CN102965182B - Method for extracting grease from schizochytrium - Google Patents
Method for extracting grease from schizochytrium Download PDFInfo
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Abstract
The invention discloses a method for extracting grease from schizochytrium. The method comprises the following steps: (1) adjusting the pH (Potential of Hydrogen) of the fermentation broth of the schizochytrium until reaching an acid stage, and then adding an organic solvent to stir, so as to realize the wall breaking of schizochytrium cells and extraction of microalgae oil; (2) adding a flocculant to the fermentation broth obtained in step (1) to flocculate, and extracting by the organic solvent to obtain crude algae oil; and (3) treating the crude algae oil by degumming, discoloring, and deacidifying/deodorizing, so as to obtain the microalgae oil rich in DHA (Docosahexaenoic Acid). According to the method disclosed by the invention, autolysis wall-breaking technology is adopted, so that the technological process in wall breaking and extraction of the schizochytrium can be simplified, the operation time in wall breaking can be shortened, the investment on equipment is decreased, and the cost is lowered; and the biological flocculant flocculation technology is carried out, therefore, the demulsification technological process is simplified, the time for layering is shortened, the layering effect is improved, the load due to wastewater discharge and water treatment caused by the demulsification process can be avoided, and the cost of water treatment can be lowered.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of extracting and refining rich grease-contained microalgae grease from fragmentation kettle algae.
Background technology
Docosahexenoic acid (Docosahexaenoic acid is called for short DHA) is the long-chain highly unsaturated fatty acid that carbochain is the longest, degree of unsaturation is the highest of finding at present.DHA is the biomembranous important structure component of body, at aspects such as infant's neurodevelopment, retina formation, intellectual developments, there is vital role, and be prostaglandin(PG), change the precursor that Prostaglandins and Leukotrienes element etc. has the self-regulation material of strong physiologically active, to preventing cardiovascular disease, anti-lipid, hypotensive, suppress tumour formation etc. and there is remarkable effect.Owing to having above-mentioned multiple important physiological function, DHA has become and has been worth high, market fatty acid product widely.
Schizochytrium limacinum, claims again to split kettle algae, belongs to a class thalassiomycetes of Mycophyta (Eumycota), Oomycete (Oomycetes), Saprolegniales (Saprolegniales), thraustochytriale section (Thraustochytriaceae), unicellular, spherical.The a large amount of active substances useful to human body of fragmentation kettle frustule accumulation, as: grease, pigment, squalene etc., wherein grease accounts for the more than 70% of dry cell weight, in total fat, docosahexenoic acid (DHA) content is very high, reach 35% ~ 40%, the fatty acid content of similar is low, easily separation and purification, and there is no fishy smell, therefore schizochytrium limacinum is a kind of one of microorganism of realizing suitability for industrialized production DHA potentiality that has.
CN 101638361A discloses a kind of method that is rich in docosahexenoic acid of extracting and refine from schizochytrium limacinum, fermented liquid flocculation treatment is carried out solid-liquid separation again, by after alkali broken wall by cell Mechanical Crushing, then broken thalline is adopted to organic solvent extraction, obtain slightly oil of DHA, the thick oil of DHA obtains DHA essential oil through coming unstuck, after alkali refining, decolouring, deodorization.The method has solved fragmentation kettle algae grease and has extracted the technical problem of refining, but this technology technology for broken wall is loaded down with trivial details, and the used time is longer, equipment investment is large, in water, algae and organic solvent hierarchical process, adopt stratification technique, this technique is bad at practical application layered effect, and the time is longer.
CN102199485A discloses a kind of method that kettle algae grease is split in extraction, and the method is that to split kettle algae mud be raw material, through homogeneous, alkali carry, enzymolysis, centrifugal, organic solvent extraction obtain splitting kettle algae oil.Raw material first carries out pyroprocessing after sodium citrate buffer solution dilution average, then carries and adopt successively neutral protease and Sumizyme MP to carry out enzymolysis through alkali, and centrifugal rear separated edible vegetable oil also adopts organic solvent extraction missible oil.The method adopts aqueous enzymatic method to carry oil tech and has solved the technical problem that fragmentation kettle algae algae oil extracts, but this technical matters is complicated, broken wall operation is various, is not suitable for suitability for industrialized production application.
In prior art, the refining of the thick oil of fragmentation kettle algae all adopts chemical refining technology, and the thick oil of DHA obtains DHA essential oil through coming unstuck, after alkali refining, decolouring, deodorization.Inevitably there are some problems in this technique, for example: part neutral oil is inevitably by saponification; Contaminated wastewater environment; While reclaiming lipid acid from byproduct Chinese honey locust, need to be through complicated processing link (hydrolysis, distillation); During especially for the refining of high acid value crude oil, grease refining consumption is large, and economical effectiveness is not good enough.And, in current breakdown of emulsion layering, adopt adds the methods such as ethanol, sodium-chlor to carry out breakdown of emulsion layering more, and this technique exists in practical application that production cost is high, complex operation, separation time is long, layered effect is unstable, ethanol reclaims difficulty and produce a large amount of problems such as effluent brine.
Summary of the invention
The object of the present invention is to provide a kind of method of extracting grease from fragmentation kettle algae, described method broken wall operating time and hierarchical process time are short, layered effect is good, has avoided the discharge of wastewater that causes because of demulsification technology and the burden of water treatment simultaneously, has reduced the cost of water treatment.
In order to achieve the above object, the present invention has adopted following technical scheme:
A method of extracting grease from fragmentation kettle algae, described method comprises the steps:
(1) regulate the pH of fragmentation kettle algae fermented liquid to acid, add organic solvent, stir, realize the broken wall of fragmentation kettle frustule and the extraction of microalgae grease;
(2) in the fermented liquid obtaining to step (1), add after flocculation agent flocculation treatment, adopt organic solvent extraction to obtain algae crude oil;
(3) algae crude oil is through coming unstuck, decolouring, and depickling/deodorization, obtains being rich in the microalgae grease of DHA.
Fragmentation kettle algae of the present invention, also claims schizochytrium limacinum.
The preparation method of the fragmentation kettle algae fermented liquid that the present invention is exemplary is:
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get;
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o 0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L;
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L;
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The present invention regulates after the pH of fermented liquid, under certain temperature and rotating speed, stir, make microalgae cell autolyze broken wall, the grease of cell interior is free out, by organic solvent, extract, when having realized the self-dissolving broken wall of fragmentation kettle frustule, effectively extract the grease in microalgae cell, greatly shortened the extraction time of microalgae grease.Autolysis method is a kind of special enzymic hydrolysis mode, and its required cytase is produced by microorganism itself.In microorganism growth metabolic process, mostly can produce the enzyme of polymer architecture on certain hydrolysis self cell walls, to growth and breeding process is gone on.Control certain condition, can bring out the cytase of microorganisms surplus or excite the vigor of self cytase, to reach the object of aqtocytolysis.In this patent, by regulating the pH value of algae liquid, control self-dissolving temperature and time, realized the self-dissolving broken wall of microalgae cell.
Described method has been simplified the technique that grease extracts greatly, and it is high to put forward oily efficiency, and technique is simple, is conducive to realize suitability for industrialized production.
Preferably, described flocculation agent is biological flocculant, preferably chitosan.
In water, algae, organic solvent hierarchical process, the present invention adopts biological flocculant chitosan to carry out biofloculation technique.Chitosan is the very abundant natural alkaline polysaccharides of a kind of reserves, the product being generated after strong lye deacetylation by chitin.Flocculate with chitosan microalgae cell, thus breakdown of emulsion completed.Chitosan and microalgae cell mainly by neutralizing effect, adsorption and net, catch bridging action and microalgae cell is had an effect, and microalgae cell is flocculated, and reaches breakdown of emulsion layered effect.Adopt biological flocculant chitosan to carry out breakdown of emulsion layering, adding the time that has greatly shortened breakdown of emulsion layering of chitosan improved the effect of breakdown of emulsion layering simultaneously, avoided the discharge of wastewater that causes because of demulsification technology and the burden of water treatment simultaneously.
Preferably, the described depickling of step (3)/deodorization adopts vacuum depickling/deodorizing technology.Vacuum depickling/deodorizing technology is under vacuum condition, to utilize steam to carry to remove FFA and volatile matter etc., it has avoided neutral oil saponification loss, reduced grease loss, and simple to operate, the consumption of steam, water and energy is all less, therefore invest lessly, and pigment and stink substance are also along with steam is removed.
Preferably, step (2) comprises the steps:
(2a) in the fermented liquid obtaining to step (1), add biological flocculant, after fully stirring evenly, stratification, extracts upper strata mixing oil phase;
(2b) to step (2a), extract in the fermented liquid mixing after oil phase and again add organic solvent, at 40 ~ 60 ℃, stir, after stratification, extract upper strata mixing oil phase;
(2c) the solvent evaporation in mixing oil phase step (2a) and step (2b) being obtained, obtains algae crude oil.
The present invention adopts three-phase layering technique to extract mixing oil phase.In the fermented liquid that step (1) is obtained, add biological flocculant, after fully stirring evenly, stratification, orlop is water, and middle level is algae-residue, and upper strata is for mixing oil phase, and described mixing oil phase is the mixture of organic solvent and algae crude oil.The fermented liquid of step (2a) is extracted after upper strata mixing oil phase, again add organic solvent, carry out reextraction.Solvent evaporation in the mixing oil phase that twice extraction obtained, obtains algae crude oil.The algae-residue in middle level can pass through solid-liquid separation, after being dried, obtains, and can be used for animal-feed and uses.
Step (1) regulates pH to 3.5 ~ 4.5, for example, regulate pH to 3.6,3.7,3.8,3.9,4.0,4., and 1,4.2,4.3,4.4, preferably 3.7 ~ 4.2.Regulate pH to 3.5 ~ 4.5 of fermented liquid, can realize the self-dissolving broken wall of fragmentation kettle algae, simplified the technical process of fragmentation kettle algae extraction, significantly reduced the broken wall operating time, reduced equipment investment.
Preferably, step (1) adopts acid for adjusting pH value, described acid to be selected from the mixture of any one or at least two kinds in hydrochloric acid, phosphoric acid, acetic acid or citric acid.Described mixture is the mixture of hydrochloric acid and phosphoric acid for example, the mixture of phosphoric acid and acetic acid, the mixture in acetic acid and citric acid, the mixture in hydrochloric acid, acetic acid and citric acid, the mixture in phosphoric acid, acetic acid and citric acid.
Preferably, in step (1) and step (2b), the volume of organic solvent is 0.8 ~ 2 times of step (1) fermentating liquid volume, for example 0.9 times, 1.1 times, 1.3 times, 1.4 times, 1.6 times, 1.8 times, 1.9 times, and preferably 1 ~ 1.5 times.
Preferably, step (1) stirs at 40 ~ 60 ℃, for example, at 42 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 59 ℃, stir, and preferably at 45 ~ 55 ℃, stirs.
Described organic solvent is selected from the mixture of any one or at least two kinds in No. six solvents, normal hexane, isohexane or iso-pentane.Described mixture is the mixture of No. six solvents and normal hexane for example, the mixture of normal hexane and isohexane, the mixture of isohexane and iso-pentane, the mixture of No. six solvents, normal hexane and isohexanes.Above-mentioned solvent is commercially available obtaining all.
The addition of biological flocculant is the fermented liquid that 100 ~ 500mg/L step (1) obtains, and " in the fermented liquid that every L step (1) obtains, adds the biological flocculant of 100 ~ 500mg ", and preferably addition is the fermented liquid that 300 ~ 500mg/L step (1) obtains.
The stir speed (S.S.) of described stirring is 80 ~ 150rpm, for example 85rpm, 95rpm, 105rpm, 110rpm, 115rpm, 125rpm, 135rpm, preferred 90 ~ 140rpm, further preferred 100 ~ 130rpm.
The described churning time of step (1) is 2 ~ 6 hours, for example 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, and preferably 2.5 ~ 5.5 hours.
The described churning time of step (2b) is 0.5 ~ 1 hour, for example 0.55 hour, 0.6 hour, 0.65 hour, 0.7 hour, 0.75 hour, 0.85 hour, 0.95 hour, and preferably 0.6 hour ~ 0.9 hour.
Described coming unstuck adopts phosphoric acid acidifying degumming technology; Preferably, described phosphoric acid acidifying degumming technology is: the algae crude oil that step (2) or step (2c) are obtained is preheating to 80 ~ 85 ℃, 0.3 ~ 0.5% the phosphoric acid that adds algae crude oil weight that step (2) or step (2c) obtain, after acidifying is come unstuck, the washing of 90 ~ 95 ℃ 2 ~ 5 times, the algae crude oil that comes unstuck after being come unstuck; The weight of described each washing water is 5 ~ 10% of the algae crude oil weight that obtains of step (2) or step (2c).The preheating temperature of described algae crude oil can be 81 ℃, 82 ℃, 83 ℃, 84 ℃.The add-on of described phosphoric acid is 0.3 ~ 0.5% of algae crude oil weight, for example 0.33%, 0.36%, 0.38%, 0.41%, 0.44%, 0.47%, 0.49%.The weight of described hot water is 5 ~ 10% of algae crude oil weight, for example 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 9.5%.
Preferably, described decolouring adopts gac or activated decoloration.
Preferably, described decoloration process is under vacuum condition, and temperature is 100 ~ 105 ℃, to the algae crude oil after coming unstuck add come unstuck after gac or the atlapulgite of algae crude oil weight 3 ~ 5% decolour, vacuum tightness is-below 0.094mPa.Described temperature for example can be 101 ℃, 102 ℃, 103 ℃, 104 ℃.Described vacuum tightness is for example-0.1mPa ,-0.2mPa ,-0.3mPa ,-0.4mPa ,-0.5mPa.Described vacuum tightness is gauge pressure.
Described depickling/deodorization adopts vacuum depickling/deodorizing technology, and described technique is: by the algae crude oil after decolouring, in temperature, be that 120 ~ 280 ℃, vacuum tightness be-below 0.094mPa, decolour 1 ~ 2 hour.Described temperature for example can be 130 ℃, 150 ℃, 180 ℃, 210 ℃, 230 ℃, 250 ℃, 270 ℃.Described bleaching time is for example 1.2 hours, 1.4 hours, 1.6 hours, 1.1 hours, 1.8 hours, 1.9 hours.Described vacuum tightness is for example-0.1mPa ,-0.2mPa ,-0.3mPa ,-0.4mPa ,-0.5mPa.Described vacuum tightness is gauge pressure.
Preferably, a kind of method of extracting grease from fragmentation kettle algae, described method comprises the steps:
(1) regulate pH to 3.5 ~ 4.5 of fragmentation kettle algae fermented liquid, add organic solvent, at 40 ~ 60 ℃, stir, realize the broken wall of fragmentation kettle frustule and the extraction of microalgae grease;
(2a) in the fermented liquid obtaining to step (1), add biological flocculant, after fully stirring evenly, stratification, extracts upper strata mixing oil phase;
(2b) to step (2a), extract in the fermented liquid mixing after oil phase and again add organic solvent, at 40 ~ 60 ℃, stir, after stratification, extract upper strata mixing oil phase;
(2c) the solvent evaporation in mixing oil phase step (2a) and step (2b) being obtained, obtains algae crude oil;
(3) algae crude oil is through coming unstuck, decolouring, and depickling/deodorization, obtains being rich in the microalgae grease of DHA.
Preferably, a kind of method of extracting grease from fragmentation kettle algae, described method comprises the steps:
(1) regulate pH to 3.5 ~ 4.5 of fragmentation kettle algae fermented liquid, add organic solvent, at 40 ~ 60 ℃, stir, realize the broken wall of fragmentation kettle frustule and the extraction of microalgae grease;
(2a) in the fermented liquid obtaining to step (1), add biological flocculant, after fully stirring evenly, stratification, extracts upper strata mixing oil phase;
(2b) to step (2a), extract in the fermented liquid mixing after oil phase and again add organic solvent, at 40 ~ 60 ℃, stir, after stratification, extract upper strata mixing oil phase;
(2c) the solvent evaporation in mixing oil phase step (2a) and step (2b) being obtained, obtains algae crude oil;
(3) algae crude oil step (2) or step (2c) being obtained is preheating to 80 ~ 85 ℃, 0.3 ~ 0.5% the phosphoric acid that adds algae crude oil weight that step (2) or step (2c) obtain, after acidifying is come unstuck, the washing of 90 ~ 95 ℃ 2 ~ 5 times, the algae crude oil that comes unstuck after being come unstuck; The weight of the water of described each washing use is 5 ~ 10% of the algae crude oil weight that obtains of step (2) or step (2c); Under vacuum condition, temperature is 100 ~ 105 ℃, to the algae crude oil after coming unstuck add come unstuck after gac or the atlapulgite of algae crude oil weight 3 ~ 5% decolour, vacuum tightness is-below 0.094mPa; By the algae crude oil after decolouring, in temperature, be that 120 ~ 280 ℃, vacuum tightness be-below 0.094mPa, decolour 1 ~ 2 hour, obtain being rich in the microalgae grease of DHA, obtain part free fatty acids simultaneously.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention adopts self-dissolving wall breaking technology, has simplified the technical process of fragmentation kettle algae extraction, has reduced the broken wall operating time, has reduced equipment investment, has reduced cost; In water, algae, organic solvent hierarchical process, adopt biological flocculant flocculation process, simplify demulsification technology flow process, reduced the hierarchical process time, improved layered effect, avoid the discharge of wastewater that causes because of demulsification technology and the burden of water treatment simultaneously, reduced the cost of water treatment;
(3) the present invention adopts microalgae wall breaking and grease to extract the technique of simultaneously carrying out, and has realized microalgae cell broken wall and grease and has extracted and carry out simultaneously, has greatly shortened the extraction time of microalgae grease;
(2) the present invention adopts physical refining technology to obtain being rich in the microalgae grease of DHA, and the refining that has reduced chemical refining consumes, and technical process is simple, product stability is good, and raw and auxiliary material is economized, and output is high, good in economic efficiency, avoided neutral oil saponification loss, refining efficiency is high, and product stability is good, can directly obtain high-quality byproduct-lipid acid, and there is no the plurality of advantages such as contaminated wastewater, particularly, for the depickling of some high acid value fragmentation kettle algae greases, its superiority is more outstanding.
Accompanying drawing explanation
Below in conjunction with accompanying drawing and by embodiment, further illustrate technical scheme of the present invention.
Fig. 1: the present invention extracts the process flow sheet of grease from fragmentation kettle algae.
Embodiment
For the present invention is described better, be convenient to understand technical scheme of the present invention, typical but non-limiting embodiment of the present invention is as follows:
Embodiment 1
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get.
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o 0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L.
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L.
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The fermented liquid 4m of fragmentation kettle algae
3, with glacial acetic acid, regulate pH to 4.5.Add 4m
3normal hexane, at 55 ~ 60 ℃, stirs 4h under 100rpm rotating speed, realizes the broken wall of fragmentation kettle frustule, and extracts microalgae grease simultaneously, then adds biological flocculant chitosan, makes chitosan concentration remain on 400mg/L.After fully stirring evenly, stratification, extracts upper strata mixing oil phase, and then adds 4m
3normal hexane, at 55 ~ 60 ℃, stirs 1h under 100rpm rotating speed, after stratification, extracts upper strata mixing oil phase.The algae-residue of lower floor, by solid-liquid separation, obtains algae-residue after being dried, and can be used for animal-feed and uses.The mixing oil phase obtaining, by evaporating solvent, obtains algae crude oil.
Algae raw oil refining technique adopts the physical refining process of advanced technology, and design parameter is as follows:
Degumming technology: adopt phosphoric acid acidifying degumming technology, algae crude oil temperature is 80 ~ 85 ℃, and phosphoric acid add-on is 0.5% of algae crude oil weight, and during washing, hot water add-on is 5% of algae crude oil weight, washes three times.
Decoloration process: temperature is 100 ~ 105 ℃, atlapulgite addition is 5% of the algae crude oil weight of coming unstuck, vacuum tightness (gauge pressure) is-below 0.094mpa.
Depickling/deodorizing technology: temperature is 200 ℃, vacuum tightness (gauge pressure) is-below 0.094mpa, the time is 2h.
By above-mentioned technique, obtain being rich in the microalgae grease of DHA.
Measuring fermented liquid biomass is 50g/L, and the total fatty acid content of extraction is that 30%, DHA content (being the ratio that DHA accounts for total fatty acids) is 43.36%.
Embodiment 2
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get.
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o 0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L.
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L.
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The fermented liquid 4m of fragmentation kettle algae
3, with glacial acetic acid, regulate pH to 3.5.Add 6m
3normal hexane, at 55 ~ 60 ℃, stirs 4h under 100rpm rotating speed, realizes the broken wall of fragmentation kettle frustule, and extracts microalgae grease simultaneously, then adds biological flocculant chitosan, makes chitosan concentration remain on 500mg/L.After fully stirring evenly, stratification, extracts upper strata mixing oil phase, and then adds 6m
3normal hexane, at 55 ~ 60 ℃, stirs 1h under 100rpm rotating speed, after stratification, extracts upper strata mixing oil phase.The algae-residue of lower floor, by solid-liquid separation, obtains algae-residue after being dried, and can be used for animal-feed and uses.The mixing oil phase obtaining, by evaporating solvent, obtains algae crude oil.
Algae raw oil refining technique adopts the physical refining process of advanced technology, and design parameter is as follows:
Degumming technology: adopt phosphoric acid acidifying degumming technology, algae crude oil temperature is 80 ~ 85 ℃, and phosphoric acid add-on is 0.3% of algae crude oil weight, and during washing, hot water add-on is 5% of algae crude oil weight, washes three times.
Decoloration process: temperature is 100 ~ 105 ℃, atlapulgite addition is 3% of the algae crude oil weight of coming unstuck, vacuum tightness (gauge pressure) is-below 0.094mpa.
Depickling/deodorizing technology: temperature is 280 ℃, vacuum tightness (gauge pressure) is-below 0.094mpa, the time is 2h.
By above-mentioned technique, obtain being rich in the microalgae grease of DHA.
Measuring fermented liquid biomass is 47g/L, and the total fatty acid content of extraction is that 32%, DHA content (being the ratio that DHA accounts for total fatty acids) is 36.81%.
Embodiment 3
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get.
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o 0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L;
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L.
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The fermented liquid 4m of fragmentation kettle algae
3, with glacial acetic acid, regulate pH to 3.5.Add 3.2m
3normal hexane, at 55 ~ 60 ℃, stirs 4h under 100rpm rotating speed, realizes the broken wall of fragmentation kettle frustule, and extracts microalgae grease simultaneously, then adds biological flocculant chitosan, makes chitosan concentration remain on 500mg/L.After fully stirring evenly, stratification, extracts upper strata mixing oil phase, and then adds 3.2m
3normal hexane, at 55 ~ 60 ℃, stirs 1h under 100rpm rotating speed, after stratification, extracts upper strata mixing oil phase.The algae-residue of lower floor, by solid-liquid separation, obtains algae-residue after being dried, and can be used for animal-feed and uses.The mixing oil phase obtaining, by evaporating solvent, obtains algae crude oil.
Algae raw oil refining technique adopts the physical refining process of advanced technology, and design parameter is as follows:
Degumming technology: adopt phosphoric acid acidifying degumming technology, algae crude oil temperature is 80 ~ 85 ℃, and phosphoric acid add-on is 0.5% of algae crude oil weight, and during washing, hot water add-on is 5% of algae crude oil weight, washes three times.
Decoloration process: temperature is 100 ~ 105 ℃, atlapulgite addition is 5% of the algae crude oil weight of coming unstuck, vacuum tightness (gauge pressure) is-below 0.094mpa.
Depickling/deodorizing technology: temperature is 260 ℃, vacuum tightness (gauge pressure) is-below 0.094mpa, the time is 2h.
By above-mentioned technique, obtain being rich in the microalgae grease of DHA.
Measuring fermented liquid biomass is 43g/L, and the total fatty acid content of extraction is that 28%, DHA content (being the ratio that DHA accounts for total fatty acids) is 35.73%.
Embodiment 4
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get.
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o 0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L.
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L.
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The fermented liquid 4m of fragmentation kettle algae
3, with lemon acid for adjusting pH to 4.0.Add 8m
3isohexane, at 40 ~ 50 ℃, stirs 6h under 80rpm rotating speed, realizes the broken wall of fragmentation kettle frustule, and extracts microalgae grease simultaneously, then adds biological flocculant chitosan, makes chitosan concentration remain on 100mg/L.After fully stirring evenly, stratification, extracts upper strata mixing oil phase, and then 8m
3isohexane, at 40 ~ 50 ℃, stirs 0.8h under 80rpm rotating speed, after stratification, extracts upper strata mixing oil phase.The algae-residue of lower floor, by solid-liquid separation, obtains algae-residue after being dried, and can be used for animal-feed and uses.The mixing oil phase obtaining, by evaporating solvent, obtains algae crude oil.
Degumming technology: adopt phosphoric acid acidifying degumming technology, algae crude oil temperature is 80 ~ 85 ℃, and phosphoric acid add-on is 0.4% of algae crude oil weight, and during washing, hot water add-on is 10% of algae crude oil weight, washes twice.
Decoloration process: temperature is 100 ~ 105 ℃, atlapulgite addition is 4% of the algae crude oil weight of coming unstuck, vacuum tightness (gauge pressure) is-below 0.094mpa.
Depickling/deodorizing technology: temperature is 120 ℃, vacuum tightness (gauge pressure) is-below 0.094mpa, the time is 1.5h.
By above-mentioned technique, obtain being rich in the microalgae grease of DHA.
Measuring fermented liquid biomass is 48g/L, and the total fatty acid content of extraction is that 26%, DHA content (being the ratio that DHA accounts for total fatty acids) is 37.25%.
Embodiment 5
Adopt fragmentation kettle algae ampere tank to be inoculated into behind inclined-plane, carry out after liquid shaking bottle cultivation, then transfer into 6m
3fermentor cultivation, the substratum under it consists of: glucose 100-130g, yeast extract paste 15-30g, peptone 10-20g, potassium primary phosphate 2-4g, magnesium sulfate 1-3g, trace element complex liquid 8-11ml, VITAMIN complex liquid 4-8ml, is settled to 1L with 0.5 * seawater; 0.5 described * seawater is that the water of the seawater of 1 volume and 1 volume is mixed to get.
The preparation method of described micro-complex liquid is as follows: Na2EDTA 6.0g, FeCl
36H
2o 0.29g, H
3bO
36.84g, MnCl
24H
2o 0.86g, ZnCl
20.06g, CoCl
26H
2o 0.026g, NiSO
46H
2o0.052g, CuSO
45H
2o 0.002g, NaMoO
42H
2o 0.005g, water is settled to 1L.
The preparation method of described VITAMIN complex liquid is as follows: VITMAIN B1 100mg, and vitamin B7 0.5mg, vitamin B12 0.5mg, water is settled to 1L.
The parameter of described fermentation culture is: 25 ~ 30 ℃ of temperature, and pH value 5.5 ~ 7.5, incubation time 72 ~ 96h, the parameter of described fermentation culture is preferably 25 ℃ of temperature, pH value 6.0, incubation time 96h, dissolved oxygen 10%.
The fermented liquid 4m of fragmentation kettle algae
3, with lemon acid for adjusting pH to 4.0.Add 5m
3no. six solvents, at 50 ~ 60 ℃, stir 2h under 150rpm rotating speed, realize the broken wall of fragmentation kettle frustule, and extract microalgae grease simultaneously, then add biological flocculant chitosan, make chitosan concentration remain on 300mg/L.After fully stirring evenly, stratification, extracts upper strata mixing oil phase, and then adds 5m
3no. six solvents, at 50 ~ 60 ℃, stir 0.5h under 150rpm rotating speed, after stratification, extract upper strata mixing oil phase.The algae-residue of lower floor, by solid-liquid separation, obtains algae-residue after being dried, and can be used for animal-feed and uses.The mixing oil phase obtaining, by evaporating solvent, obtains algae crude oil.
Degumming technology: adopt phosphoric acid acidifying degumming technology, algae crude oil temperature is 80 ~ 85 ℃, and phosphoric acid add-on is 0.4% of algae crude oil weight, and during washing, hot water add-on is 8% of algae crude oil weight, washes five times.
Decoloration process: temperature is 100 ~ 105 ℃, atlapulgite addition is 4% of the algae crude oil weight of coming unstuck, vacuum tightness (gauge pressure) is-below 0.094mpa.
Depickling/deodorizing technology: temperature is 280 ℃, vacuum tightness (gauge pressure) is-below 0.094mpa, the time is 1h.
By above-mentioned technique, obtain being rich in the microalgae grease of DHA.
Measuring fermented liquid biomass is 39g/L, and the total fatty acid content of extraction is that 24%, DHA content (being the ratio that DHA accounts for total fatty acids) is 38.14%.
Applicant's statement, the present invention illustrates method detailed of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned method detailed, does not mean that the present invention must rely on above-mentioned method detailed and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to the selection of the interpolation of the equivalence replacement of each raw material of product of the present invention and ancillary component, concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (25)
1. from fragmentation kettle algae, extract a method for grease, it is characterized in that, described method comprises the steps:
(1) regulate the pH of fragmentation kettle algae fermented liquid to acid, add organic solvent, at 40～60 ℃, stir, realize the broken wall of fragmentation kettle frustule and the extraction of microalgae grease;
(2a) in the fermented liquid obtaining to step (1), add biological flocculant, after fully stirring evenly, stratification, extracts upper strata mixing oil phase;
(2b) to step (2a), extract in the fermented liquid mixing after oil phase and again add organic solvent, at 40～60 ℃, stir, after stratification, extract upper strata mixing oil phase;
(2c) the solvent evaporation in mixing oil phase step (2a) and step (2b) being obtained, obtains algae crude oil;
(3) algae crude oil is through coming unstuck, decolouring, and depickling/deodorization, obtains being rich in the microalgae grease of DHA;
Described flocculation agent is chitosan.
2. the method for claim 1, is characterized in that, the described depickling of step (3)/deodorization adopts vacuum depickling/deodorizing technology.
3. the method for claim 1, is characterized in that, step (1) regulates pH to 3.5～4.5.
4. method as claimed in claim 3, is characterized in that, step (1) regulates pH to 3.7～4.2.
5. the method for claim 1, is characterized in that, step (1) adopts acid for adjusting pH value, described acid to be selected from the mixture of any one or at least two kinds in hydrochloric acid, phosphoric acid, acetic acid or citric acid.
6. the method for claim 1, is characterized in that, step (1) stirs at 45～55 ℃.
7. the method for claim 1, is characterized in that, in step (1) and step (2b), the volume of organic solvent is 0.8～2 times of step (1) fermentating liquid volume.
8. method as claimed in claim 7, is characterized in that, in step (1) and step (2b), the volume of organic solvent is 1～1.5 times of step (1) fermentating liquid volume.
9. the method for claim 1, is characterized in that, described organic solvent is selected from the mixture of any one or at least two kinds in No. six solvents, normal hexane, isohexane or iso-pentane.
10. the method for claim 1, is characterized in that, the addition of described chitosan is the fermented liquid that 100～500mg/L step (1) obtains.
11. methods as claimed in claim 10, is characterized in that, the addition of described chitosan is the fermented liquid that 300～500mg/L step (1) obtains.
12. the method for claim 1, is characterized in that, the stir speed (S.S.) of described stirring is 80～150rpm.
13. methods as claimed in claim 12, is characterized in that, the stir speed (S.S.) of described stirring is 90～140rpm.
14. methods as claimed in claim 13, is characterized in that, the stir speed (S.S.) of described stirring is 100～130rpm.
15. the method for claim 1, is characterized in that, the described churning time of step (1) is 2～6 hours.
16. methods as claimed in claim 15, is characterized in that, the described churning time of step (1) is 2.5～5.5 hours.
17. the method for claim 1, is characterized in that, the described churning time of step (2b) is 0.5～1 hour.
18. methods as claimed in claim 17, is characterized in that, the described churning time of step (2b) is 0.6 hour～0.9 hour.
19. the method for claim 1, is characterized in that, described in come unstuck and adopt phosphoric acid acidifying degumming technology.
20. methods as claimed in claim 19, it is characterized in that, described phosphoric acid acidifying degumming technology is: the algae crude oil that step (2c) is obtained is preheating to 80～85 ℃, 0.3～0.5% the phosphoric acid that adds algae crude oil weight that step (2c) obtains, after acidifying is come unstuck, the washing of 90～95 ℃ 2～5 times, the algae crude oil that comes unstuck after being come unstuck; The weight of described each washing water is 5～10% of the algae crude oil weight that obtains of step (2c).
21. the method for claim 1, is characterized in that, described decolouring adopts gac or activated decoloration.
22. methods as claimed in claim 21, it is characterized in that, described decoloration process is under vacuum condition, and temperature is 100～105 ℃, to the algae crude oil after coming unstuck add come unstuck after gac or the atlapulgite of algae crude oil weight 3～5% decolour, vacuum tightness is-below 0.094MPa.
23. the method for claim 1, it is characterized in that, described depickling/deodorization adopts vacuum depickling/deodorizing technology, and described technique is: by the algae crude oil after decolouring temperature be 120～280 ℃, vacuum tightness for-below 0.094MPa, depickling/deodorization 1～2 hour.
24. the method for claim 1, is characterized in that, step (2b) extracts after upper strata mixing oil phase, are positioned at the algae-residue in middle level by solid-liquid separation, after being dried, obtain.
25. the method for claim 1, is characterized in that, described method comprises the steps:
(1) regulate pH to 3.5～4.5 of fragmentation kettle algae fermented liquid, add organic solvent, at 40～60 ℃, stir, realize the broken wall of fragmentation kettle frustule and the extraction of microalgae grease;
(2a) in the fermented liquid obtaining to step (1), add biological flocculant, after fully stirring evenly, stratification, extracts upper strata mixing oil phase;
(2b) to step (2a) extraction, mix in the fermented liquid after oil phase and again add organic solvent, at 40～60 ℃, stir, after stratification, extract upper strata mixing oil phase, the algae-residue in middle level, by solid-liquid separation, obtains after being dried;
(2c) the solvent evaporation in mixing oil phase step (2a) and step (2b) being obtained, obtains algae crude oil;
(3) algae crude oil step (2c) being obtained is preheating to 80～85 ℃, adds 0.3～0.5% phosphoric acid of algae crude oil weight that step (2c) obtains, after acidifying is come unstuck, and the washing of 90～95 ℃ 2～5 times, the algae crude oil that comes unstuck after being come unstuck; The weight of described each washing water is 5～10% of the algae crude oil weight that obtains of step (2c); Under vacuum condition, temperature is 100～105 ℃, to the algae crude oil after coming unstuck add come unstuck after gac or the atlapulgite of algae crude oil weight 3～5% decolour, vacuum tightness is-below 0.094MPa; By the algae crude oil after decolouring temperature be 120～280 ℃, vacuum tightness for-below 0.094MPa, depickling/deodorization 1～2 hour, obtains being rich in the microalgae grease of DHA, obtains part free fatty acids simultaneously.
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| CN103275877B (en) * | 2013-06-09 | 2015-07-22 | 新奥科技发展有限公司 | Method for collecting schizochytrium limacinum |
| CN103351946B (en) * | 2013-06-15 | 2015-04-08 | 青岛海智源生命科技有限公司 | Preparation method of moderately processed ARA (Arachidonic acid) microalgae grease |
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| CN107372864A (en) * | 2016-05-17 | 2017-11-24 | 嘉必优生物技术（武汉）股份有限公司 | Composition, preparation method and application containing DHA grease |
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| CN111234912A (en) * | 2018-11-28 | 2020-06-05 | 中国科学院大连化学物理研究所 | Preparation method of microalgae edible oil |
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| CN111286400A (en) * | 2020-02-25 | 2020-06-16 | 南京乔运康生物科技有限公司 | Preparation method of microalgae oil DHA product capable of reducing 3-chloro-1, 2-propanediol content |
| CN113100357A (en) * | 2021-05-26 | 2021-07-13 | 广州优百特科技有限公司 | Fermented fat source feed raw material and preparation method and application thereof |
| CN114343021B (en) * | 2021-12-22 | 2024-04-05 | 嘉必优生物技术（武汉）股份有限公司 | Application of DHA algae oil for improving fishy smell in edible oil |
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