（3）Conclusion：
Detected by GC-MS, aspergillus niger(Aspergillus nigerXj) content of furfural is in hypha extract
1.039%。
The above described is only a preferred embodiment of the present invention, not making any formal limitation to the present invention, appoint
What is without departing from technical solution of the present invention content, and what the technical spirit according to the present invention was made to above example any simply repaiies
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Claims (4)
1. a kind of method that Aspergillus niger strain produces furfural, comprises the following steps：
（1）Actication of culture
By aspergillus niger xj（Aspergillus nigerxj）Bacterial strain is connected in PDA solid slope culture mediums, 27 DEG C of quiescent cultures
3-5 days, activate 2-3 times, saved backup in 4 DEG C；
（2）The preparation of spore suspension
By the bacterial strain activated, with containing spore under the sterile washing of 0.05% Tween 80,100 mL with bead are transferred to sterile
In water, shaken again on vortex oscillator after stirring, fully break up spore, be filtered to remove mycelia with 4 layers of sterile lens paper, be made
Homogeneous spore suspension；
（3）The preparation of seed liquor
By spore suspension, with 8-12%（v/v）Inoculum concentration is inoculated in the 10L fermentation tanks equipped with PDA liquid seed culture mediums,
26-28 DEG C, 100-120rpm, throughput is controlled in 500-700L/h, and canister culture 2-3 days obtains seed liquor；
（4）Fermented and cultured
Take seed liquor 10-15%（v/v）It is inoculated in the 80L fermentation tanks equipped with PDA fermentation mediums, 26-28 °C, mixing speed
80rpm, throughput is controlled in 2.5m3/ h, big tank culture terminates fermentation in 3-5 days, and 4 layers of sterile gauze are filtered to remove zymotic fluid, receives
Collect mycelium, produce.
2. a kind of Aspergillus niger strain as claimed in claim 1 produces the system of the method, wherein PDA solid slope culture mediums of furfural
Preparation Method is as follows：Potato 200g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 20g, and agar 20g, water is supplied
1000mL, pH are natural；Sterilising conditions：121 DEG C, 30min.
3. the method that a kind of Aspergillus niger strain as claimed in claim 1 or 2 produces furfural, wherein PDA liquid seed culture mediums
Preparation method it is as follows：Potato 200g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 20g, and water is supplied
1000mL, pH are natural；Sterilising conditions：121 DEG C, 30min.
4. a kind of Aspergillus niger strain as claimed in claim 3 produces the preparation side of the method, wherein PDA fermentation mediums of furfural
Method is as follows：Potato 14000g strippings and slicings, 30min is boiled with water, filters to take juice, plus glucose 1400g, and water supplies 70000mL, pH
Naturally, 121 DEG C, sterilize 30min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004193A (en) * | 2018-12-04 | 2019-07-12 | 贵州大学 | A method of utilizing 5,5 ' of aspergillus niger separation and Extraction-oxo two (methylene), two furans -2- formaldehyde |
| CN110511198A (en) * | 2019-08-31 | 2019-11-29 | 贵州大学 | A method of utilizing aspergillus niger spore powder separation and Extraction 5- methylol-furancarboxylic acid |
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2017
- 2017-06-12 CN CN201710438937.0A patent/CN107022585A/en active Pending
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| CN103031350A (en) * | 2012-05-06 | 2013-04-10 | 贵州大学 | Method for producing PUFA (polyunsaturated fatty acid) |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004193A (en) * | 2018-12-04 | 2019-07-12 | 贵州大学 | A method of utilizing 5,5 ' of aspergillus niger separation and Extraction-oxo two (methylene), two furans -2- formaldehyde |
| CN110511198A (en) * | 2019-08-31 | 2019-11-29 | 贵州大学 | A method of utilizing aspergillus niger spore powder separation and Extraction 5- methylol-furancarboxylic acid |
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