CN106801075A - A kind of production method of rhamnolipid - Google Patents
A kind of production method of rhamnolipid Download PDFInfo
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- CN106801075A CN106801075A CN201710097464.2A CN201710097464A CN106801075A CN 106801075 A CN106801075 A CN 106801075A CN 201710097464 A CN201710097464 A CN 201710097464A CN 106801075 A CN106801075 A CN 106801075A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The present invention provides a kind of production method of rhamnolipid, and it is to addition cellulase in manioc waste and pseudomonas aeruginosa simultaneous saccharification and fermentation production rhamnolipid.Production method proposed by the present invention, adds cellulase and pseudomonas aeruginosa simultaneously in course of reaction, realize direct simultaneous saccharification and fermentation production rhamnolipid, shortens the reaction time, advantageously reduces production cost；Chemicals use cost is not reduced after chemical pretreatment, both in whole course of reaction, and whole technical process environmental protection is caused again；Rhamnolipid is produced compared to by raw material of glucose, can significantly improve efficiency of pcr product by direct simultaneous saccharification and fermentation production rhamnolipid by raw material of manioc waste.Manioc waste only adds cellulase and pseudomonas aeruginosa on the premise of without two enzymes method starch enzymatic conversion, and rhamnolipid yield is up to 11.49g/L.
Description
Technical field
The invention belongs to field of organic polymer compound, and in particular to a kind of preparation method of rhamnolipid.
Background technology
Cassava is to plant one of the crops in torrid areas and part subtropical zone extensively, and raw material sources are wide, resource
It is abundant.Tapioca root meat is rich in starch, and main in industrial production is Raw material processing starch or alcohol with it.Manioc waste is with wood
Potato is solid waste remaining after Raw material processing product, and its content of starch is higher, while also containing a certain amount of wood fibre
, there is sizable recycle value in plain and a small amount of protein.Though make the research of feed applications using manioc waste at present,
But still unrealized extensive industrialization is utilized, most of enterprise still selects directly to be abandoned as discarded object, and this is not only right
The huge waste of resource, is also the huge pollution to environment.Manioc waste can after effectively being degraded because of contained crude fibre warp in recent years
To turn waste into wealth, save resources again environmental protection and be subject to the extensive concern of domestic and foreign scholars, grinding in terms of manioc waste recycling
Studying carefully has turned into a big focus.If the starch and cellulose in manioc waste can be changed into Portugal by screening microorganism and enzyme preparation
Grape sugar, then daily use chemicals raw material, food additives and newtype drug etc. are produced through microbial fermentation, just can be with cheap cost of material
Considerable economic benefit is brought, so as to realize effective degraded and the recycling of manioc waste.
Rhamnolipid is the secondary metabolite produced under certain condition by microorganism, by 1~2 rhamnose and difference
Beta-hydroxy fatty acid two parts connection composition of carbon chain lengths.Rhamnolipid is a kind of new anion Bio-surface active
Agent, with the functions such as emulsification, solubilising, dispersion, cohesion and reduction interfacial tension, and low toxicity, easily biological-degradable, thus in oil
The fields such as chemical industry, biological medicine, food and environmental protection have broad application prospects.The preparation of rhamnolipid is mainly by micro-
Biofermentation, then extracted from zymotic fluid and obtain.The strain of rhamnolipid is produced with pseudomonas aeruginosa (Pseudomonas
Aeruginosa it is) most commonly seen.The carbon source of numerous species can fermenting and producing rhamnolipid, from petrochemistry derivative to day
Right raw material, most common of which is vegetable oil, sugar and glycerine.At present, the Main Bottleneck of rhamnolipid is obtained through microbial fermentation
Be carbon source high cost and rhamnolipid yield it is relatively low, which has limited the large-scale production and application of rhamnolipid.Reduce
The cost of fermentation substrate turns into the Main way of rhamnolipid exploitation.Manioc waste is with it as a kind of carbon source cheap and easy to get
Raw material biosynthesis rhamnolipid, not only reduces the cost of production rhamnolipid, and reduces the treatment cost of manioc waste,
Realize the recycling of manioc waste.
The content of the invention
For the weak point that this area is present, the purpose of the present invention is to propose to a kind of production method of rhamnolipid, use
Manioc waste produces rhamnolipid on the premise of without any pretreatment by simultaneous saccharification and fermentation, and this technique can be significantly
Improve the utilization rate of manioc waste set of resources.
Concrete technical scheme to achieve the above object is：
A kind of production method of rhamnolipid, is to addition cellulase and pseudomonas aeruginosa in manioc waste, synchronous sugar
Change fermenting and producing rhamnolipid.
Compared by the saccharification and fermentation test to manioc waste, simultaneous saccharification and fermentation compare through two enzymes method be saccharified after access copper
Green pseudomonad fermentation or manioc waste access pseudomonas aeruginosa after enzymolysis and are fermented, and have superior technique effect.
Further, the manioc waste is industrial manioc waste, and water content is 8~15%, and the granularity of manioc waste is by 40
Mesh sieve.
Wherein, the pseudomonas aeruginosa first passes around fluid nutrient medium culture and fermentation medium culture, is subsequently adding
Manioc waste, the ratio that manioc waste accounts for fermentation medium is 2~6w/v%.
Wherein, the composition that the fluid nutrient medium contains is：Peptone 10.0g/L, beef extract 3.0g/L, NaCl 5.0g/
L；
The composition that fermentation medium contains is：MgSO4·7H2O 0.125g/L, NaNO31.5g/L, KH2PO4 0.125g/
L, yeast extract 0.5g/L.
Wherein, the addition cellulase, cellobiase and pseudomonas aeruginosa seed suspension.Fiber in reaction system
The addition of plain enzyme is 3.75FPU/g- manioc wastes, and the addition of cellobiase is 5.65CBU/g- manioc waste (cellulases
Middle cellobiase content is relatively low, it is necessary to additionally add cellobiase).Wherein, the pseudomonas aeruginosa seed suspension connects
Enter the access amount of fermentation medium for 1~1.5% (v/v), the condition of culture is 35~38 DEG C of temperature, 144~168h of time.
Wherein, after fermentation ends, by zymotic fluid separation of solid and liquid, clear liquid is collected, regulation clear liquid pH is standing after 1.6~2.2,
Treat that flocculent deposit occurs, clear liquid is extracted with isometric ethyl acetate, collect the upper solution of extraction.
Preferably, supernatant is collected after zymotic fluid being centrifuged into 15min under 12000 × g centrifugal conditions, is adjusted with HCl solution
Section supernatant pH is 1.6~2.2, is stood overnight at 4 DEG C, is extracted with isometric ethyl acetate, collects the upper solution of extraction,
Use rotary evaporator vacuum distillation.
The beneficial effects of the present invention are：
Manioc waste is to extract the industrial solid castoff after starch, and stock number is big, cheap；The shallow lake contained in manioc waste
Powder and cellulose can be converted into glucose through effectively degraded, may be used to produce rhamnolipid.In production method of the invention, cassava
Slag is saccharified without two enzymes method, and most starch and cellulose are converted into grape by only adding the cellulase of low consumption
Sugar, is conducive to process simplification.
Production method proposed by the present invention, adds cellulase and pseudomonas aeruginosa simultaneously in course of reaction, realize
Direct simultaneous saccharification and fermentation production rhamnolipid, shortens the reaction time, advantageously reduces production cost；In whole course of reaction
Not after chemical pretreatment, chemicals use cost had both been reduced, whole technical process environmental protection had been caused again；Compared to grape
Sugar produces rhamnolipid for raw material, can obtain product by direct simultaneous saccharification and fermentation production rhamnolipid by raw material of manioc waste
Rate is significantly improved.Manioc waste only adds cellulase and pseudomonas aeruginosa on the premise of without two enzymes method starch enzymatic conversion,
Rhamnolipid yield is up to 11.49g/L.
Brief description of the drawings
Fig. 1 is the process chart of rhamnolipid production method of the present invention.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
In embodiment, manioc waste is to extract the industrial solid castoff after starch, moisture content 8~12%.
In embodiment, unless otherwise specified, the means for being used are this area conventional technology.
Embodiment 1
Flow is shown in Fig. 1.
1) the size-reduced machine of manioc waste is crushed, and industrial vibration is sieving through 40 mesh sieves.
2) fluid nutrient medium (g/L) is prepared：Peptone 10.0, beef extract 3.0, NaCl 5.0.Culture is obtained for accessing
Pseudomonas aeruginosa (preserving number CGMCC1.1785) seed suspension of fermentation system.
3) fermentation medium (g/L) is prepared：MgSO4·7H2O 0.125, NaNO31.5, KH2PO40.125, yeast is extracted
Thing 0.5.
To the manioc waste that 5% (w/v) is added in fermentation medium, and with 2mol/L NaOH solution regulation systems pH extremely
6.0, zymotic fluid is obtained.Zymotic fluid is poured into 250mL aerobic fermentations blake bottle (triangular flask of the perpendicular baffle plate of band), bottling amount is
50mL, sterilizes after bottleneck ParafilmTM.It is 3.75FPU/g- manioc wastes then to consumption is added in aseptic zymotic fluid
Cellulase, the cellobiase of 5.65CBU/g- manioc wastes and inoculum concentration are hanged for the pseudomonas aeruginosa seed of 1% (v/v)
Liquid.168h is reacted during aerobic fermentation blake bottle is placed in into 37 DEG C, the constant-temperature table of 180rpm.
4) rhamnolipid in zymotic fluid is extracted, zymotic fluid is centrifuged 15min under 12000 × g centrifugal conditions
After collect supernatant.Supernatant pH to 2.0 is adjusted with 6mol/L HCl, is stood overnight at 4 DEG C, treat that flocculent deposit occurs, used
Isometric ethyl acetate extraction clear liquid 3 times, collects 3 upper solutions of extraction, uses rotary evaporator vacuum distillation, obtains sandlwood
Glycolipid slightly carries product.
The rhamnolipid crude yield of acquisition is 11.49g/L.
Embodiment 2
The size-reduced machine of manioc waste is crushed, and is sieving through 40 mesh sieves.Culture obtains the P. aeruginosa for accessing fermentation system
Bacterium (CGMCC 1.1785) seed suspension.Fermentation medium is prepared, to the manioc waste that 5% (w/v) is added in fermentation medium, and
With 2mol/L NaOH solution regulation systems pH to 6.0, zymotic fluid is obtained.Zymotic fluid is poured into 250mL aerobic fermentation blake bottles
In (band baffle plate), bottling amount is 50mL, is sterilized after bottleneck ParafilmTM.It is then to consumption is added in aseptic zymotic fluid
The cellulase of 3.75FPU/g- manioc wastes, the cellobiase of 5.65CBU/g- manioc wastes and inoculum concentration are the copper of 3% (v/v)
Green pseudomonad seed suspension.144h is reacted during aerobic fermentation blake bottle is placed in into 37 DEG C, the constant-temperature table of 180rpm.It is then right
Rhamnolipid in zymotic fluid is extracted.
Other operations are with embodiment 1.The rhamnolipid crude yield of acquisition is 11.08g/L.
Embodiment 3：
The size-reduced machine of manioc waste is crushed, and industrial vibration is sieving through 40 mesh sieves.Culture is obtained for accessing fermentation system
Pseudomonas aeruginosa (CGMCC 1.1785) seed suspension.Fermentation medium is prepared, to 2.5% (w/ of addition in fermentation medium
V) manioc waste, and with 2mol/L NaOH solution regulation systems pH to 6.0, zymotic fluid is obtained.Zymotic fluid is poured into 250mL good
In aerobe fermentation blake bottle (band baffle plate), bottling amount is 50mL, is sterilized after bottleneck ParafilmTM.Then to aseptic zymotic fluid
Middle addition consumption is that cellulase, the cellobiase of 5.65CBU/g- manioc wastes and the inoculum concentration of 3.75FPU/g- manioc wastes are
The pseudomonas aeruginosa seed suspension of 1% (v/v).It is anti-during aerobic fermentation blake bottle is placed in into 37 DEG C, the constant-temperature table of 180rpm
Answer 96h.Then the rhamnolipid in zymotic fluid is extracted.
Other operations are with embodiment 1.The rhamnolipid crude yield of acquisition is 5.98g/L.
Comparative example 1：
Culture obtains pseudomonas aeruginosa (CGMCC 1.1785) the seed suspension for accessing fermentation system.Prepare fermentation
Culture medium, to the glucose that 30g/L is added in fermentation medium, and with 2mol/L NaOH solution regulation system pH to 6.0, makes
Obtain zymotic fluid.Zymotic fluid is poured into 250mL aerobic fermentations blake bottle (band baffle plate), bottling amount is 50mL, bottleneck sealed membrane
Sterilized after sealing.Then to the pseudomonas aeruginosa seed suspension that 1% (v/v) is accessed in aseptic zymotic fluid.By aerobic fermentation
Blake bottle is placed in 37 DEG C, reacts 144h in the constant-temperature table of 180rpm.Then the rhamnolipid in zymotic fluid is extracted, is obtained
The rhamnolipid crude yield for obtaining is 1.98g/L.
Discuss：Premenstruum (premenstrua) experiment learns that manioc waste can obtain the glucose of about 30g/L through cellulase conversion, but directly
It is that substrate produces rhamnolipid with the glucose of 30g/L, yield is relatively low.Comparative example 2：
The size-reduced machine of manioc waste is crushed, and industrial vibration is sieving through 40 mesh sieves.Manioc waste is saccharified through two enzymes method：20% cassava
Slag uses amylase liquefaction 1h (CaCl2 is added before enzyme-added), starch enzyme dosage 20U/g manioc wastes, regulation gelatinization at 85 DEG C
Liquid pH to 4.0 (10%H2SO4), carbohydrase is added according to 150U/g manioc wastes, and be saccharified 2-3h in 60 DEG C, obtains manioc waste starch
Saccharified liquid.
Culture obtains pseudomonas aeruginosa (CGMCC 1.1785) the seed suspension for accessing fermentation system.Prepare 2 times
Concentration fermentation medium：MgSO4·7H2O 0.25, NaNO33.0, KH2PO40.25, yeast extract 1.0.It is aerobic to 250mL
2 times of concentration fermentation mediums of 25mL are added in fermented and cultured bottle (with baffle plate), 12.5g manioc wastes saccharified liquid and 15g is added
Water, and with 2mol/L NaOH solution regulation systems pH to 6.0, zymotic fluid is obtained.The bottleneck of aerobic fermentation blake bottle is sealed
Film sterilizes after sealing.Then in aseptic zymotic fluid add consumption for 3.75FPU/g- manioc wastes cellulase,
The cellobiase and inoculum concentration of 5.65CBU/g- manioc wastes are the pseudomonas aeruginosa seed suspension of 1% (v/v).By aerobic hair
Ferment blake bottle is placed in 37 DEG C, reacts 168h in the constant-temperature table of 180rpm.Then the rhamnolipid in zymotic fluid is extracted,
The rhamnolipid crude yield of acquisition is 5.65g/L.
Discuss：Manioc waste accesses pseudomonas aeruginosa and is fermented again after being saccharified through two enzymes method, can cause initial reaction stage
Concentration of glucose in solution is too high, is unfavorable for the growth of strain.
Comparative example 3：
The size-reduced machine of manioc waste is crushed, and industrial vibration is sieving through 40 mesh sieves.Manioc waste is digested：10% manioc waste is 48
DEG C, digest 96h under 180rpm, cellulase addition is that 3.55FPU/g- manioc wastes, cellobiase addition are
5.65CBU/g- manioc wastes.Culture obtains being hanged for pseudomonas aeruginosa (CGMCC 1.1785) seed for accessing fermentation system
Liquid.Prepare 2 times of concentration fermentation mediums：MgSO4·7H2O 0.25, NaNO33.0, KH2PO40.25, yeast extract 1.0.To
2 times of concentration fermentation mediums of 25mL are added in 250mL aerobic fermentations blake bottle (with baffle plate), 25mL manioc wastes enzymolysis is added
Liquid, and with 2mol/L NaOH solution regulation systems pH to 6.0, zymotic fluid is obtained.The bottleneck of aerobic fermentation blake bottle is sealed
Film sterilizes after sealing.Then to the pseudomonas aeruginosa seed suspension that 1% (v/v) is accessed in aseptic zymotic fluid.By aerobic hair
Ferment blake bottle is placed in 37 DEG C, reacts 168h in the constant-temperature table of 180rpm.Then the rhamnolipid in zymotic fluid is extracted,
The rhamnolipid crude yield of acquisition is 1.30g/L.
Discuss：Manioc waste accesses pseudomonas aeruginosa after enzymolysis and is fermented, and can cause the Portugal in initial reaction stage solution
Grape sugar excessive concentration, suppresses the growth of pseudomonas aeruginosa.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of production method of rhamnolipid, it is characterised in that be to adding cellulase and P. aeruginosa in manioc waste
Bacterium simultaneous saccharification and fermentation produces rhamnolipid.
2. production method according to claim 1, it is characterised in that the manioc waste is industrial manioc waste, and water content is 8
~15%, the granularity of manioc waste is by 40 mesh sieves.
3. production method according to claim 1, it is characterised in that the pseudomonas aeruginosa first passes around Liquid Culture
Base culture and fermentation medium culture, are subsequently adding manioc waste, and the ratio that manioc waste accounts for fermentation medium is 2~6w/v%.
4. production method according to claim 3, it is characterised in that the composition that the fluid nutrient medium contains is：Albumen
Peptone 10.0g/L, beef extract 3.0g/L, NaCl 5.0g/L；
The composition that fermentation medium contains is：MgSO4·7H2O 0.125g/L, NaNO31.5g/L, KH2PO40.125g/L, ferment
Female extract 0.5g/L.
5. production method according to claim 1, it is characterised in that the addition cellulase, cellobiase and copper
Green pseudomonad seed suspension.The addition of reaction system cellulase is 3.75FPU/g- manioc wastes, cellobiase
Addition is 5.65CBU/g- manioc wastes.
6. the production method according to any one of Claims 1 to 5, it is characterised in that the pseudomonas aeruginosa seed hangs
The access amount that liquid accesses fermentation medium is 1~1.5% (v/v), the condition of culture is 35~38 DEG C of temperature, the time 144~
168h。
7. the production method according to any one of Claims 1 to 5, it is characterised in that after fermentation ends, by zymotic fluid solid-liquid
Separate, collect clear liquid, regulation clear liquid pH is standing after 1.6~2.2, treats that flocculent deposit occurs, and is extracted with isometric ethyl acetate
Clear liquid, collects the upper solution of extraction.
8. production method according to claim 7, it is characterised in that zymotic fluid is centrifuged under 12000 × g centrifugal conditions
Supernatant is collected after 15min, it is 1.6~2.2 to adjust supernatant pH with HCl solution, is stood overnight at 4 DEG C, uses isometric second
Acetoacetic ester is extracted, and collects the upper solution of extraction, uses rotary evaporator vacuum distillation.
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Cited By (4)
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| CN109183452A (en) * | 2018-09-04 | 2019-01-11 | 黄敏 | The preparation of pseudomonas aeruginosa extractive from fermentative and the application in dyeing and printing adhesive agent |
| CN109680526A (en) * | 2018-09-03 | 2019-04-26 | 黄敏 | The preparation of pseudomonas aeruginosa extractive from fermentative and the application in printing and dyeing color fixing agent |
| CN111440742A (en) * | 2020-04-08 | 2020-07-24 | 天津科技大学 | Pseudomonas aeruginosa mutant strain for petroleum hydrocarbon degradation |
| CN117126904A (en) * | 2023-10-26 | 2023-11-28 | 万华化学集团股份有限公司 | Method for circularly and continuously fermenting rhamnolipid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680526A (en) * | 2018-09-03 | 2019-04-26 | 黄敏 | The preparation of pseudomonas aeruginosa extractive from fermentative and the application in printing and dyeing color fixing agent |
| CN109183452A (en) * | 2018-09-04 | 2019-01-11 | 黄敏 | The preparation of pseudomonas aeruginosa extractive from fermentative and the application in dyeing and printing adhesive agent |
| CN111440742A (en) * | 2020-04-08 | 2020-07-24 | 天津科技大学 | Pseudomonas aeruginosa mutant strain for petroleum hydrocarbon degradation |
| CN117126904A (en) * | 2023-10-26 | 2023-11-28 | 万华化学集团股份有限公司 | Method for circularly and continuously fermenting rhamnolipid |
| CN117126904B (en) * | 2023-10-26 | 2024-02-02 | 万华化学集团股份有限公司 | Method for circularly and continuously fermenting rhamnolipid |
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| CN106801075B (en) | 2020-09-15 |
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