CN109852642A - A method of the enriching polyunsaturated fatty acid from the crude oil that microbial fermentation obtains - Google Patents
A method of the enriching polyunsaturated fatty acid from the crude oil that microbial fermentation obtains Download PDFInfo
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Abstract
The present invention relates to the bio-separation of fatty acid and enzyme technology fields, more particularly to a kind of method for obtaining enriching polyunsaturated fatty acid in unrefined crude oil from microbial fermentation, comprising the following steps: (1) crude oil obtained that will ferment obtains free fatty acid by enzymatic hydrolysis；(2) difference for utilizing alcohol and unsaturated fatty acid ester fat acid and saturated fatty acid reaction rate removes part saturated fatty acid, and remaining unreacted desired fatty acid is extracted；(3) multiple-grade molecular distillation is recycled to be enriched with to obtain the polyunsaturated fatty acid of high-purity in the fatty acid that step (2) obtains.Above step is using sepiolite as filtering agent, and based on isolating and purifying above, the method for the present invention that enrichment coupling polyunsaturated fatty acid is formed obtains crude oil as raw material using microbial fermentation, PUFAs >=95% can be made.The present invention is that a kind of hydrolysis and enrichment method by green high-efficient obtains the polyunsaturated fatty acid method of high-purity, creative using crude oil as initial feed, eliminates algae oil refining workshop section, process environment is friendly, cleaning, and greatly improves yield.
Description
Technical field
Polyunsaturated fatty acid is isolated and purified from the crude oil that microbial fermentation obtains the present invention relates to a kind of, via enzyme
Method hydrolysis, method of the enzyme process except heteroacid and multiple-grade molecular distillation enriching polyunsaturated fatty acid.
Background technique
Polyunsaturated fatty acid (PUFAs) plays the role of great, docosahexaenoic acid therein to human health
Two five olefin(e) acid of (cis-4,7,10,13,16,19-Docosahexaenoic Acid, DHA) He Ershi carbon
(Docosapentaenoic acid, DPA) is especially prominent.On the one hand, DHA is commonly called as " docosapentaenoic acid ", can be directly entered brain,
It is the important composition ingredient of fatty acid in nervous system, has significant function to the development of children's power and nerve, brain function can be improved
Can, promote brain development, improve eyesight, prevention cardiovascular disease, anticancer etc..On the other hand, DPA has effects that anticancer,
All there is prophylactic function to kinds cancers such as gastric cancers, also there is anti-inflammatory efficacy, by influencing the expression of cell factor or enzyme, drop
Low pro-inflammatory cytokine activity, to treat the diseases such as rheumatic arthritis, psoriasis, systemic loupus erythematosus to a certain extent.
It isolates and purifies to obtain the polyunsaturated fatty acid of high-purity from crude oil, raw material sources are particularly critical.PUFAs is passed
Uniting, there are many poisonous and harmful substances in abyssal fishes, abyssal fishes for food source, such as dioxin (Dioxin), toxaphene
Deng the heavy metal element that the mankind discharge in ocean also can leverage the quality of DHA in the cylinder accumulation of ocean fish.DPA is deposited
It is in the grease of some marine mammals, such as the content of DPA is produced in 5%-6% due to complex disposal process in seal oil
Product fishy smell is difficult to remove and the enrichment of food chain cause in deep sea fish oil heavy metal ion and organic pollutant content compared with
Height, there are security risks.But with the product that fungi and algae synthesize be practically free of EPA, it is pollution-free, environmental-friendly, pure,
Stability is good, fishy smell is small, cholesterol-free, and in various algae, Dinophyceae has the PUFAs of high-content, in recent years with fermentation
Obtained algae oil substitution deep sea fish oil has become a kind of development trend as the source of DHA and DPA.
PUFAs is enriched with by the algae oil that fermentation obtains, is all using the algae oil of refining as initial feed in traditional handicraft, essential oil is
It is obtained by crude oil by workshop sections such as degumming, depickling, decoloration, debrominates.Such as Chinese patent CN104394702B, for being constantly enriched with
A kind of method of oil generated by microalgae, with DHA ethyl ester.Algae oil refining process, it is necessary to using a large amount of strong acid and strong base with
And organic solvent, and workshop section's complicated and time consumption, so that cost is excessively high.Therefore, the invention using crude oil as raw material,
Using the selection specificity direct hydrolysis of lipase hydrolysis enzyme, process is simple, and percent hydrolysis is high, on the one hand, hydrolysis is dissociated
Fatty acid, on the other hand, reached degumming, depickling, decolorizing effect, meanwhile, by multiple-grade molecular distillation enrichment reach debrominate
Effect.Directly using crude oil as initial feed, the refining workshop section of algae oil is eliminated, process is simple, and it is environmental-friendly, it is at low cost.
The hydrolysis of algae oil is enriched in workshop section, requires to be removed by filtration fraction solids and impurity, in traditional workshop section all
It is to be filtered using atlapulgite, active carbon and diatomite.The invention cleans by filtering agent of sepiolite, Hai Pao
Stone is a kind of magnesia silicates clay compound in the Nature, and chemical formula is Mg8Si12O30(OH)4(OH2)4·8H2O
(SEP4H2O, 8H2O), since sepiolite is that have large specific surface area and layered porous by SiO2Tetrahedron and Mg-O eight
3 D stereo special construction made of the vegetative grafting of face, on its surface, there is also more acidity [SiO4] alkalinity [MgO6] center, from
And make sepiolite that there is stronger absorption property, adsorption-edulcoration is come with this, is worked well, it is white that traditional activity can be substituted completely
Soil, active carbon and diatomite.Selected sepiolite derives from the domestic sepiolite mineral products in xiangtan, hunan province city.
It is enriched with PUFAs from the algae crude oil that microbial fermentation obtains, via hydrolysis, separation, enrichment.On the one hand,
Algae oil hydrolyzes traditional handicraft and is mainly hydrolyzed by chemical method, is limited in that, in workshop section using a large amount of strong acid and strong base and
Organic solvent, reaction temperature is high, and a large amount of waste water is generated during washing process, pollutes environment.On the other hand, hydrolysis obtains
Free fatty acid must obtain polyunsaturated fatty acid by enrichment, traditional enrichment method specifically include that urea adduct method,
Metal salts as precipitator, low-temperature freezing, supercritical gas extraction method etc..Cai Shuanfei et al. handles how unsaturated with urea adduct method
The content of fatty acid methyl ester, methyl esters PUFA is increased to 86.7% by 47.7%, still, using a large amount of organic in urea clathrate workshop section
Solvent, and urea has to filter out, yield is low；Zheng Gongming etc. combines salting out method and urea adduct method, in marine animal
The PUFAs such as DPA are purified, and the ratio of the PUFAs such as final DPA has reached the 70%-75% of acid, still, salt object in the process
Matter can not reuse, and cause larger pollution to environment；Wang Xiaoling et al. utilizes crystallisation, under cryogenic to a kind of insect
In PUFAs be enriched with, at -8 DEG C, 17.08% and 6.59% has been respectively increased in the content of two kinds of PUFAs in raw material, still,
A large amount of organic solvents are used in the process, have residual, yield is lower；Supercritical gas extraction method, V. Riha et al. utilize this method
The DHA product of non-free type is purified, for PUFA total content up to 96%, disadvantage is that equipment cost is higher than conventional method.
To sum up, traditional hydrolysis and enrichment method or polluted environment or be equipment cost height using a large amount of organic solvent,
Low yield.Therefore, hydrolysis and the enrichment method for seeking a kind of green high-efficient, isolate and purify the polyunsaturated fat for obtaining high-purity
Acid has highly important researching value and application prospect.
Summary of the invention
The invention belongs to the bio-separation of fatty acid and enzyme technology field, the object of the present invention is to provide one kind from micro-
In crude oil made from biofermentation, the polyunsaturated fatty acid side of high-purity is obtained by the hydrolysis and enrichment method of green high-efficient
Method, process environment is friendly, cleaning, and greatly improves yield.
To achieve the above object, the invention adopts the following technical scheme:
From crude oil made from microbial fermentation, the polyunsaturated fatty acid method for obtaining high-purity is isolated and purified, fermentation is made
The crude oil obtained obtains free fatty acid by enzymatic hydrolysis, and hydrolase is reusable；Utilize alcohol and desired fatty acid and miscellaneous
The difference of sour reaction rate carries out esterification removal of impurities with lipase-catalyzed, and saturated fatty acid esterification is enriched in ester, and will be remaining
Unreacted desired fatty acid extracts, wherein the alcohol is n-hexyl alcohol, Decanol, lauryl alcohol, tetradecyl alchohol etc., hydrolysis and enzyme process are enriched with
The lipase used can be selected from Novozym435 lipase, Palatase, Alcaligenes sp. fat
Enzyme and Candida sp.99-125 lipase, preferably Candida sp.99-125 lipase, the lipase are according to China
It is prepared by the method for patent of invention CN1948470A；The fatty acid that enzyme process is enriched with is enriched with to obtain height using multiple-grade molecular distillation
The polyunsaturated fatty acid of purity.Hydrolysis is enriched with reaction solution obtained in workshop section by filtering agent of sepiolite and obtains crude product,
Selected sepiolite content 25%, 40%, 55%, 75%, 90%, preferential 75% content.Itself specific steps are as follows:
(1) hydrolysis: oil-water ratio 1:0.2-0.6, enzyme amount: 50-300 unit/grease, 20 DEG C -50 DEG C of reaction temperature, reaction
56 h-90 h of time, 300 rpm/min-500 rpm/min of mixing speed.According to percent hydrolysis in hydrolytic process, rouge is suitably added
Fat acid neutralizing agent isolates hydrolase after reaction, and hydrated sheath is acidified, stands, and extracts, and washes, water removal, sepiolite filtering
And evaporation, obtain free fatty acid.
(2) enzyme process is enriched with: the mass ratio of free fatty acid and alcohol is 1:0.166-0.3, and enzyme amount is the 6%- of sour quality
15%, 2 h-6 h of reaction time, 30 DEG C -50 DEG C of reaction temperature, n-hexane solvent appropriate, 200 rpm/min- of mixing speed
300 rpm/min.Conversion ratio reaches 75-85%, the effect of esterification removal of impurities is reached, after reaction by reaction solution sepiolite mistake
Filter, centrifuge separation, lipase is reusable, and remaining unreacted desired fatty acid is extracted, rich for lower step molecular distillation
Collection.
(3) molecular distillation is enriched with: the free fatty acid in step (2) is distilled in molecular distillation apparatus, level-one distillation,
Evaporator temperature: 50 DEG C -85 DEG C, cryosurface temperature: 30 DEG C -50 DEG C, vacuum degree: 2.5-4.5*10-1mbar；Retain first order molecular
Heavy constituent after distillation carries out secondary molecules distillation, evaporator temperature: 85 DEG C -115 DEG C, cryosurface temperature: and 45 DEG C -75 DEG C, very
Reciprocal of duty cycle: 2.5-3.5*10-2 mbar；Heavy constituent after retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature:
115 DEG C -140 DEG C, cryosurface temperature: 55 DEG C -90 DEG C, vacuum degree: 2.5-3.5*10-2 Mbar, after retaining three-level molecular distillation
Heavy constituent carries out quaternary molecule distillation, evaporator temperature: 140 DEG C -170 DEG C, cryosurface temperature: and 65 DEG C -90 DEG C, vacuum degree:
2.5-3.5*10-3 Mbar, the heavy constituent after retaining quaternary molecule distillation carry out Pyatyi molecular distillation, evaporator temperature: 170 DEG C-
200 DEG C, cryosurface temperature: 65 DEG C -90 DEG C, vacuum degree: 1.5-2.5*10-3 Mbar retains light component, and sepiolite absorbs and filter,
Obtain the polyunsaturated fatty acid of high-purity.
Compared with prior art, the invention has the following beneficial effects:
The present invention is that a kind of hydrolysis and enrichment method by green high-efficient obtains the polyunsaturated fatty acid method of high-purity, mistake
Journey is environmental-friendly, cleaning, and greatly improves yield.
(1) compared with conventional chemical methods hydrolysis, enzymatic hydrolysis is avoided using organic solvent, efficient green, reaction condition temperature
With algae oil percent hydrolysis reaches 90-99%.
(2) the invention using crude oil as initial feed, eliminate the refining workshop section of algae oil, process is simple, environment
Close friend greatly reduces cost of material；
(3) the invention using sepiolite as filtering agent, take full advantage of regional advantages, it is at low cost, work well.
(4) the use multiple-grade molecular distillation of the invention is connected, it will be apparent that improves PUFAs purity, PUFAs content
Reach 95-99%.
(5) enzyme process enrichment and molecular distillation technique are carried out under conditions of being in low temperature or high vacuum, entire technique mistake
Journey efficiently avoids the oxidation of PUFAs, to greatly ensure that the quality of product.
Specific embodiment
Embodiment 1
(1) will ferment 500 g(DHA+DPA >=55% of crude oil obtained), 150 mL of deionized water, 20 mL hydrolyze enzyme solution mixing,
35 DEG C of reaction temperature, 60 h of reaction time, 350 rpm/min of mixing speed.When percent hydrolysis reaches 25 %, 12.5 mL hydrogen of addition
In potassium oxide and fatty acid adds 12.5 mL potassium hydroxide, when percent hydrolysis reaches 85%, most when percent hydrolysis reaches 65%
After 12.5 mL potassium hydroxide are added.Final percent hydrolysis reaches 95.8%, and reaction solution is acidified through Centrifugical extraction after hydrolysis, and washing removes
Water, the sepiolite filtering of 75% content, evaporation obtain free fatty acid.
(2) 240 g of free fatty acid of step (1), 43.2 g of lauryl alcohol are taken, enzyme amount is the 8% of sour quality, reaction time 4
H, 35 DEG C of reaction temperature, the n-hexane solvent of 200 mL, 260 rpm/min of mixing speed.Conversion ratio is 81.5%, and reaction terminates
Reaction solution is filtered with sepiolite afterwards, is centrifugated, lipase is reusable, and by remaining unreacted fatty acid purification by liquid extraction
For crude product.
(3) step (2) crude product is isolated and purified into molecular distillation apparatus, level-one distillation, evaporator temperature: 60
DEG C, cryosurface temperature: 35 DEG C, vacuum degree: 3.0*10-1 Mbar, 0.2 kg/h of sample rate；Retain the weight of first order molecular distillation
Component carries out secondary molecules distillation, evaporator temperature: 90 DEG C, cryosurface temperature: and 50 DEG C, vacuum degree: 2.5*10-2 Mbar, sample introduction
0.2 kg/h of rate；The heavy constituent for retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature: 120 DEG C, cryosurface
Temperature: 60 DEG C, vacuum degree: 2.5*10-2 Mbar, 0.2 kg/h of sample rate；The heavy constituent for retaining three-level molecular distillation carries out four
Grade molecular distillation, evaporator temperature: 140 DEG C, cryosurface temperature: 70 DEG C, vacuum degree: 3.5*10-3 Mbar, sample rate 0.2
kg/h；The heavy constituent for retaining quaternary molecule distillation carries out Pyatyi molecular distillation, evaporator temperature: 190 DEG C, cryosurface temperature: 80
DEG C, vacuum degree: 1.5*10-3 Mbar, 0.2 kg/h of sample rate retain light component, and after being filtered with sepiolite, centrifuge separation is obtained
To the polyunsaturated fatty acid of high-purity, after esterification, through gas chromatographic detection, obtaining DHA content is that 79.8%, DPA content is
15.9%, it the results are shown in Table one.
Embodiment 2
(1) will ferment 500 g(DHA+DPA >=55% of crude oil obtained), 200 mL of deionized water, 20 mL hydrolyze enzyme solution mixing,
40 DEG C of reaction temperature, 65 h of reaction time, 350 rpm/min of mixing speed.When percent hydrolysis reaches 30%, 12.5 mL hydrogen are added
In potassium oxide and fatty acid adds 12.5 mL potassium hydroxide when percent hydrolysis reaches 65%, when percent hydrolysis reaches 85%,
It is eventually adding 12.5 mL potassium hydroxide.Final percent hydrolysis reaches 96.2%, and reaction solution is through Centrifugical extraction, acidification, water after hydrolysis
It washes, removes water, the sepiolite filtering of 75% content, evaporation obtains free fatty acid.
(2) 250 g of free fatty acid of step (1), 45 g of lauryl alcohol are taken, enzyme amount is the 8% of sour quality, reaction time 4
H, 40 DEG C of reaction temperature, the n-hexane solvent of 200 mL, 260 rpm/min of mixing speed.Conversion ratio is 82.1%, and reaction terminates
Reaction solution is filtered with sepiolite afterwards, is centrifugated, lipase is reusable, and by remaining unreacted fatty acid purification by liquid extraction
For crude product.
(3) step (2) crude product is isolated and purified into molecular distillation apparatus, level-one distillation, evaporator temperature: 65
DEG C, cryosurface temperature: 35 DEG C, vacuum degree: 2.5*10-1 Mbar, 0.2 kg/h of sample rate；Retain the weight of first order molecular distillation
Component carries out secondary molecules distillation, evaporator temperature: 100 DEG C, cryosurface temperature: and 50 DEG C, vacuum degree: 2.5*10-2 Mbar, into
0.2 kg/h of sample rate；The heavy constituent for retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature: 125 DEG C, condensation
Face temperature: 60 DEG C, vacuum degree: 2.5*10-2 Mbar, 0.2 kg/h of sample rate；The heavy constituent for retaining three-level molecular distillation carries out
Quaternary molecule distillation, evaporator temperature: 145 DEG C, cryosurface temperature: 70 DEG C, vacuum degree: 3.5*10-3 Mbar, sample rate 0.2
kg/h；The heavy constituent for retaining quaternary molecule distillation carries out Pyatyi molecular distillation, evaporator temperature: 195 DEG C, cryosurface temperature: 80
DEG C, vacuum degree: 1.5*10-3 Mbar, 0.2 kg/h of sample rate retain light component, and after being filtered with sepiolite, centrifuge separation is obtained
To the polyunsaturated fatty acid of high-purity, after esterification, through gas chromatographic detection, obtaining DHA content is that 80.9%, DPA content is
15.6%, it the results are shown in Table one.
Embodiment 3
(1) will ferment crude oil 500g(DHA+DPA >=55% obtained), 200 mL of deionized water, 20 mL hydrolyze enzyme solution mixing, instead
Answer 40 DEG C of temperature, 70 h, 350 rpm/min of mixing speed when reaction.When percent hydrolysis reaches 30%, 12.5mL potassium hydroxide is added
Fatty acid is neutralized to add 12.5 mL potassium hydroxide when percent hydrolysis reaches 66% and be eventually adding when percent hydrolysis reaches 86%
12.5 mL potassium hydroxide.Final percent hydrolysis reaches 97.5%, and reaction solution is acidified through Centrifugical extraction after hydrolysis, washes, water removal, 75%
The sepiolite of content filters, and evaporation obtains free fatty acid.
(2) 250 g of free fatty acid of step (1), 41.5 g of lauryl alcohol are taken, enzyme amount is the 8% of sour quality, reaction time 4
H, 40 DEG C of reaction temperature, the n-hexane solvent of 200mL, 260 rpm/min of mixing speed.Conversion ratio is 83.5%, after reaction
Reaction solution is filtered with sepiolite, is centrifugated, lipase is reusable, and is by remaining unreacted fatty acid purification by liquid extraction
Crude product.
(3) step (2) crude product is isolated and purified into molecular distillation apparatus, level-one distillation, evaporator temperature: 70
DEG C, cryosurface temperature: 40 DEG C, vacuum degree: 2.5*10-1 Mbar, 0.2 kg/h of sample rate；Retain the weight of first order molecular distillation
Component carries out secondary molecules distillation, evaporator temperature: 105 DEG C, cryosurface temperature: and 55 DEG C, vacuum degree: 2.5*10-2 Mbar, into
0.2 kg/h of sample rate；The heavy constituent for retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature: 125 DEG C, condensation
Face temperature: 65 DEG C, vacuum degree: 2.5*10-2 Mbar, 0.2 kg/h of sample rate；The heavy constituent for retaining three-level molecular distillation carries out
Quaternary molecule distillation, evaporator temperature: 150 DEG C, cryosurface temperature: 70 DEG C, vacuum degree: 3.5*10-3 Mbar, sample rate 0.2
kg/h；The heavy constituent for retaining quaternary molecule distillation carries out Pyatyi molecular distillation, evaporator temperature: 195 DEG C, cryosurface temperature: 85
DEG C, vacuum degree: 1.5*10-3 Mbar, 0.2 kg/h of sample rate retain light component, and after being filtered with sepiolite, centrifuge separation is obtained
To the polyunsaturated fatty acid of high-purity, after esterification, through gas chromatographic detection, obtaining DHA content is that 81.1%, DPA content is
15.8%, it the results are shown in Table one.
Embodiment 4
(1) will ferment 500 g(DHA+DPA >=55% of crude oil obtained), 200 mL of deionized water, 20 mL hydrolyze enzyme solution mixing,
40 DEG C of reaction temperature, 76 h of reaction time, 350 rpm/min of mixing speed.When percent hydrolysis reaches 30%, 12.5 mL hydrogen-oxygens are added
Change in potassium and fatty acid adds 12.5 mL potassium hydroxide, when percent hydrolysis reaches 86%, finally when percent hydrolysis reaches 66%
12.5 mL potassium hydroxide are added.Final percent hydrolysis reaches 97.8%, and reaction solution is acidified through Centrifugical extraction after hydrolysis, and washing removes
Water, the sepiolite filtering of 75% content, evaporation obtain free fatty acid.
(2) 250 g of free fatty acid of step (1), 41.5 g of lauryl alcohol are taken, enzyme amount is the 8% of sour quality, reaction time 5
H, 45 DEG C of reaction temperature, the n-hexane solvent of 200 mL, 260 rpm/min of mixing speed.Conversion ratio is 83.8%, and reaction terminates
Reaction solution is filtered with sepiolite afterwards, is centrifugated, lipase is reusable, and by remaining unreacted fatty acid purification by liquid extraction
For crude product.
(3) step (2) crude product is isolated and purified into molecular distillation apparatus, level-one distillation, evaporator temperature: 75
DEG C, cryosurface temperature: 40 DEG C, vacuum degree: 2.5*10-1 Mbar, 0.2 kg/h of sample rate；Retain the weight of first order molecular distillation
Component carries out secondary molecules distillation, evaporator temperature: 110 DEG C, cryosurface temperature: and 55 DEG C, vacuum degree: 2.5*10-2 Mbar, into
0.2 kg/h of sample rate；The heavy constituent for retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature: 130 DEG C, condensation
Face temperature: 65 DEG C, vacuum degree: 2.5*10-2 Mbar, 0.2 kg/h of sample rate；The heavy constituent for retaining three-level molecular distillation carries out
Quaternary molecule distillation, evaporator temperature: 155 DEG C, cryosurface temperature: 65 DEG C, vacuum degree: 3.0*10-3 Mbar, sample rate 0.2
kg/h；The heavy constituent for retaining quaternary molecule distillation carries out Pyatyi molecular distillation, evaporator temperature: 195 DEG C, cryosurface temperature: 80
DEG C, vacuum degree: 1.5*10-3 Mbar, 0.2 kg/h of sample rate retain light component, and after being filtered with sepiolite, centrifuge separation is obtained
To the polyunsaturated fatty acid of high-purity, after esterification, through gas chromatographic detection, obtaining DHA content is that 81.6%, DPA content is
16.1%, it the results are shown in Table one.
Embodiment 5
(1) will ferment 500 g(DHA+DPA >=55% of crude oil obtained), 200 mL of deionized water, 20 mL hydrolyze enzyme solution mixing,
45 DEG C of reaction temperature, 85 h of reaction time, 350 rpm/min of mixing speed.When percent hydrolysis reaches 30%, 12.5 mL hydrogen-oxygens are added
Change in potassium and fatty acid adds 12.5 mL potassium hydroxide, when percent hydrolysis reaches 86%, finally when percent hydrolysis reaches 66%
12.5 mL potassium hydroxide are added.Final percent hydrolysis reaches 98.1%, and reaction solution is acidified through Centrifugical extraction after hydrolysis, and washing removes
Water, the sepiolite filtering of 75% content, evaporation obtain free fatty acid.
(2) 250 g of free fatty acid of step (1), 41.5 g of lauryl alcohol are taken, enzyme amount is the 8% of sour quality, reaction time 6
H, 45 DEG C of reaction temperature, the n-hexane solvent of 220 mL, 260 rpm/min of mixing speed.Conversion ratio is 84.6%, and reaction terminates
Reaction solution is filtered with sepiolite afterwards, is centrifugated, lipase is reusable, and by remaining unreacted fatty acid purification by liquid extraction
For crude product.
(3) step (2) crude product is isolated and purified into molecular distillation apparatus, level-one distillation, evaporator temperature: 85
DEG C, cryosurface temperature: 45 DEG C, vacuum degree: 2.5*10-1 Mbar, 0.2 kg/h of sample rate；Retain the weight of first order molecular distillation
Component carries out secondary molecules distillation, evaporator temperature: 115 DEG C, cryosurface temperature: and 50 DEG C, vacuum degree: 2.5*10-2 Mbar, into
0.2 kg/h of sample rate；The heavy constituent for retaining secondary molecules distillation carries out three-level molecular distillation, evaporator temperature: 140 DEG C, condensation
Face temperature: 60 DEG C, vacuum degree: 2.5*10-2 Mbar, 0.2 kg/h of sample rate；The heavy constituent for retaining three-level molecular distillation carries out
Quaternary molecule distillation, evaporator temperature: 165 DEG C, cryosurface temperature: 70 DEG C, vacuum degree: 3.0*10-3 Mbar, sample rate 0.2
kg/h；The heavy constituent for retaining quaternary molecule distillation carries out Pyatyi molecular distillation, evaporator temperature: 200 DEG C, cryosurface temperature: 70
DEG C, vacuum degree: 1.5*10-3 Mbar, 0.2 kg/h of sample rate retain light component, and after being filtered with sepiolite, centrifuge separation is obtained
To the polyunsaturated fatty acid of high-purity, after esterification, through gas chromatographic detection, obtaining DHA content is that 82.1%, DPA content is
16.5%, it the results are shown in Table one.
Table one
Claims (8)
- It, will 1. one kind isolates and purifies the polyunsaturated fatty acid method for obtaining high-purity from crude oil made from microbial fermentation Crude oil made from fermenting obtains free fatty acid by enzymatic hydrolysis, and crude oil and water are mixed in a certain ratio in hydrolysis enzymatic Under, efficient green reaction, reaction condition is mild, and algae oil percent hydrolysis is high, reaction solution filtered with sepiolite after reaction, from Heart separation, enzyme are reusable；Using the difference of alcohol and desired fatty acid and heteroacid reaction rate, ester is carried out with lipase-catalyzed Eliminate it is miscellaneous, by saturated fatty acid esterification be enriched in ester, and by remaining unreacted desired fatty acid extract；Enzyme process is enriched with to obtain Fatty acid recycle multiple-grade molecular distillation be enriched with to obtain the polyunsaturated fatty acid of high-purity.
- 2. isolating and purifying to obtain high-purity how unsaturated rouge according to claim 1 from crude oil made from microbial fermentation Fat acid method, it is characterised in that: by enzymatic hydrolysis, enzyme process enrichment is enriched with to obtain more insatiable hungers of high-purity with multiple-grade molecular distillation And fatty acid.
- 3. according to from algae oil made from microbial fermentation, isolating and purifying to obtain the how unsaturated rouge of high-purity described in claim 1 Fat acid method, hydrolase are Lipase Powder or enzyme solution, and the lipase that hydrolysis and enzyme process enrichment use can be selected from Novozym435 lipase, Palatase, Alcaligenes sp. lipase and Candida Sp.99-125 lipase, preferably Candida sp.99-125 lipase, the lipase are according to Chinese invention patent Prepared by the method for CN1948470A, hydrolysis enzyme amount is 50-300 unit/grease, and the enzyme amount of enzymatic removal of impurities is the 6%- of sour quality 15%。
- 4. according to from crude oil made from microbial fermentation, isolating and purifying to obtain high-purity polyunsaturated fat described in claim 1 Sour method, in hydrolysing step, oil-water ratio is 1:0.2-0.6 arbitrarily than column, enzyme amount: 50-300 unit/grease, reaction temperature 30 DEG C -50 DEG C, 56 h-90 h of reaction time, 300 rpm/min-500 rpm/min of mixing speed, according to hydrolysis in hydrolytic process Rate suitably adds fatty acid neutralizing agent, and the fat acid neutralizing agent is NaOH or KOH.
- 5. compared with conventional chemical methods hydrolysis, enzymatic hydrolysis is avoided using organic solvent, efficiently according to claim 4 the method Green, reaction condition is mild, and algae oil percent hydrolysis reaches 90-99%.
- 6. according to from algae oil made from microbial fermentation, isolating and purifying to obtain the how unsaturated rouge of high-purity described in claim 1 Fat acid method, in enzyme process enrichment, the mass ratio of free fatty acid and alcohol is 1:0.166-0.3 arbitrarily than column, and enzyme amount is sour quality 6%-15%, 2 h-6 h of reaction time, 30 DEG C -50 DEG C of reaction temperature, n-hexane solvent appropriate, 200 rpm/ of mixing speed min-300 rpm/min。
- 7. according to from crude oil made from microbial fermentation, isolating and purifying to obtain the how unsaturated rouge of high-purity described in claim 1 Fat acid method is distilled in molecular distillation apparatus, level-one distillation, evaporator temperature: 50 DEG C -85 DEG C, cryosurface temperature: 30 DEG C -50 DEG C, vacuum degree: 2.5-4.5*10-1mbar；Heavy constituent after retaining first order molecular distillation carries out secondary molecules distillation, evaporator temperature Degree: 85 DEG C -115 DEG C, cryosurface temperature: 45 DEG C -75 DEG C, vacuum degree: 2.5-3.5*10-2 mbar；After retaining secondary molecules distillation Heavy constituent carry out three-level molecular distillation, evaporator temperature: 115 DEG C -140 DEG C, cryosurface temperature: 55 DEG C -90 DEG C, vacuum degree: 2.5-3.5*10-2 Mbar, the heavy constituent after retaining three-level molecular distillation carry out quaternary molecule distillation, evaporator temperature: 140 DEG C- 170 DEG C, cryosurface temperature: 65 DEG C -90 DEG C, vacuum degree: 2.5-3.5*10-3Mbar, the heavy constituent after retaining quaternary molecule distillation Pyatyi molecular distillation is carried out, evaporator temperature: 170 DEG C -200 DEG C, cryosurface temperature: 65 DEG C -90 DEG C, vacuum degree: 1.5-2.5* 10-3Mbar, retains light component, and sepiolite adsorption filtration obtains the polyunsaturated fatty acid of high-purity.
- 8. being connected according to claim 7 the method using multiple-grade molecular distillation, it will be apparent that improve PUFAs purity, PUFAs contains Amount reaches 95-99%, and molecular distillation enrichment method, temperature is low, heated time is short, separation degree is high, energy consumption is low, environmental-friendly, point It is physical process from process, does not use organic solvent in separation process, separated object matter can be protected well not contaminated, obtained Pure safe product, and molecular distillation carries debrominate function, obtained polyunsaturated fatty acid is obvious compared to former algae oil taste Reduce.
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