CN105771791A - Efficient extraction method of environment-friendly biosurfactant sophorolipid - Google Patents
Efficient extraction method of environment-friendly biosurfactant sophorolipid Download PDFInfo
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Abstract
The invention discloses an efficient extraction method of environment-friendly biosurfactant sophorolipid. The efficient extraction method comprises the following steps of (1) adjusting the pH value of fermented sophorolipid fermentation broth to be 6.0-6.5; (2) performing microfiltration on the pH value-adjusted sophorolipid fermentation broth through a ceramic membrane so as to remove thallus and a part of macromolecules, and filtering to obtain bacteria-removed fermentation broth; (3) removing impure proteins in the bacteria-removed fermentation broth obtained in step (2) with ethanol solution, and filtering to remove protein precipitation, thus obtaining impure protein-removed sophorolipid solution; (4) concentrating the impure protein-removed sophorolipid solution obtained in step (3) by compression to a set concentration to obtain a liquid sophorolipid product; (5) drying the sophorolipid liquid product obtained in step (4) to obtain a solid sophorolipid product. The efficient extraction method of the environment-friendly biosurfactant sophorolipid is simple and safe in production process, easy to operate and control, and very suitable for industrialized mass production.
Description
Technical field
The invention belongs to the preparing technical field of biosurfactant, be specifically related to the industrial production technology of a kind of high efficiency extraction environment protection biological surfactant sophorolipid.
Background technology
Surfactant (Surfactants) is the compound that a class just can significantly reduce surface tension of liquid when very low concentrations, and its molecule is generally made up of nonpolar lipophilic group (being mainly hydrocarbon chain or its substituent) and the hydrophilic group of polarity.Its special architectural characteristic makes it have the multiple actions such as moistening, dispersion, emulsifying, foaming, washing, thus is widely used in almost each field of modern industry.Most surfactants of application all adopt with oil for raw material and are chemically synthesized at present, and this not only to consume non-renewable petroleum resources in a large number, and inevitably bring serious pollution and destruction to environment for human survival.Sophorolipid is the environmentally-friendly surfactant being utilized reproducible saccharide and plant wet goods resource to produce by microorganism as a kind of biosurfactant.Sophorolipid, except the total characteristic with surfactant, also has the characteristics such as good emulsifying, dispersion, solubilising, has hypotoxicity, and being compatible with the environment property is good, it is easy to the advantages such as biodegradation.The good characteristic of sophorolipid makes it have broad application prospects in food, daily use chemicals, oil exploitation, environmental conservation, medicine and other fields.
Along with the fast development of human economic society, petroleum resources are day by day exhausted, and environmental protection pressure increases the weight of increasingly, and the market demand of the reproducible biosurfactant sophorolipid of environmental protection will expand rapidly.
Sophorolipid is a kind of to have hydrophilic and oleophylic double properties surfactant, and its product separation, extraction and purification difficulty are relatively big, are also the key factors causing sophorolipid production cost higher.Mostly concentrating on about the research of production sophorolipid both at home and abroad at present adopts cheap raw materials for production to improve the aspects such as sophorolipid fermentation production rate, and it is less about the relevant report developing the extraction purification technology being suitable for industrialized production, its report is also only, after being partial to employing organic solvent diluting fermentation liquid extraction sophorolipid, obtain product by concentrating under reduced pressure.This technology needs to consume substantial amounts of organic solvent (ethyl acetate or high concentration ethanol), and technical process degree of danger is higher, no matter ethyl acetate or high concentration ethanol belong to inflammable explosive article, and highly volatile is comparatively unfavorable for the safety in production of extensive continuous print.
Therefore the safe and efficient purification technology being suitable for sophorolipid industrialized production is developed significant for realizing heavy industrialization sophorolipid.
Summary of the invention
The shortcoming that, danger coefficient height high for existing sophorolipid extraction purification technical costs is unfavorable for large-scale continuous production, the present invention has researched and solved an above-mentioned difficult problem by a series of, it is provided that a kind of sophorolipid Industrialized processing technique with low-concentration ethanol degeneration removing impurity by means of precipitation albumen degerming with microfiltration of ceramic membrane.
The technical solution used in the present invention is as follows:
A kind of method of high efficiency extraction sophorolipid, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes
Regulate the pH value of the sophorolipid fermentation liquid fermented to 6.0～6.5；
(2) microfiltration of ceramic membrane is degerming and macromole extracellular polysaccharide and protein
Adopt ceramic membrane to carry out microfiltration the sophorolipid fermentation liquid after regulating pH value, remove the thalline in fermentation liquid and macromole extracellular polysaccharide and protein, after filtration, obtain degerming fermentation liquid；
(3) ethanol precipitation removing protein and inorganic salt
Adopt the little molecule foreign protein in fermentation liquid degerming in low-concentration ethanol solution removal step (2), be filtered to remove little molecule foreign protein precipitation and part inorganic salt, the sophorolipid solution of the foreign protein that is removed；
(4) concentrating under reduced pressure
The sophorolipid solution decompression removing foreign protein in step (3) is concentrated to setting concentration, namely obtains sophorolipid fluid product；
(5) dry
Sophorolipid fluid product in step (4) is dried and obtains sophorolipid solid product.
In step (1), the initial pH of the sophorolipid fermentation liquid that described fermentation completes is 3.0～3.5, because sophorolipid molecule dissolubility when this pH is relatively low, namely stand has a large amount of sophorolipid to precipitate, the layering of fermentation liquid is unfavorable for that follow-up microfiltration of ceramic membrane is degerming, therefore, first sophorolipid fermentation liquid is neutralized adjustment pH by the present invention makes sophorolipid can be completely dissolved before microfiltration, and precipitate with ethanol degerming for ceramic membrane filter provides advantage.
Preferably, the NaOH solution adopting 2～10N adjusts sophorolipid fermentation liquid pH.
Preferably, in the sophorolipid fermentation liquid that described fermentation completes, the content of sophorolipid is 120～350g/L.In described sophorolipid fermentation liquid, the content assaying method of sophorolipid adopts the method for total sugar-reducing sugar to measure, and wherein total sugar adopts anthrone method to measure, and reducing sugar adopts SBA-40D type bio-sensing automatic analyzer to measure.
In step (2), before carrying out microfiltration of ceramic membrane, analyze and research through great many of experiments, so that the microfiltration effect of ceramic membrane is more excellent, it is preferred that the sophorolipid fermentation liquid after regulating pH value is suitably diluted, the multiple of dilution is 1～3 times, or need not dilute.
The character feature of the present invention popular feature according to sophorolipid fermentation liquid and sophorolipid self: sophorolipid fermenting and producing bacterium is candida mycoderma, thalline is relatively large sized, it is dissolved in fermentation liquid after fermentation fluid viscosity at sophorolipid relatively low simultaneously, these character features determine and the filtering technique that high-efficiency and continuous is prone to extensive employing can be adopted to remove thalline and macromolecular polysaccharide and albumen, therefore the present invention selects microfiltration of ceramic membrane technology, although prior art there is also the technology of other multiple filtration sterilizations or other isolation technics, sheet frame is such as adopted to add kieselguhr filtration sterilization, bactofugation etc., but through lot of experiment validation and analysis, the industrialization sophorolipid of the present invention extracts and adopts microfiltration of ceramic membrane degerming, the productivity of product and purity and its stability can be significantly improved, it is suitable for industrial promoting the use of.
Preferably, the aperture of described ceramic membrane is 0.05～0.5 μm.The sophorolipid fermentation liquid prioritizing selection ceramic membrane filter technology of the present invention, the character feature according to sophorolipid fermentation liquid popular feature and sophorolipid, the aperture of ceramic membrane is arranged on 0.05～0.5 μm.Through substantial amounts of experimentation, during by aperture less than 0.05 μm, the filter effect of thalline is better, but the extraction ratio of sophorolipid is relatively low；When aperture is more than 0.5 μm, although sophorolipid retained less to make extraction rate reached arrive maximum, but thalline removal effect significantly reduces, and affects the purity of product, does not reach desirable filter effect.According to a large amount of practical study results, its aperture is set to 0.05～0.5 μm.
Preferably, the use condition of described ceramic membrane is: pressure 0.2～0.3MPa, temperature 25～50 DEG C.
In step (3), after fermentation liquor filtration sterilization, its impurity is mainly solubility small protein and a small amount of inorganic salt.The technology removing water solublity foreign protein at present mainly has salt precipitation, isoelectric point precipitation, organic solvent precipitation method etc. multiple available.The present invention is compared by substantial amounts of research according to the characteristic of filtrate after sophorolipid fermentation liquid ceramic membrane filter, and the finally preferred alcoholic solution sedimentation method are as the method precipitating foreign protein in sophorolipid extraction purification process.Its prominent advantage is in that and saltouts or isoelectric point precipitation ratio, does not introduce other inorganic salts thus reducing follow-up desalting steps to save cost raising extraction efficiency in operation；Comparing with other organic solvents (trichloroacetic acid, methanol, acetonitrile etc.), ethanol safety in process of production is higher and the impact of sophorolipid product quality is minimum.Therefore being be suitable in sophorolipid industrial processes the optimization technique of remove impurity albumen by the bright employing alcoholic solution sedimentation method of big quantity research comparison sheet, its extraction efficiency height, good product quality, relative cost are low.
The present invention adopts the alcoholic solution of low concentration 50～75% (v/v) that foreign protein carries out degeneration precipitation and removes.In the filtrate after ceramic membrane filter, add alcoholic solution so that it is volume fraction reaches 30～60%, stand flocculation sediment 50～70min (preferred 60min), plate-and-frame filtration or be centrifuged off protein precipitation, sophorolipid solution must be clarified.The pH value of concentration of alcohol and solution is very big to precipitate with ethanol influential effect, through lot of experiment validation and analysis, after adding alcoholic solution in fermentation liquid, when the volume fraction of alcoholic solution is 30～60%, it is possible to remove the protein up to more than 90%.When volume fraction is less than 30%, the clearance of protein is relatively low, and when more than 60%, the basic effect with its volume fraction 30～60% of the removal effect of protein is consistent, and when ethanol content is higher, relatively costly.It addition, through experimental verification, adopt precipitate with ethanol protein can't affect the extraction of sophorolipid.
Preferably, the assay method of described protein impurities removal effect is Kjeldahl's method (with reference to GB/T5009.5-2010).
In step (4), the condition of concentrating under reduced pressure is: temperature controls at 40～90 DEG C, and vacuum degree control is at-0.1～-0.08MPa.
Preferably, concentrating under reduced pressure stops concentration operation to when the mass fraction of sophorolipid solution is 40～60%.
The invention has the beneficial effects as follows:
(1) operating procedure that the present invention neutralizes initially with sophorolipid fermentation liquid, makes sophorolipid can be completely dissolved before microfiltration, forms homogeneous sophorolipid solution, and this operation improves effective extraction ratio of sophorolipid.Then the feature according to sophorolipid fermentation liquid and sophorolipid physicochemical property, adopts that ceramic membrane filter is degerming, macromole and ethanol degeneration removing impurity by means of precipitation albumen and inorganic salt.
(2) different biosurfactants has different separation, extraction and method for concentration.In fermentation liquid, sophorolipid Bio-surface active often concentration is relatively low and have hydrophilic, oleophilic properties, therefore, the separation of sophorolipid, extraction and concentration difficulty are relatively big, but instant invention overcomes the shortcoming that in prior art, sophorolipid extraction purification technical costs height, danger coefficient height are unfavorable for large-scale continuous production.
(3) production technology of the present invention simply, safely, be easy to operate and control, be especially suitable for industrialization large-scale production, improve its economic benefit.
(4) present invention obtains the solid product of rufous, makes the extraction ratio height of this product, purity higher by the extracting method of the present invention.
Accompanying drawing explanation
Fig. 1 is product photo in embodiment 1.
Detailed description of the invention
Below the technical scheme in the embodiment of the present invention is described with carrying out complete fine rule, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1
A kind of method of high efficiency extraction sophorolipid, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes
In sophorolipid fermentation liquid, the content of sophorolipid is at about 120g/L, adopts the NaOH solution of 2N to adjust sophorolipid fermentation liquid pH to 6.0.
(2) microfiltration of ceramic membrane is degerming
By the sophorolipid fermentation liquid after neutralization either directly through the thalline in the ceramic micro filter film removing fermentation liquid of certain pore size, filter after fermentation liquid and become clarification.The aperture of described ceramic membrane is at 0.5 μm；The use condition of described ceramic membrane is: pressure 0.25MPa, temperature 25 DEG C.
(3) ethanol precipitation removing protein
Gained filtrate adds in step (2) ethanol (70%v/v) so that it is volume fraction reaches 50%, stands flocculation sediment 60min, and plate-and-frame filtration removes protein precipitation, and protein removal rate reaches 85%, obtains clear filtrate.
(4) concentrating under reduced pressure
Gained sophorolipid solution decompression concentration 50% (w/w) in step (3) is namely obtained the sophorolipid fluid product that content is 50%, and wherein, the condition of described concentrating under reduced pressure is: temperature controls at 70 DEG C, and vacuum degree control is at-0.09MPa.Its outward appearance is rufous clarification thick liquid.See accompanying drawing 1.
(5) dry
After being dried by gained thick liquid in step (4), obtain sophorolipid solid product.Outward appearance is red brown solid.
By the excellent of the solid product that the method obtains, extraction ratio is 98%, and purity is 90%.
Embodiment 2
A kind of method of high efficiency extraction sophorolipid, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes
In sophorolipid fermentation liquid, the content of sophorolipid is at about 180g/L, adopts the NaOH solution of 5N to adjust sophorolipid fermentation liquid pH to 6.3.
(2) microfiltration of ceramic membrane is degerming
Remove thalline by the ceramic micro filter film of certain pore size after sophorolipid fermentation liquid after neutralization is diluted twice with deionized water, filter after fermentation liquid and become clarifying.The aperture of described ceramic membrane is at 0.2 μm；The use condition of described ceramic membrane is: pressure 0.28MPa, temperature 30 DEG C.
(3) ethanol precipitation removing protein
Gained filtrate adds in step (2) ethanol (65%v/v) so that it is volume fraction reaches 55%, stands flocculation sediment 60min, and plate-and-frame filtration removes protein precipitation, and protein removal rate reaches 90%, obtains clear filtrate.
(4) concentrating under reduced pressure
Gained sophorolipid solution decompression concentration 50% (w/w) in step (3) is obtained rufous clarification thick liquid, and wherein, the condition of described concentrating under reduced pressure is: temperature controls at 80 DEG C, and vacuum degree control is at-0.09MPa.
(5) dry
After being dried by gained thick liquid in step (4), obtain sophorolipid solid product.Outward appearance is red brown solid.
By the excellent of the solid product that the method obtains, extraction ratio is 95%, and purity is 95%.
Embodiment 3
A kind of method of high efficiency extraction sophorolipid, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes
In sophorolipid fermentation liquid, the content of sophorolipid is at about 240g/L, adopts the NaOH solution of 10N to adjust sophorolipid fermentation liquid pH to 6.5.
(2) microfiltration of ceramic membrane is degerming
Remove thalline by the ceramic micro filter film of certain pore size after sophorolipid fermentation liquid after neutralization is diluted three times with deionized water, filter after fermentation liquid and become clarifying.The aperture of described ceramic membrane is at 0.1 μm；The use condition of described ceramic membrane is: pressure 0.25MPa, temperature 33 DEG C.
(3) ethanol precipitation removing protein
Gained filtrate adds in step (2) ethanol (68%v/v) so that it is volume fraction reaches 55%, stands flocculation sediment 60min, and plate-and-frame filtration removes protein precipitation, and protein removal rate reaches 80%, obtains clear filtrate.
(4) concentrating under reduced pressure
Gained sophorolipid solution decompression concentration 50% (w/w) in step (3) is obtained rufous clarification thick liquid, and wherein, the condition of described concentrating under reduced pressure is: temperature controls at 85 DEG C, and vacuum degree control is at-0.08MPa.
By the excellent of the solid product that the method obtains, extraction ratio is 90%, and purity is 97%.
(5) dry
After being dried by gained thick liquid in step (4), obtain sophorolipid solid product.Outward appearance is red brown solid.
Embodiment 4
A kind of method of high efficiency extraction sophorolipid, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes
In sophorolipid fermentation liquid, the content of sophorolipid is at about 120g/L, adopts the NaOH solution of 5N to adjust sophorolipid fermentation liquid pH to 6.0.
(2) microfiltration of ceramic membrane is degerming
Remove thalline by the ceramic micro filter film of certain pore size after sophorolipid fermentation liquid after neutralization is diluted twice with deionized water, filter after fermentation liquid and become clarifying.The aperture of described ceramic membrane is at 0.05 μm；The use condition of described ceramic membrane is: pressure 0.28MPa, temperature 35 DEG C.
(3) ethanol precipitation removing protein
Gained filtrate adds in step (2) ethanol (70%v/v) so that it is volume fraction reaches 50%, stands flocculation sediment 60min, and plate-and-frame filtration removes protein precipitation, and protein removal rate reaches 80%, obtains clear filtrate.
(4) concentrating under reduced pressure
Gained sophorolipid solution decompression concentration 30% (w/w) in step (3) is namely obtained the sophorolipid fluid product that content is 30%, and wherein, the condition of described concentrating under reduced pressure is: temperature controls at 80 DEG C, and vacuum degree control is at-0.08MPa.Its outward appearance is rufous clarification thick liquid.
(5) dry
After being dried by gained thick liquid in step (4), obtain sophorolipid solid product.Outward appearance is red brown solid.
By the excellent of the solid product that the method obtains, extraction ratio is 87%, and purity is 98%.
The performance of the sophorolipid product of the present invention is in Table 1.
Table 1 sophorolipid properties of product table of the present invention
Claims (10)
1. a method for high efficiency extraction sophorolipid, is characterized in that, comprises the following steps:
(1) sophorolipid fermentation liquid neutralizes: regulate the pH value of the sophorolipid fermentation liquid fermented to 6.0～6.5；
(2) microfiltration of ceramic membrane is degerming and macromole extracellular polysaccharide and protein: adopts ceramic membrane to carry out microfiltration the sophorolipid fermentation liquid after regulating pH value, removes the thalline in fermentation liquid and macromole extracellular polysaccharide and protein, obtain degerming fermentation liquid after filtration；
(3) ethanol precipitation removing protein and inorganic salt: adopt the little molecule foreign protein in fermentation liquid degerming in alcoholic solution removal step (2), is filtered to remove little molecule foreign protein precipitation and part inorganic salt, the sophorolipid solution of the foreign protein that is removed；
(4) concentrating under reduced pressure: the sophorolipid solution decompression removing foreign protein in step (3) is concentrated to setting concentration, namely obtains sophorolipid fluid product；
(5) dry: the sophorolipid fluid product in step (4) to be dried and obtains sophorolipid solid product.
2. the method for claim 1, is characterized in that: in step (1), and the initial pH of the sophorolipid fermentation liquid that described fermentation completes is 3.0～3.5.
3. the method for claim 1, is characterized in that: in step (1), adopts the NaOH solution of 2～10N to adjust sophorolipid fermentation liquid pH.
4. the method for claim 1, is characterized in that: in step (1), and in the sophorolipid fermentation liquid that described fermentation completes, the content of sophorolipid is 120～350g/L.
5. the method for claim 1, is characterized in that: in step (2), before carrying out microfiltration of ceramic membrane, is diluted by the sophorolipid fermentation liquid after regulating pH value, and the multiple of dilution is 1～3 times.
6. the method for claim 1, is characterized in that: in step (2), and the aperture of described ceramic membrane is 0.05～0.5 μm；Preferably, the use condition of described ceramic membrane is: pressure 0.2～0.3MPa, temperature 25～50 DEG C.
7. the method for claim 1, is characterized in that: in step (3), and the volume fraction of described alcoholic solution is 50～75%.
8. the method for claim 1, is characterized in that: in step (3), adds in the mixed solution after alcoholic solution in fermentation liquid, and the volume fraction of alcoholic solution is 30～60%.
9. the method for claim 1, is characterized in that: in step (3), after adding alcoholic solution, stands flocculation sediment 50～70min.
10. the method for claim 1, is characterized in that: in step (4), concentrating under reduced pressure stops concentration operation to when the mass fraction of sophorolipid solution is 40～60%.
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Cited By (4)
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| CN114736368A (en) * | 2021-01-08 | 2022-07-12 | 尚科生物医药（上海）有限公司 | Method for extracting and purifying gamma-polyglutamic acid from fermentation liquor |
| CN115141236A (en) * | 2022-07-15 | 2022-10-04 | 南京理工大学 | Separation method of sophorolipid |
| CN115260254A (en) * | 2022-08-30 | 2022-11-01 | 陕西德冠生物科技有限公司 | Industrialized purification method of sophorolipid |
| CN115992184A (en) * | 2023-03-23 | 2023-04-21 | 科乐美(广州)生物科技有限公司 | Biological fermentation surfactant, cosmetic and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736368A (en) * | 2021-01-08 | 2022-07-12 | 尚科生物医药（上海）有限公司 | Method for extracting and purifying gamma-polyglutamic acid from fermentation liquor |
| CN115141236A (en) * | 2022-07-15 | 2022-10-04 | 南京理工大学 | Separation method of sophorolipid |
| CN115260254A (en) * | 2022-08-30 | 2022-11-01 | 陕西德冠生物科技有限公司 | Industrialized purification method of sophorolipid |
| CN115992184A (en) * | 2023-03-23 | 2023-04-21 | 科乐美(广州)生物科技有限公司 | Biological fermentation surfactant, cosmetic and preparation method thereof |
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