CN103194398A - Citric-acid high-yield strain and screening method thereof - Google Patents
Citric-acid high-yield strain and screening method thereof Download PDFInfo
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Abstract
The invention relates to the field of biological fermentation and also relates to a high-yield strain which is prepared by the fact that Aspergillus niger spore obtained by a space mutation manner is subjected to high-glucose-high-acid-resistance directional screening and the mutation breeding is realized by designing a high-efficiency scientific screening scheme. The high-yield strain is named as TN-A09 and is biologically preserved, wherein the preservation number of the strain is CGMCC No. 5751; the high-yield strain is a citric-acid production strain with the high-glucose and high-acid resistance, high acid yield, and high conversion rate; and when the high-yield strain is used for producing citric acid in a fermentation manner, the average acid yield is 18%, the fermentation period is 60 hours, the conversion rate is 99%, all of which are largely higher than those of citric acid produced by fermenting an Aspergillus niger liquid in the prior art.
Description
Technical field
The present invention relates to the biological fermentation field, relate to the aspergillus niger spore that a strain adopts space flight mutagenesis means to obtain, and obtain high yield citric acid bacterial strain by the directed screening of the high sugared peracid of tolerance.
Background technology
Citric acid (Citric acid) has another name called Citric Acid, is white granular or white crystalline powder, and relative density is 1.6550, odorless has pleasant strong tart flavour, the entrance frankness, tart flavour without issue, safety non-toxic is widely used as the acidic flavoring agent of food and drink, and the production of citric acid is mainly by producing the fermentation of Aspergillus niger of citric acid in the global range, aspergillus niger belongs to deuteromycetes, generally only carrying out vegetative propagation, begun to new sporulation by spore-germination, is a life cycle.Aspergillus niger utilizes carbohydrate fermentation to generate citric acid, and its biosynthetic pathway now generally believes and is: glucose generates pyruvic acid through EMP, the degraded of HMP approach, and pyruvic acid oxidative decarboxylation on the one hand generates acetyl-CoA, on the other hand through CO
2Immobilized reactant generates oxaloacetic acid, and oxaloacetic acid and acetyl-CoA condensation generate citric acid.
The tank fermentation method that the industrial production of citric acid adopts aspergillus niger to carry out usually, raw material is done and corn based on potato.The intensive degree of world's citric acid industry is very high, external in removing, external main citric acid manufacturers has only 5～6 families (U.S., Austria, Canada, Brazil etc.), reach 940,000 tons to China's citric acid output in 2011, accounting for about 70% of Gross World Product, is genuine big producing country from output.The intensive world lead level that reached of China's citric acid industry.Along with the industrial expansion people to containing the increase in demand of citric acid product, and the acid producing ability of Aspergillus niger strain is a bottleneck of restriction citric acid production ability.Therefore the Aspergillus niger strain of screening acquisition high yield citric acid is significant.
Having both at home and abroad much and report about the research of citric acid production bacterium aspergillus niger breeding, mainly is to adopt physics and chemical means to carry out selection by mutation.Various mutagenic compound have the singularity of its effect, and people also can't select optimum mutagenic compound at a certain purpose, generally all are rule of thumb to prove mutagenesis effect factor preferably in real work.Since beginning with the microorganisms producing citric acid at the beginning of last century, people have carried out the mutagenesis research of decades to the bacterial classification of producing citric acid.China is accumulating rich experience aspect the breeding of citric acid production bacterium aspergillus niger, thinks more effective with single physical method or chemical process with the method ratio of complex mutation.At present the comparatively effective mutagen of aspergillus niger breeding is had: gamma-radiation, x-ray, ultraviolet ray, laser, nitrosoguanidine (NTG), nitrosourea, ethylamine etc. also have stream of energetic electrons, electromagnetic field etc. in addition.Utilize these mutagens in the aspergillus niger breeding work, to obtain many achievements.
Late 1950s and the beginning of the sixties, people such as Jin Qirong have carried out the bacterial classification research of citric acid fermentation.Shanghai industrial microorganism institute, the wild aspergillus niger 628 that obtains through acid plate isolation from soil is as starting strain, through complex mutations such as 60Co-gamma-rays and sulfuric acid diethyl ester repeatedly, obtained high yield citric acid bacterial strain Co827.It can directly utilize the fermentation of potato dry powder, produces acid and reaches 12～13%, and average conversion is 95%, fermentation period 54～64h.Wuxi polytechnical university is starting strain with aspergillus niger H-142, by gamma-rays, sulfuric acid diethyl ester, high temperature separately or complex mutation handle, obtain the HQL-601 bacterial classification by high temperature, peracid and the high directed screening that oozes culture condition.Its leavening temperature is 40～41 ℃, and cycle 60～64h, 20% potato dry powder shake bottle and produce acid 13%, and its citric acid purity obviously is better than existing zymophyte.
Hao Jie uses low energy ion and injects the selection by mutation technology, existing citric acid fermentation bacterium is improved, and improved the prescreening method that produces citric acid high yield bacterium.Inject through N+ repeatedly, obtained the superior strain of the maize raw material fermentation production of citric acid of 2 strain inheritance stabilities in the laboratory seed selection, HN-2004 shake flask fermentation 96h wherein on average produces acid 13.8%.Although the product acid of HN-2004 has improved 50% than original starting strain, but still there is the problem that fermentation period is long, glucose acid invert ratio is low.This shows, use single mutagen often to be difficult to reach its intended purposes.Wang Jun etc. have attempted using two kinds of nuclear technique means first and simultaneously aspergillus niger have been carried out mutagenesis, and confirmed employing 60Co-γ irradiation and compound N in the breeding of aspergillus niger
+The feasibility of method for implanting has obtained 1 strain fermentation period 72h, has produced acid 13.68%, transformation efficiency reached 103.45% M3 for bacterial strain CN05.
Ion implantation is the current techique that material surface is handled, and at present, uses the ion implantation improvement of crop cultivar that carries out to obtain success, at present can be roughly with ion implantation four reaction process such as energy deposition, momentum transfer, particle injection and charge-exchange that are divided into.Its mechanism of action is comparatively complicated, is difficult to get across with single-mode.Energetic ion injects except having the feature that energy deposition causes body injury, also has the cascade damage that exchange of kinetic energy produces, show as the disappearance of genetic material atom transfer, rearrangement or gene, the damage that the biomolecules transfer transport that also has chemical damage that slowing down ion, displaced atom and the reaction of background elements compounding cause and charge-exchange to cause causes.From ion implantation lethality rate curve etc. as can be seen, this mutagenesis means are different from traditional method in mechanism.Low energy ion injects and has characteristics such as physiological damage is little, mutation rate height, can obtain higher positive mutation rate.Therefore can use it for the breed improvement of animal and microorganism
At present, citric acid fermentation bacterium aspergillus niger has experienced the mutagenesis screening of over half a century, the aspergillus niger strain that is used for the suitability for industrialized production citric acid mostly passes through the processing of physics and chemical mutagen, increased the resistance of aspergillus niger to some mutagenic compound to some extent, then be difficult to existing bacterial classification is improved if re-use same method.Physics and chemical mutagen commonly used are produced certain resistance, therefore how to have adopted more advanced technique means to realize that the selection by mutation that citric acid is produced the bacterium aspergillus niger becomes present problem demanding prompt solution.
Summary of the invention
The present invention is directed to existing citric acid and produce the technical bottleneck that the selection by mutation of bacterium aspergillus niger runs into, a kind of aspergillus niger spore that adopts space flight mutagenesis means to obtain is provided, by tolerating the directed screening of high sugared peracid, thereby design the mutagenic and breeding of science screening scheme realization efficiently and obtain superior strain, this bacterial strain called after TN-A09, the contriver has carried out biological preservation to it, its preserving number is CGMCC No.5751, this bacterial strain is the sugared peracid of anti-height, it is high to produce acid, the citric acid production bacterial strain that transformation efficiency is high, utilize its fermentation production of citric acid, the acid of average product reaches 18%, fermentation period 60 hours, transformation efficiency 99% is far above existing aspergillus niger liquid fermenting citric acid state of the art.
Production bacterial strain of the present invention, be after aspergillus niger spore is carried recoverable spacecraft and carries out space flight, the directed screening of the sugared peracid of the anti-height of process obtains, the contriver has carried out biological preservation to it, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, its preserving number is CGMCCNo.5751, and its state is survival.
The present invention carries out the method for space flight for the selection by mutation of citric acid generation bacterium aspergillus niger to carry recoverable spacecraft first.Not only can avoid aspergillus niger to the resistance of some mutagenic compound, and bigger selection space is arranged, obtain comparatively good bacterial classification easilier.
After obtaining above-mentioned bacterial classification, the screening of bacterial strain is the associated steps behind the induction mutation of bacterium, also is the committed step that determines strain excellent seed selection efficient.Microorganism cells colony is through after the mutagenic treatment, though the frequency that sudden change takes place than high many of the frequency of spontaneous mutation, but in the colony of whole cell, its frequency is still very low, the cell absolute number that namely morphs is very low.What suddenly change on the DNA chain is at random, and the frequency that needed mutant strain occurs is just lower, seeks required mutant just as looking for a needle in a haystack.Therefore reasonably screening procedure and method are extremely important in the seed selection of aspergillus niger strain.Mutagenesis is at random, but selection is directed.Need after the mutagenesis to screen detection method by correct, sensitivity, fast, from not becoming in a large number and bearing the colony that becomes and pick out remaining very a spot of forward mutation.
In the mutagenic and breeding of existing aspergillus niger, people often indicate the size of circle as the primary dcreening operation foundation with yellow on the indicative plate substratum of tetrabromo-mcresolsulfonphthalein, eliminate on a large scale, to improve screening efficiency, but find in actual applications, tetrabromo-mcresolsulfonphthalein obviously influences the growth of aspergillus niger and produces acid, can not objectively reflect the acid energy of product of bacterial strain.Therefore, for minimum workload, in the shortest time, obtain maximum screening effect, thereby the present inventor designs the target that the mutagenic and breeding of science screening scheme realization efficiently obtains superior strain.The final acquisition sugared peracid of anti-height and the citric acid bacterial classification that product acid is high, transformation efficiency is high, energy consumption is low, production cost is low have important actual production and are worth.
Concrete technical scheme of the present invention is:
One, the preparation of space flight mutagenesis aspergillus niger xerospore
1, aspergillus niger Co827 is inoculated in PDA (potato culture) inclined-plane, static cultivation is 4 days under 35 ℃ of conditions, the inclined-plane covers with behind the spore with the aseptic water washing spore to the aseptic triangular flask with granulated glass sphere, and every inclined-plane is washed in the triangular flask, wraps up with gauze;
2, the triangular flask that will fill spore suspension was put on the shaking table vibration 2 hours, rotating speed 270r/min, and 25 ℃ of temperature are fully broken up spore;
3, will the go out vacuum filtration bottle of bacterium is put in the Bechtop, and the opening power vacuum pump is started working, and spore suspension is poured in the funnel on the filter flask, and the suction filtration process should cover four layers of sterile gauze at funnel, and the suction filtration process continues 3 minutes;
4, suction filtration finishes, and takes off the suction filtration film with aseptic nipper, puts into aseptic empty culture dish, moves into dry 24h in the normal temperature dryer after sterilizing; Described moisture eliminator is cleaned twice through 70wt% ethanol in advance and is reached surface sterilization, and in Bechtop through ultra violet lamp 30 minutes, sterilized fully in the moisture eliminator internal space;
5, the spore that drying is good blows in the aseptic sample bottle with the inoculation shovel in Bechtop, and sample bottle is filled, and builds lid, and sample is ready;
6, retain the dry good spore sample of part and place drying and sterile seal pipe to be placed in the moisture eliminator, place identical time with space flight mutagenesis, sample in contrast in room temperature;
Wherein PDA culture medium preparation method of the present invention is peeling potatoes, and 200 grams are cut into piece and boil 30min, then with supplying water after the filtered through gauze to 1000mL, adds glucose 20 grams then, agar powder 15 grams, and 113 ℃ of sterilization 15min fall dull and stereotyped.
The sample that above-mentioned steps 5 obtains carries out space flight by carrying recoverable spacecraft, and mutagenic obtained xerospore is aimed strain through the bacterial strain of the directed screening acquisition of the sugared peracid of anti-height, and concrete screening method is:
1, high sugared peracid culture medium preparation
High sugared peracid substratum:
A liquid is made: peeling potatoes, and 200 grams are cut into piece and boil 30min, then with supplying water after the filtered through gauze to 1000mL, 121 ℃ of sterilization 15min, standby;
B liquid is made: glucose 200-220 gram, Citric Acid, usp, Anhydrous Powder 170-190 gram, MgSO
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, agar powder 22 grams are dissolved in 500ml distilled water, and 113 ℃ of sterilization 15min are standby;
A liquid and B liquid are heated to 50 ℃ respectively, B liquid is fully melted after, get A liquid, B liquid respectively and mix for each 500 milliliters, pour in the culture dish, cool off the standby high sugared peracid substratum that is.
2, the directed screening of the sugared peracid of anti-height
Xerospore 100 μ g shake up into spore suspension after getting space flight mutagenesis in the 100ml sterilized water, then 10 times of gradient dilutions to 10
-4, from 10
-3, 10
-4Extent of dilution is respectively got 100 μ l and is evenly coated on the sugared peracid substratum of above-mentioned height, and 35 ℃ of static cultivation 96h observe media surface and grow white small colonies, are the bacterial strain with the sugared peracid performance of anti-height.
Owing to contain the agar powder composition in the B liquid, make whole substratum after being lower than 40 degree, become solid, so spore directly can be coated on its surface.
On the basis of the bacterial strain of the sugared peracid performance of anti-height of above-mentioned acquisition, the contriver has further carried out the bacterial strain of screening to obtain wherein to be suitable for the most, and its concrete steps are as follows:
Produce the preliminary screening of citric acid performance: Semen Maydis powder liquefaction clear liquid substratum is made:
Getting Semen Maydis powder liquefaction clear liquid makes: get the Semen Maydis powder that 100 grams were pulverized 60 mesh sieves, add 300 ml waters, stir and be heated to 60 ℃, add alpha-amylase, as high-temperature, calculate with the every gram Semen Maydis powder of 10-15 enzyme activity unit, be warming up to 90 ℃, keep temperature to stir liquefaction and continue 4h, use the filter cloth press filtration, measure total sugar concentration.
Get 1000 milliliters of this Semen Maydis powder liquefaction clear liquids, add glucose and make its total sugar concentration reach 15%-18%, add MgSO simultaneously
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, agar powder 22 grams, 113 ℃ of sterilization 15min, standby.
Produce the screening of acid energy: take the bacterium colony of in the sugared peracid substratum of height, growing and it is transferred to 35 ℃ of static cultivation 72h in the Semen Maydis powder liquefaction clear liquid substratum, observe media surface transparent circle and colony diameter ratio greater than 5 bacterium colony, with such colony lift 35 ℃ of static cultivation 96h to the inclined-plane of PDA substratum, it is standby to treat that the surface covers with spore.Why selecting media surface transparent circle and colony diameter ratio greater than 5 bacterium colony, is because the bacterial strain production performance in this bacterium colony is better than other bacterial strain, is more suitable for the application in producing.
Shaking a bottle Semen Maydis powder liquefaction clear liquid substratum makes:
Getting Semen Maydis powder liquefaction clear liquid makes: get the Semen Maydis powder that 100 grams were pulverized 60 mesh sieves, add 300 ml waters, stir and be heated to 60 ℃, add alpha-amylase, calculate with the every gram Semen Maydis powder of 10-15 enzyme activity unit, be warming up to 90 ℃, keep temperature to stir liquefaction and continue 4h, use the filter cloth press filtration, measure total sugar concentration.
Get 1000 milliliters of this Semen Maydis powder liquefaction clear liquids, add glucose and make its total sugar concentration reach 15%-16%, MgSO
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams.113 ℃ of sterilization 15min are standby in per 50 milliliters of 500 milliliters of triangular flasks of packing into.
Get above-mentioned standby spore, be inoculated in 50 milliliters of Semen Maydis powder clear liquid substratum in above-mentioned 500 milliliters of triangular flasks, 35 ℃, per minute 300 changes, and measures the concentration of total acid in the substratum behind the shaking culture 72h, compare with the mutagenesis initial strain, to relatively produce the high bacterial strain of acid concentration with initial strain and carry out the PDA inclined-plane and go down to posterity, passed for 10 generations after, get respectively 2,4,6,8,10 generation the inclined-plane, shake bottle again and produce the acid test, acid yield is stable and produce the highest bacterial strain of acid as alternative.
The contriver utilizes the alternative bacterial strain of above-mentioned acquisition to carry out continuous 30 batches of 500 tons of citric acid fermented tests, and the result shows that average product acid reaches 18%, fermentation period 60 hours, and transformation efficiency 99% is far above existing aspergillus niger liquid fermenting citric acid state of the art.
The contriver has carried out biological preservation to the above-mentioned bacterial strain that filters out, and depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, and its preserving number is CGMCC No.5751, and its state is survival.
In sum, citric acid superior strain provided by the present invention, this bacterial strain called after TN-A09, the contriver has carried out biological preservation to it, and this bacterial strain is the sugared peracid of anti-height, produces the citric acid production bacterial strain that acid is high, transformation efficiency is high, utilizes its fermentation production of citric acid, the acid of average product reaches 18%, fermentation period 60 hours, transformation efficiency 99% is far above existing aspergillus niger liquid fermenting citric acid state of the art.
Preservation information
The preservation time: on February 10th, 2012
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center
Deposit number: CGMCC No.5751
Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Classification name: aspergillus niger Aspergillus niger
Embodiment
Further specify the present invention below in conjunction with embodiment, can make those skilled in the art more fully understand the present invention, but not limit the present invention in any way.
Embodiment 1:
The preparation of space flight mutagenesis aspergillus niger xerospore
1, aspergillus niger Co827 is inoculated in PDA (potato culture) inclined-plane, static cultivation is 4 days under 35 ℃ of conditions, the inclined-plane covers with behind the spore with the aseptic water washing spore to the aseptic triangular flask with granulated glass sphere, and every inclined-plane is washed in the triangular flask, wraps up with gauze;
2, the triangular flask that will fill spore suspension was put on the shaking table vibration 2 hours, rotating speed 270r/min, and 25 ℃ of temperature are fully broken up spore;
3, will the go out vacuum filtration bottle of bacterium is put in the Bechtop, and the opening power vacuum pump is started working, and spore suspension is poured in the funnel on the filter flask, and the suction filtration process should cover four layers of sterile gauze at funnel, and the suction filtration process continues 3 minutes;
4, suction filtration finishes, and takes off the suction filtration film with aseptic nipper, puts into aseptic empty culture dish, moves into dry 24h in the normal temperature dryer after sterilizing; Described moisture eliminator is cleaned twice through 70wt% ethanol in advance and is reached surface sterilization, and in Bechtop through ultra violet lamp 30 minutes, sterilized fully in the moisture eliminator internal space;
5, the spore that drying is good blows in the aseptic sample bottle with the inoculation shovel in Bechtop, and sample bottle is filled, and builds lid, and sample is ready;
6, retain the dry good spore sample of part and place drying and sterile seal pipe to be placed in the moisture eliminator, place identical time with space flight mutagenesis, sample in contrast in room temperature;
Wherein PDA culture medium preparation method is peeling potatoes, and 200 grams are cut into piece and boil 30min, then with supplying water after the filtered through gauze to 1000mL, adds glucose 20 grams then, agar powder 15 grams, and 113 ℃ of sterilization 15min fall dull and stereotyped.
Embodiment 2:
The sample that step 5 obtains in above-described embodiment carries out space flight by carrying recoverable spacecraft.
Embodiment 3:
The directed screening of the sugared peracid of anti-height
High sugared peracid substratum:
A liquid is made: peeling potatoes, and 200 grams are cut into piece and boil 30min, then with supplying water after the filtered through gauze to 1000mL, 121 ℃ of sterilization 15min, standby;
B liquid is made: glucose 200-220 gram, Citric Acid, usp, Anhydrous Powder 170-190 gram, MgSO
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, agar powder 22 grams are dissolved in 500ml distilled water, and 113 ℃ of sterilization 15min are standby;
A liquid and B liquid are heated to 50 ℃ respectively, B liquid is fully melted after, get A liquid, B liquid respectively and mix for each 500 milliliters, pour in the culture dish, cool off the standby high sugared peracid substratum that is.
2, the directed screening of the sugared peracid of anti-height
Xerospore 100 μ g shake up into spore suspension after getting space flight mutagenesis in the 100ml sterilized water, then 10 times of gradient dilutions to 10
-4, from 10
-3, 10
-4Extent of dilution is respectively got on the high sugared peracid substratum after 100 μ l evenly coat above-mentioned curing, and 35 ℃ of static cultivation 96h observe media surface and grow white small colonies, are the bacterial strain with the sugared peracid performance of anti-height.
Embodiment 4: the primary dcreening operation that produces acid energy
Semen Maydis powder liquefaction clear liquid substratum is made:
Getting Semen Maydis powder liquefaction clear liquid makes: get the Semen Maydis powder that 100 grams were pulverized 60 mesh sieves, add 300 ml waters, stir and be heated to 60 ℃, adding high-temperature calculates with the every gram Semen Maydis powder of 10-15 enzyme activity unit, be warming up to 90 ℃, keep temperature to stir liquefaction and continue 4h, use the filter cloth press filtration, measure total sugar concentration.
Get 1000 milliliters of this Semen Maydis powder liquefaction clear liquids, add glucose and make its total sugar concentration reach 15-18wt%, add MgSO simultaneously
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, agar powder 22 grams, 113 ℃ of sterilization 15min namely get Semen Maydis powder liquefaction clear liquid substratum, and are standby.
Produce the screening of acid energy: take the bacterial strain with the sugared peracid performance of anti-height of in the sugared peracid substratum of height, growing that obtains among the embodiment 3, it is transferred to 35 ℃ of static cultivation 72h in the above-mentioned Semen Maydis powder liquefaction clear liquid substratum, observe media surface transparent circle and colony diameter ratio greater than 5 bacterium colony, with such colony lift 35 ℃ of static cultivation 96h to the inclined-plane of PDA substratum, it is standby to treat that the surface covers with spore.
Embodiment 5: shake the multiple sieve that bottle produces acid energy
Shaking a bottle Semen Maydis powder liquefaction clear liquid substratum makes:
Getting Semen Maydis powder liquefaction clear liquid makes: get the Semen Maydis powder that 100 grams were pulverized 60 mesh sieves, add 300 ml waters, stir and be heated to 60 ℃, add high-temperature, calculate with the every gram Semen Maydis powder of 10-15 enzyme activity unit, be warming up to 90 ℃, keep temperature to stir liquefaction and continue 4h, use the filter cloth press filtration, measure total sugar concentration.
Get 1000 milliliters of this Semen Maydis powder liquefaction clear liquids, add glucose and make its total sugar concentration reach 15%-16%, add MgSO simultaneously
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, 113 ℃ of sterilization 15min are standby in per 50 milliliters of 500 milliliters of triangular flasks of packing into.
Get among the embodiment 4 and to produce acid circle and bacterium colony ratio greater than 5 bacterial strain, spore on the PDA inclined-plane, be inoculated in 50 milliliters of Semen Maydis powder clear liquid substratum in above-mentioned 500 milliliters of triangular flasks, 35 ℃, per minute 300 changes, measure the concentration of total acid in the substratum behind the shaking culture 72h, compare with the mutagenesis initial strain, to relatively produce the high bacterial strain of acid concentration with initial strain carries out the PDA inclined-plane and goes down to posterity, after passing for 10 generations, get 2 respectively, 4,6,8,10 generation the inclined-plane, shake bottle again and produce the acid test, it is identical that the result produces the citric acid level during with initial 0 generation, and the highest bacterial classification of acid yield is as alternative.The contriver has carried out biological preservation to the above-mentioned bacterial strain that filters out, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, its preserving number is CGMCC No.5751, its state is survival, utilize its fermentation production of citric acid, on average produce acid and reach 18%, fermentation period 60 hours, transformation efficiency 99% is far above existing aspergillus niger liquid fermenting citric acid state of the art.
Claims (5)
1. a strain citric acid superior strain, it is characterized in that: this bacterial strain called after TN-A09, its preserving number is CGMCC No.5751.
2. superior strain according to claim 1, it is characterized in that: this bacterial strain is by the aspergillus niger mutagenesis of code name Co827.
3. the preparation method of the described superior strain of claim 1, it is characterized in that: concrete steps are as follows:
(1), aspergillus niger Co827 is inoculated in the PDA inclined-plane, static cultivation is 4 days under 35 ℃ of conditions, and the inclined-plane covers with behind the spore with the aseptic water washing spore to the aseptic triangular flask with granulated glass sphere, and every inclined-plane is washed in the triangular flask, wraps up with gauze;
(2), the triangular flask that will fill spore suspension put on the shaking table vibration 2 hours, rotating speed 270r/min, 25 ℃ of temperature are fully broken up spore;
(3), the vacuum filtration bottle of the bacterium of will going out puts in the Bechtop, the opening power vacuum pump is started working, and spore suspension is poured in the funnel on the filter flask, the suction filtration process should cover four layers of sterile gauze at funnel, the suction filtration process continues 3 minutes;
(4), suction filtration finishes, and takes off the suction filtration film with aseptic nipper, puts into aseptic empty culture dish, moves into dry 24h in the normal temperature dryer after the sterilization; Described moisture eliminator is cleaned twice through 70wt% ethanol in advance and is reached surface sterilization, and in Bechtop through ultra violet lamp 30 minutes, sterilized fully in the moisture eliminator internal space;
(5), spore that drying is good blows in the aseptic sample bottle with the inoculation shovel in Bechtop, and sample bottle is filled, and builds lid, sample is ready;
The sample that above-mentioned steps (5) obtains carries out space flight by carrying recoverable spacecraft, and mutagenic obtained xerospore is aimed strain through the bacterial strain of the directed screening acquisition of the sugared peracid of anti-height, and its preserving number is CGMCC No.5751.
4. method according to claim 3 is characterized in that:
The directed screening of the sugared peracid of described anti-height adopts high sugared peracid culture medium culturing directed screening, and concrete steps are:
Xerospore 100 μ g shake up into spore suspension after getting space flight mutagenesis in the 100ml sterilized water, then 10 times of gradient dilutions to 10
-4, from 10
-3, 10
-4Extent of dilution is respectively got 100 μ l and is evenly coated on the sugared peracid substratum of above-mentioned height, and 35 ℃ of static cultivation 96h observe media surface and grow white small colonies, are the bacterial strain with the sugared peracid performance of anti-height.
5. method according to claim 4, it is characterized in that: the sugared peracid medium preparation of described high method is as follows:
A liquid is made: peeling potatoes, and 200 grams are cut into piece and boil 30min, then with supplying water after the filtered through gauze to 1000mL, 121 ℃ of sterilization 15min, standby;
B liquid is made: glucose 200-220 gram, Citric Acid, usp, Anhydrous Powder 170-190 gram, MgSO
47H
2The O1.5 gram, K
2HPO
43.6, ammonium sulfate 2 grams, agar powder 22 grams are dissolved in 500ml distilled water, and 113 ℃ of sterilization 15min are standby;
A liquid and B liquid are heated to 50 ℃ respectively, B liquid is fully melted after, get A liquid, B liquid respectively and mix for each 500 milliliters, pour in the culture dish, cool off the standby high sugared peracid substratum that is.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367359A (en) * | 2016-11-16 | 2017-02-01 | 中国林业科学研究院林产化学工业研究所 | Aspergillus niger and application thereof to preparing citric acid from fermented acorns |
| CN107058209A (en) * | 2017-05-26 | 2017-08-18 | 天津科技大学 | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder |
| CN108410743A (en) * | 2018-05-15 | 2018-08-17 | 日照金禾博源生化有限公司 | A kind of aspergillus niger wheat bran culture medium and its preparation method |
| CN110791439A (en) * | 2019-10-10 | 2020-02-14 | 天津科技大学 | Recombinant aspergillus niger strain for fermentation production of malic acid by genetic engineering construction and application |
| CN112322505A (en) * | 2020-11-30 | 2021-02-05 | 日照金禾博源生化有限公司 | Method for producing citric acid by using space mutation aspergillus niger |
| CN113667607A (en) * | 2020-05-15 | 2021-11-19 | 中粮生物科技股份有限公司 | Aspergillus niger mutant strain and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367359A (en) * | 2016-11-16 | 2017-02-01 | 中国林业科学研究院林产化学工业研究所 | Aspergillus niger and application thereof to preparing citric acid from fermented acorns |
| CN106367359B (en) * | 2016-11-16 | 2019-07-30 | 中国林业科学研究院林产化学工业研究所 | A kind of aspergillus niger and its application in citric acid is prepared in fermentation acorn |
| CN107058209A (en) * | 2017-05-26 | 2017-08-18 | 天津科技大学 | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder |
| CN108410743A (en) * | 2018-05-15 | 2018-08-17 | 日照金禾博源生化有限公司 | A kind of aspergillus niger wheat bran culture medium and its preparation method |
| CN110791439A (en) * | 2019-10-10 | 2020-02-14 | 天津科技大学 | Recombinant aspergillus niger strain for fermentation production of malic acid by genetic engineering construction and application |
| CN113667607A (en) * | 2020-05-15 | 2021-11-19 | 中粮生物科技股份有限公司 | Aspergillus niger mutant strain and application thereof |
| CN112322505A (en) * | 2020-11-30 | 2021-02-05 | 日照金禾博源生化有限公司 | Method for producing citric acid by using space mutation aspergillus niger |
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Effective date of registration: 20161031 Address after: 276500 Laiyang Road, Juxian Economic Development Zone, Shandong, No. 209, No. Patentee after: Rizhao Rijin Boyuan Biochemistry Co., Ltd. Patentee after: Tianjin University of Science & Technology Address before: 276800 west section of Xing Hai Road, Shandong, Rizhao City Patentee before: Rizhao Luxin RZBC Co., Ltd. Patentee before: Tianjin University of Science & Technology |