CN102277230B - Method of optimum rapid solvent extracting treatment for raising extraction amount of microalgae grease - Google Patents
Method of optimum rapid solvent extracting treatment for raising extraction amount of microalgae grease Download PDFInfo
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Abstract
The invention relates to a method of an optimum rapid solvent extracting treatment for raising the extraction amount of microalgae grease. According to a design scheme of an orthogonal experiment, an optimum extraction solvent, the extraction temperature, the extraction time and the extraction cycle frequency of microalgae extraction by an accelerated solvent can be determined, so that the effect of microalgae grease extraction with high efficiency can be achieved. The concrete operation processes comprise: cultivating microalgae cells, centrifugally collecting the concentrated microalgae cells, freezing and drying to obtain the dry product of microalgae cells. The dry product of microalgae cells is extracted for microalgae grease by using a ASE 100 accelerated solvent extraction, a design scheme of L25(56) orthogonal experiment is used for acquiring the optimum condition for extracting the microalgae grease, under the condition of the optimum extracting treatment, the extraction yield of the obtained microalgae grease is higher than that of the routine soxhlet extraction method by 39.08%-47.09%. The method of the invention has the advantages of simple equipment, simple operation, fast grease extraction speed, high extraction yield and the like.
Description
Technical field
The present invention relates to a kind of fast method from the biological material specimens oil and grease extracting, specifically is to belong to the optimizing accelerated solvent extraction to handle the technical field that improves the microalgae grease percentage extraction.
Background technology
Little algae is that volume is small; simple in structure; the unicellular algae that growth and breeding is fast; known little algae kind reaches more than 10 ten thousand kinds at present; various dissimilar little algaes have specific biology and ecological characteristics; they can be by various biochemical reactions and the pathways metabolism that takes place in the cell; synthetic many high value cytoactive things with unique texture and exceptional function, peptidic substrate for example, protein; lipid acid; polysaccharide, natural pigment, VITAMIN etc.; therefore; little algae is applied in food by extensive exploitation; medicine; makeup, genetically engineered; biomass fuel, aquaculture; industry fields such as environment protection have the excellent development application prospect.Separation and Extraction, purifying natural actives from little algae are the important channels of realizing the algae higher value application.
After 20th century, broke out oil crisis the seventies, the mankind began to seek renewable energy source.Exhausted day by day at fossil oil, that environmental pollution threatens human survival gradually today, the exploitation of the renewable energy source of cleaning has become strategic direction and the emphasis of various countries' research and development
[5]Little algae has that biomass is big, growth cycle is short, easily cultivate, the fat content advantages of higher.By the regulation and control little algae of amplification cultivation and the biomass of gathering, extract microalgae grease, further by transesterification, can prepare the bio-fuel-oil with good combustion and environmental protection characteristic again, therefore, little algae is the good biological material that produces the green clean energy of recyclability.
On the other hand, contain various polyunsaturated fatty acids (the polyunsaturated fatty acid useful to HUMAN HEALTH in the microalgae grease, PUFA), linolenic acid for example, docosahexenoic acid (docosahexaenoic acid, DHA) and timnodonic acid (eicosapentaenoic acid, EPA).Polyunsaturated fatty acid is in the vital role of nutrition and medical field, cause people's extensive concern day by day, particularly docosahexenoic acid and timnodonic acid, studies show that they have many important physical functions, as improve the brain memory ability, keep twenty-twenty vision, control heart disease, arteriosclerosis, rheumatic arthritis, asthma and diabetes, therefore effects such as reducing blood-fat, anti-blood aggegation, anti-inflammatory, anticancer and immunomodulatory have become the focus of research and development.
Fish oil is the main source of polyunsaturated fatty acids such as EPA and DHA traditionally, but there is stench flavor in the polyunsaturated fatty acid that extracts from fish oil, and steps such as last handling process will be through oily water separation, concentrate, distillation, refining, deodorization, complex process, valuable product
[36]Little algae has the ability of unsaturated fatty acidss such as synthetic EPA and DHA, the relative content of EPA and DHA is higher than the content in the fish oil far away in the frond, and the not stench flavor of the PUFA product that from frustule, extracts, do not contain the cholesterol composition, take in the shortcoming of a large amount of cholesterol when having avoided taking fish-oil capsule.The EPA and the DHA that contain high level in some algae have respectively reduced EPA and the mutually mutual interference of DHA in single leaching process.In addition, some algae can directly eat, and has avoided the oxygenolysis in the purification process.Therefore, utilize little algae production EPA and DHA to have good prospect.
Utilize little algae to prepare bio-fuel-oil or extract the polyunsaturated fatty acid that purifying contains no matter be, at first should extract the lubricant component of little algae, further adopt relevant program then, prepare target product, therefore, seeking to operate method fast and convenient, that extraction is complete, efficient is high, is the key of development and use microalgae grease.The microalgae cell individuality is little, and cell walls is more abundant usually, extracts difficulty of lubricant component, needs to adopt special effective extracting process just can reach the high effect of extraction.
The oil component of separation and Extraction biological sample, the technology of Cai Yonging and method have soxhlet extraction, organic solvent method, acid heat method etc. traditionally.Though these methods are more simple and easy to do, the common process length consuming time of separation and Extraction, for example soxhlet extraction often needs a few hours, and for little algae sample of oil extraction difficulty, need operation just can finish extraction process in tens hours, and percentage extraction is low.And key problem of these traditional grease extracting method existence, namely to consume the organic solvent of a large amount of toxic effects in the extraction process, this not only causes the analysis cost height, and organic solvent damages HUMAN HEALTH easily and produces environmental pollution.In addition, Microwave Digestion, ultrasonic extraction method and supercritical extraction etc., the present separation and Extraction that also is applied to the oil component of biological sample.Though these technological methods have shortened the time of separation and Extraction to a certain extent and can improve to a certain degree extraction efficiency, but use and still have the problem that needs to consume the more amount organic solvent in the process of these technological method separation and Extraction biological sample greases, and produce the other problem simultaneously, for example certain slightly the algae kind adopt the efficient of ultrasonic extraction method oil and grease extracting very low, even can't extract the grease of cell.Supercritical fluid extraction (SFE) usually needs a kind of cosolvent, and its range of application is narrower, and operator need have the higher super technical ability of doing just can obtain reliable result.
Therefore, for little algae of special biological sample, need seek and set up a kind of easy, oil extraction technology and method fast and effectively.
ASE (Accelerated Solvent Extraction) solvent extration fast is a kind of advanced person's solvent extraction technology, its principle is to utilize raising temperature and pressure boost to accelerate the efficient of traditional extraction treatment, raising oil extraction, use conventional solvent that solid or semi-solid sample are extracted, by setting up optimized accelerated solvent extraction treatment condition, can reach the time of abundant shortening extraction, and reach the usage quantity of obvious reduction extraction solvent and the effect that improves the efficient of oil extraction, be specially adapted to the efficient extraction of microalgae grease.
Summary of the invention
Technical problem to be solved by this invention is exactly at background technology recited above, by the orthogonal experiment design, determine best extraction solvent, extraction temperature, extraction time and the extraction cycle number of times of accelerated solvent extraction microalgae grease, mouse out the method for effective extraction microalgae grease.Up to now, still do not have the optimizing of employing accelerated solvent extraction and handle, improve the relevant report of microalgae grease percentage extraction.
Research material and method that the present invention adopts:
Microalgae cell is amplification cultivation under the suitable culture condition, the frustule of gathering, and algae liquid is centrifugal to be concentrated, and handles through lyophilize again, obtains can be used for the microalgae cell dry product of microalgae grease content analysis.Get the microalgae cell dry product of a certain amount of dry weight, be under the 1500psi in extracting pressure, carrying out accelerated solvent extraction (ASE) handles, resulting extraction liquid is through Rotary Evaporators volatilization desolventizing, obtain the weight of microalgae cell grease, microalgae cell fat content (%)=microalgae cell grease weight/microalgae cell dry product weight.Investigate different extraction solvents, extraction temperature, extraction time and extraction cycle time said conditions to the influence of microalgae cell oil extraction, determine best microalgae cell oil extraction condition.
The present invention solves the problems of the technologies described above the technical scheme that adopts:
Adopted orthogonal experiment design (the orthogonal table L of five levels, four factors
25(5
6)), use variance analysis method simultaneously, investigate the main effect factor (extraction solvent, extraction temperature, extraction time and extraction cycle number of times) to the influence of microalgae grease extraction, analyzing these main effect factor pair microalgae grease extractions influences size sequence and determines optimized extraction conditions.Below be experimental design, experimental result and analysis.
Table one, accelerated solvent extraction (ASE) are handled the empirical factor water-glass
Table two, accelerated solvent extraction (ASE) are handled interpretation reckoner (orthogonal table L
25(5
6))
Table three, accelerated solvent extraction (ASE) are handled the experimental variance analytical table
Conclusion: adopt different extraction solvents and under the extraction conditions of various various combinations, microalgae grease content (%) numerical value has tangible difference, variation range shows that different extraction conditionss exists very big difference to the effect of extracting of microalgae grease between 2.98-21.36%.In 5 kinds of extraction solvents investigating, chloroform, normal hexane, sherwood oil is poor to the effect of extracting of microalgae grease, and dehydrated alcohol and acetone are good to the effect of extracting of microalgae grease.The size of the extreme difference value R that calculates from table two experiment test value has reflected the size of corresponding factor effect, the factor that the extreme difference value is big, mean that its different levels is bigger to the difference that the index amount of investigating causes, principal element normally, the factor that the extreme difference value is little, meaning that its different levels is less to the difference that the index amount of investigating causes, generally is than secondary cause.This experimental analysis shows, the extreme difference value R that extraction solvent A, extraction temperature B, extraction time C and extraction cycle number of times D cause is respectively 25.91,38.85,16.44 and 16.67, therefore, press extreme difference value size, in accelerated solvent extraction (ASE) treating processes, the primary and secondary order of factor that influences the effect of extracting of microalgae grease can be arranged as successively: extraction temperature B＞extraction solvent A＞extraction cycle number of times D＞extraction time C.
The The results of analysis of variance that accelerated solvent extraction (ASE) is handled experiment further clearlys show, the effect of extracting of the microalgae grease of extraction temperature B has remarkable influence (P value=0.000515), next is that the effect of extracting of the microalgae grease of extraction solvent A also has very remarkable influence (P value=0.003855), and extraction time C influence to the effect of extracting of microalgae grease when significance level is a=0.05 is significant (P value=0.035094).Comparatively speaking, the influence of the effect of extracting of extraction cycle number of times D microalgae grease is smaller, when significance level is a=0.05 not significantly (P value=0.081996).
Extreme difference value R and the The results of analysis of variance of comprehensive orthogonal experiment data consider that from the index parameter of the effect of extracting of the best microalgae grease of favourable generation in accelerated solvent extraction (ASE) treating processes, the collocation of the suitable condition parameter factors numerical value that should adopt should be A
4,5B
5C
5D
3, or A
4,5B
5C
4D
3, and further from extraction solvent toxicity relative size and whether save time and cost consideration, be A with the collocation of adopting conditional parameter factor numerical value then
4B
5C
4D
3Optimum (because comparatively speaking, the toxicity of ethanol is less than acetone, and price is lower, and the extreme difference value R when the extraction time is 16 or 20 minutes is about the same, therefore considers that from saving the extraction time it is suitable that optimum extraction time is decided to be 16 minutes).Namely selecting dehydrated alcohol or acetone for use is extraction solvent, and extraction temperature is at 175 ℃, and the extraction time is 16 minutes, and the extraction cycle number of times is 3 o'clock, and the microalgae grease extraction quantity is maximum, and effect of extracting is best, is conducive to obtain more target product.
Advantage of the present invention is: have that equipment is simple, easy and simple to handle, oil extraction speed is fast, percentage extraction height, advantage such as the extraction solvent toxicity of employing is little.
The objective of the invention is:
In order to seek to be used in the method for efficient extraction microalgae grease, by adopting conditions such as optimum extraction solvent, extraction temperature, extraction time and extraction cycle number of times in accelerated solvent extraction (ASE) treating processes, reach fast, effectively extract the effect of microalgae grease.
Further specific embodiment is demonstrate,proved
By adopting the multilevel efficient orthogonal experimental technique of multiplefactor, conditions such as the optimum extraction solvent that should adopt, extraction temperature, extraction time and extraction cycle number of times have been obtained in accelerated solvent extraction (ASE) treating processes.In order further to verify the validity of the optimum extraction condition of favourable index parameter value (microalgae grease content), repeatedly measured the fat content of little algae under the optimum extraction condition.Below be further specific embodiment card:
A, employing optimizing accelerated solvent extraction facture extraction of the present invention:
1, with the dehydrated alcohol is extraction solvent
Implement illustration 1: take by weighing little algae dry product 5.0578 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with the dehydrated alcohol, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.9805 gram, and the fat content of little algae is 19.39%.
Implement illustration 2: take by weighing little algae dry product 5.0637 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with the dehydrated alcohol, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 1.0356 grams, and the fat content of little algae is 20.45%.
Implement illustration 3: take by weighing little algae dry product 5.0504 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with the dehydrated alcohol, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.9908 gram, and the fat content of little algae is 19.62%.
Implement illustration 4: take by weighing little algae dry product 5.0838 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with the dehydrated alcohol, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.9779 gram, and the fat content of little algae is 19.24%.
Implement illustration 5: take by weighing little algae dry product 5.0735 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with the dehydrated alcohol, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.9739 gram, and the fat content of little algae is 19.20%.
2, with acetone be extraction solvent
Implement illustration 1: take by weighing little algae dry product 5.014 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with acetone, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.981 gram, and the fat content of little algae is 19.57%.
Implement illustration 2: take by weighing little algae dry product 5.0616 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with acetone, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 1.0694 grams, and the fat content of little algae is 21.13%.
Implement illustration 3: take by weighing little algae dry product 5.0521 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with acetone, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 1.0012 grams, and the fat content of little algae is 19.82%.
Implement illustration 4: take by weighing little algae dry product 5.2892 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with acetone, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 1.0763 grams, and the fat content of little algae is 20.35%.
Implement illustration 5: take by weighing little algae dry product 5.0856 grams, put into 30 milliliters sample extraction pond, carrying out accelerated solvent extraction (ASE) handles, be extraction solvent with acetone, extraction temperature is at 175 ℃, and the extraction time is 16 minutes, the extraction cycle number of times is 3, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 1.0658 grams, and the fat content of little algae is 20.96%.
B, the conventional soxhlet extraction extraction of employing:
Comparative examples card 1: take by weighing little algae dry product 5.00 grams, add the dehydrated alcohol extraction solvent, behind the static extracting 18 hours, it is 75 ℃ in temperature, with the soxhlet extraction dynamic extraction of routine 6 hours, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.541 gram, and the fat content of little algae is 10.82%.
Comparative examples card 2: take by weighing little algae dry product 5.00 grams, add the acetone extract solvent, behind the static extracting 18 hours, it is 75 ℃ in temperature, with the soxhlet extraction dynamic extraction of routine 6 hours, resulting extraction liquid is through the Rotary Evaporators solvent evaporated, and the microalgae grease weight that extraction obtains is 0.596 gram, and the fat content of little algae is 11.92%.
Can be found out by above enforcement illustration result repeatedly, adopt accelerated solvent extraction of the present invention (ASE) optimal treatment condition extraction microalgae grease, the oil extraction rate is very stable, not only saves the extracting operation time greatly, and the effect of extracting of microalgae grease is significantly increased.Compare with the soxhlet extraction of routine, when being extraction solvent with the dehydrated alcohol, extraction efficiency can improve 43.63-47.09%, and for the extraction solvent extraction efficiency can improve 39.08-43.58%, therefore, the method for extraction microalgae grease of the present invention is worth adopting with acetone.
Claims (1)
1. the method that improves the microalgae grease extraction quantity is handled in the quick solvent extraction of optimizing, it is characterized in that by adopting the quick solvent extraction of optimizing to handle, with one of dehydrated alcohol or acetone as extraction solvent, extracting pressure 1500psi, 175 ℃ of extraction temperature, 16 minutes extraction time, extraction cycle number of times 3.
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| CN102816636A (en) * | 2012-07-25 | 2012-12-12 | 江苏科技大学 | Extraction method for microalgae grease |
| CN102925283B (en) * | 2012-10-23 | 2014-01-22 | 中国热带农业科学院香料饮料研究所 | Preparation method of pepper oleoresin |
| US8591912B1 (en) | 2013-02-28 | 2013-11-26 | Kiran L. Kadam | Algae extraction process |
| CN103203117B (en) * | 2013-03-26 | 2015-11-18 | 国家海洋局第三海洋研究所 | A kind of method of material in pressurized liquid extraction fermentable culture |
| CN108862285B (en) * | 2018-06-26 | 2020-05-19 | 浙江岩谷科技有限公司 | Method for rapidly preparing aerogel by accelerating solvent extraction under normal pressure |
| CN108751206B (en) * | 2018-06-26 | 2020-04-07 | 浙江岩谷科技有限公司 | Method for rapidly preparing aerogel by variable-pressure accelerated solvent extraction |
| CN108584965B (en) * | 2018-06-26 | 2021-07-16 | 浙江岩谷科技有限公司 | Method for rapidly preparing aerogel by accelerating solvent extraction |
| CN113116804B (en) * | 2019-12-31 | 2023-03-21 | 新生活化妆品科技(上海)有限公司 | Cosmetic material composition, method for producing same, and cosmetic |
| CN113769437A (en) * | 2021-10-22 | 2021-12-10 | 武汉轻工大学 | Method for rapidly extracting high-quality bioactive components from microalgae cells and application thereof |
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| WO2010007328A1 (en) * | 2008-07-18 | 2010-01-21 | Valagro Carbone Renouvelable Poitou-Charentes | Method for obtaining algae extracts and use of these extracts |
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| CN101106913A (en) * | 2005-01-26 | 2008-01-16 | 努特诺瓦营养产品及食品成分有限公司 | Production and use of an antioxidant extract from crypthecodinium sp. |
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