CN107874253A - A kind of spirulina glycolipid preparation method rich in acid and gamma-linolenic - Google Patents
A kind of spirulina glycolipid preparation method rich in acid and gamma-linolenic Download PDFInfo
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- CN107874253A CN107874253A CN201610873024.7A CN201610873024A CN107874253A CN 107874253 A CN107874253 A CN 107874253A CN 201610873024 A CN201610873024 A CN 201610873024A CN 107874253 A CN107874253 A CN 107874253A
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- spirulina
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- glycolipid
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- linolenic
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- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 79
- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 79
- 229940082787 spirulina Drugs 0.000 title claims abstract description 78
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 229930186217 Glycolipid Natural products 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 239000002253 acid Substances 0.000 title abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 49
- 239000000194 fatty acid Substances 0.000 claims abstract description 49
- 229930195729 fatty acid Natural products 0.000 claims abstract description 49
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 34
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- 239000001656 lutein Substances 0.000 claims abstract description 6
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- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 claims abstract description 6
- 235000020664 gamma-linolenic acid Nutrition 0.000 claims description 75
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 claims description 75
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 claims description 66
- 229960002733 gamolenic acid Drugs 0.000 claims description 66
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 41
- 239000000741 silica gel Substances 0.000 claims description 39
- 229910002027 silica gel Inorganic materials 0.000 claims description 39
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 33
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 4
- 229910052740 iodine Inorganic materials 0.000 claims description 4
- 239000011630 iodine Substances 0.000 claims description 4
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- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 claims description 2
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- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims 1
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
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- 239000007788 liquid Substances 0.000 description 5
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- 239000000203 mixture Substances 0.000 description 4
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 3
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- 150000002632 lipids Chemical class 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
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- 125000001931 aliphatic group Chemical group 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- SZHOJFHSIKHZHA-UHFFFAOYSA-N tridecanoic acid Chemical compound CCCCCCCCCCCCC(O)=O SZHOJFHSIKHZHA-UHFFFAOYSA-N 0.000 description 2
- FROLUYNBHPUZQU-IIZJPUEISA-N (2R,3R,4S,5R)-2-(hydroxymethyl)-6-[3-[3-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxypropoxy]propoxy]oxane-3,4,5-triol Chemical compound OC[C@H]1OC(OCCCOCCCOC2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@H]1O FROLUYNBHPUZQU-IIZJPUEISA-N 0.000 description 1
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- 241000192700 Cyanobacteria Species 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
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- 240000008916 Oenothera biennis Species 0.000 description 1
- 241000530636 Spirulina subsalsa Species 0.000 description 1
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- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 229940045761 evening primrose extract Drugs 0.000 description 1
- 229940089020 evening primrose oil Drugs 0.000 description 1
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- 239000013505 freshwater Substances 0.000 description 1
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- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a kind of spirulina glycolipid preparation method rich in acid and gamma-linolenic, this method is using spirulina powder as raw material, using alcohol steep, high speed centrifugation, vacuum filtration, vacuum concentration, vacuum freeze drying, column chromatographic isolation and purification, gas Chromatographic Determination, the spirulina glycolipid rich in acid and gamma-linolenic is finally given.The selected reagent of extracting and developing purifying of the present invention is the food processing aid for allowing addition to use in food industry, and green non-poisonous, extraction conditions are gently and the spirulina glycolipid recovery rate rich in acid and gamma-linolenic is high；The present invention is rich in the spirulina glycolipid MGDG of acid and gamma-linolenic using macroporous absorbent resin and silica gel column chromatography separating purification, separation except realizing the spirulina glycolipid rich in acid and gamma-linolenic, the components such as lutein, chlorophyll can be also obtained, can apply and be used as food color；Gained of the invention is rich in the spirulina glycolipid MGDG of acid and gamma-linolenic, and the content of its acid and gamma-linolenic accounts for the 45~51% of total fatty acids.
Description
Technical field
The present invention relates to technical field of health care food, and in particular to prepared by a kind of spirulina glycolipid rich in gamma-Linolenic acid
Method.
Background technology
Spirulina (spirulina) is a kind of rudimentary plant, and Cyanophyta, Oscillariaceae are belonged in classification[1], in the world oneself
Know that spirulina shares 36 kinds, mostly fresh water species, at present, the spirulina kind of large-scale production has spirulina maxim
(Spirulina maximum), blunt top spirulina (Spirulina platensis) and Spirulina subaalsa (Spirulina
subsalsa)。
Spirulina is rich in a variety of nutriments, wherein rich in protein, and composition of amino acid is appropriate, it is considered to be
Protein content highest, top-quality food known to the mankind at present.And contain various bioactivators in spirulina,
Wherein glycolipid content enriches, including single galactolipin diacetyl glyceride (MGDG, mono-galactosyl-
Diaclyglycerol), double galactolipin diacetyl glyceride (DGDG, digalactosyl-diaclyglycerol) are thio different
Rhamnose diacetyl glyceride (SQDG, sulfoqiuvonosyldiaclyglycerol), there is reducing blood lipid, anti-inflammatory, antitumor
Isoreactivity, the molecular structure of these three greases are as shown in Figure 1.The gamma-Linolenic acid that spirulina is rich in is primarily present in MGDG Sn-
1 or Sn-2 positions, it is the main functional activity group of glycolipid.
Gamma-Linolenic acid (GLA, Gamma linolenic Acid) belongs to the serial polyunsaturated fatty acids of ω -6[2], have
Blood pressure is adjusted, reduces cholesterol, prevention diabetes, cancer and other effects[3].Gamma-Linolenic acid is distributed mainly on plant and microorganism
In, wherein it is oenothera biennis and Common Borage to study most commonly used, in evening primrose oil containing in 7~10%, borage oil containing 18~
26%, because domestic and international market demand increased in recent years, to seek substitutable resources, domestic and international researcher is found, scale
Substantial amounts of gamma-Linolenic acid is rich in the spirulina of culture, accounts for 1.1~1.5% of dry cell weight or so, account for total fatty acids 18~
21%[4], it is seen then that spirulina is to obtain gamma-Linolenic acid potential source, has development prospect well.
At present, the patent and pertinent literature prepared for the spirulina glycolipid rich in gamma-Linolenic acid is studied, the extraction mentioned
Purification process is concentrated mainly on supercritical carbon dioxide extraction method, urea adduct method etc..But the former is high to equipment requirement, and carries
Rate is taken not to be significantly increased；The latter has the residual such as urea, methanol, easily forms suspect carcinogen, is not directly applicable to food
Or health products production.Therefore, the present invention is Extraction solvent from ethanol that is green safe, ratifying to use in the food industry, hair
Understand a kind of easy-operating extracting mode of safety, while the spirulina glycolipid to being rich in gamma-Linolenic acid in ethanol crude extract enters
Row isolates and purifies, and is enriched with gamma-Linolenic acid.
The content of the invention
For the disadvantages mentioned above of existing method, it is an object of the invention to provide a kind of green low poison solvent extracting and developing
Spirulina glycolipid preparation method of the purifying rich in gamma-Linolenic acid, to improve the extraction efficiency of gamma-Linolenic acid and purity.
To achieve the above object, the present invention carries out spiral shell under mild conditions from food-grade ethanol as Extraction solvent
The thick fat extraction of algae is revolved, and is isolated and purified using the food processing aid for allowing addition to use in food industry, to obtain richness
Spirulina glycolipid containing gamma-Linolenic acid.The method includes extracting thick fat composition from spirulina, the technique that optimization influences extraction efficiency
Parameter, the spirulina glycolipid rich in gamma-Linolenic acid is isolated and purified, analyze total fatty acids therein and GLA contents.
Specifically, present invention firstly provides a kind of spirulina glycolipid preparation method rich in gamma-Linolenic acid, this method bag
Include following steps：
S1：Alcohol steep, spirulina powder and ethanol are pressed 1:6~10 (g/mL) ratio is well mixed, and constant temperature stirring carries
Take, Extracting temperature can be adjusted between room temperature~60 DEG C, and extraction time is 30~120min；
S2：The thick fat of spirulina obtains, and the crude extract after ethanol extracts, be centrifuged at a high speed algae-residue and supernatant, supernatant
Liquid produces the thick fat extract of spirulina using removing organic solvent is concentrated in vacuo after vacuum freeze drying；
S3：Column chromatographic isolation and purification, the thick fat extract of spirulina that column chromatographic isolation and purification step S2 is obtained, applied sample amount with
Column chromatography silica gel amount presses 1:30~200 (g/g) loadings, column chromatography mobile phase are ethanol and n-hexane mixed solvent.
Specifically, the column chromatographic isolation and purification step is：
S3-1：Post is filled, silica gel and appropriate eluent are tuned into suspension, using wet method dress post；
S3-2：Loading, the thick fat extract of spirulina redissolve pure in ethanol/n-hexane mixed solvent, column chromatography silica gel separation
Change the thick fat extract of spirulina, do (wet) method loading, applied sample amount presses 1 with column chromatography silica gel amount:30~200 (g/g)；Loading
Amount presses more preferably 1 with column chromatography silica gel amount:200(g/g)；
S3-3：Elution, ethanol/n-hexane mixed system gradient elution is used after filling post loading, ethanol/n-hexane ratio is
1:Adjusted between 1~10 (v/v), 0.5~3.0 column volume is eluted, by volume collection eluent, 50~100mL/ bottles；
Preferably, Extracting temperature is 35-50 DEG C in S1, and the ratio of spirulina powder and ethanol is 1：6~8.It is furthermore preferred that spiral shell
The ratio of spirulina powder and ethanol is 1：7.
Preferably, described spirulina is any one in blunt top spirulina, spirulina maxim and Spirulina subaalsa.
Preferably, described extracting and developing purification solvent ethanol and n-hexane are food-grade ethanol and n-hexane.
Preferably, the described filler that isolates and purifies is 200-400 mesh column chromatography silica gels.
Preferably, ethanol and n-hexane the in the mixed solvent ethanol and n-hexane ratio are 1:1~10 (v/v).
Further, present invention additionally comprises the step of gas Chromatographic Determination total fatty acids and gamma -linolenic acid content, specifically
For：
S4：The eluent that S3 is collected carries out thin-layered chromatography analysis, iodine vapor dyeing, determines glycolipid MGDG in eluent
In position；
S5:The eluent that S4 is collected, which is concentrated in vacuo, removes organic solvent, after vacuum freeze drying, produces rich in γ-Asia
The spirulina glycolipid of numb acid, then carry out directly turning esterification reaction of organic acid, so by total fatty acids in GC-MS measure eluents and
The content of gamma-Linolenic acid.
Further, present invention additionally comprises macroporous absorbent resin to remove step, and specially described macroporous absorbent resin takes off
Except in the thick fat extract of spirulina pigment, 1.25~2.50mg/mL of sample concentration, 2~3 column volumes of loading, eluant, eluent choosing
70~100% ethanol are selected, 2~6 column volumes is eluted, pigment spirulina crude extract must be removed.
Preferably, the pigment includes lutein and chlorophyll etc.；The macroporous absorbent resin be D101, HP20 and
One kind in HP2MGL.
Further, present invention additionally comprises the lutein and chlorophyll further to recycle step.
Beneficial effect
First, the selected reagent of extracting and developing purifying of the present invention is the food for allowing addition to use in food industry
Processing aid, green non-poisonous, extraction conditions are gently and the spirulina glycolipid recovery rate rich in gamma-Linolenic acid is high；Secondly, using big
Macroporous adsorbent resin and silica gel column chromatography separating purification are rich in the spirulina glycolipid MGDG of gamma-Linolenic acid, and γ-Asia is rich in except realizing
The separation of the spirulina glycolipid of numb acid, can also obtain the components such as lutein, chlorophyll, can apply and be used as food color；This hair
Bright gained is rich in the spirulina glycolipid MGDG of gamma-Linolenic acid, and the content of its gamma-Linolenic acid accounts for the 45~51% of total fatty acids.
Brief description of the drawings
Fig. 1 monogalactosyl diglycerides (MGDG), digalactosyl diglyceride (DGDG), thio isorhamnose diglyceride
(SQDG) molecular structure；
Fig. 2 applied sample amounts and column chromatography silica gel amount ratio 1:The thin-layer chromatographic analysis result of eluent when 200 (g/g)；
Fig. 3 applied sample amounts and column chromatography silica gel amount ratio 1:The thin-layer chromatographic analysis result of eluent when 100 (g/g)；
Fig. 4 applied sample amounts and column chromatography silica gel amount ratio 1:The thin-layer chromatographic analysis result of eluent when 50 (g/g)；
Fig. 5 applied sample amounts and column chromatography silica gel amount ratio 1:The thin-layer chromatographic analysis result of eluent when 30 (g/g)；
The thin-layer chromatographic analysis result of silica gel column chromatography separating purification sample after Fig. 6 removing pigments.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means.For example, methyl esterification of fatty acid reaction, gas phase color
Spectrum-GC-MS (GC-MS) and GC-FID analysis test methods etc., outer unless specified otherwise, the present invention is to this and is not limited.
The preparation of experiment material
Console mode supercentrifuge (Beckman), vacuum rotary evaporator (BUCHI), gas chromatograph-mass spectrometer (GC-MS)
(Agilent 7890B/5977A GC-MS, chromatographic column HP-88：60m × 0.25mm × 0.2 μm), ethanol (food-grade), spiral
Algae (water content≤7.0%), TLC Silica gel 60 (Merck kGaA, Germany).
Experimental method
1. the analysis determining method of thick fat extract and aliphatic acid
(1) direct methyl esterization
With reference to Lieve etc.[5]Method is simultaneously appropriately modified, and weighs 10mg algae powder, sequentially adds 200uL chloroform-methanol
Mixed liquor (2:1, V/V), the hydrochloric acid-methanol solution (v/v) of 300uL 5%, 20uL 2mg/mL tridecanoic acid solution, mix, 85
DEG C reaction 1h, after cooling add 1mL n-hexanes, be vortexed mix after stand 1h extracted, pipette 200uL n-hexane layers, add
5uL 1mg/mL pentadecanes carry out analysis measure as internal standard by GC-MS.
GC-MS analysis conditions：Carrier gas is high-pure helium, using not shunt mode sample introduction, the μ L of sample size 1.00, and injection port and auxiliary
It is 250 DEG C to help heter temperature.Temperature programming condition：50 DEG C of initial temperature is kept for 2 minutes, then with 25 DEG C/min of speed
Rate is warming up to 175 DEG C, and is kept for 5 minutes；210 DEG C then are warming up to 7 DEG C/min of speed again, is kept for 2 minutes；Finally with 2
DEG C/min speed be warming up to 230 DEG C, kept for 1 minute.
(2) Bligh-Dyer methods
The extracting method of thick fat extract refers to Bligh-Dyer methods, and is suitably modified, specific as follows：Weigh 100mg
Dry algae powder, liquid nitrogen grinding 3 times, add 6mL chloroform-methanols (2:1, v/v), 30 DEG C of constant-temperature table 260rpm hatching extraction 1h, add
Enter 1.5mL 0.7%KCl isolating proteins, lower floor's organic phase is drawn in centrifugation, and surplus solution adds 2mL chloroform-methanols (2:1,v/v)
Extraction 2 times is repeated, merges organic phase, freeze-drying, which is weighed, after concentration calculates total lipid content.Take again and carry out direct esterification on a small quantity
Reaction, measure aliphatic acid and GLA content are analyzed by GC-MS.
2. glycolipid analyzes and identifies
The spirulina glycolipid of gamma-Linolenic acid is rich in by thin-layer chromatography chromatography analysis, solvent is chloroform：Methanol：Water
=4:1:0.1 (v/v/v), iodine vapor dyeing.Qualitative analysis list galactosyl diglyceride (MGDG), digalactosyl glycerine two
Ester (DGDG) and thio isorhamnose base glycerol diester (SQDG).
Embodiment 1 is rich in the spirulina glycolipid preparation method 1 of gamma-Linolenic acid
S1 weighs 10.00g algae powder, is placed in reaction bulb, by 1:7 (mL/g) liquid ratios add ethanol, and after mixing, 50 DEG C are stirred
Extraction 1h is mixed, repeats extraction 2 times.
Crude extracts of the S2 after ethanol extracts, be centrifuged at a high speed algae-residue and supernatant, and supernatant is steamed using vacuum rotating
Instrument concentration removing organic solvent is sent out, the thick fat extract of spirulina is produced after vacuum freeze drying.
S3-1 fills post, silica gel and appropriate eluent is tuned into suspension, using wet method dress post；
S3-2-3 applied sample amount 651.0mg, applied sample amount and column chromatography silica gel amount ratio 1:200 (g/g), silicagel column volume are
522.8mL, first with 1:5 (v/v) ethanol/n-hexane elute 0.5 column volume, then with 1:1 (v/v) ethanol/n-hexane elutes 3 posts
Volume.
S4 collects eluent and carries out thin-layered chromatography analysis, iodine vapor dyeing, determines position of the glycolipid in eluent.
The eluent of collection is concentrated in vacuo by S5 removes organic solvent, and after vacuum freeze drying, progress directly turns esterification
Reaction, then total fatty acids and the content of gamma-Linolenic acid in eluent are determined by GC-MS.
As a result as shown in Fig. 2 and table 1, after thick fat extract sample is by silicagel column, it is possible to achieve chlorophyll and MGDG,
DGDG's and SQDG is efficiently separated, and gamma-Linolenic acid is primarily present in MGDG, and wherein gamma-Linolenic acid highest can in total fatty acids
Up to 50.34%, up to 73.40%, it is significantly higher than gamma-Linolenic acid in loading sample and accounts for total fatty acids and thick total fatty acid content
Ratio (the loading sample of extract：Gamma-Linolenic acid/total fatty acids=25.58%, total fatty acids/thick fat extract=
42.18%), applied sample amount and column chromatography silica gel amount ratio 1:When 200 (g/g), the spirulina sugar rich in gamma-Linolenic acid can be obtained
Fat MGDG.The sample of table 1 is 1 with column chromatography silica gel amount ratio：Total fatty acids and total GLA content yield when 200
Note：Sample number into spectrum is consistent with being numbered in Fig. 2, and 1-5 is sequentially to collect the eluent by silicagel column, due to eluting
The time come is different, is collected into sample 1-5 successively.
Embodiment 2
The spirulina glycolipid rich in gamma-Linolenic acid, other concrete operations are further isolated and purified using silica gel column chromatography
Step such as embodiment 1.Applied sample amount and column chromatography silica gel amount ratio 1:100(g/g).
As a result as shown in Fig. 3 and table 2, after thick fat extract sample is by silicagel column, it is possible to achieve chlorophyll and MGDG,
DGDG's and SQDG is efficiently separated, and gamma-Linolenic acid is primarily present in MGDG, and wherein gamma-Linolenic acid highest can in total fatty acids
Up to 46.91%, up to 64.43%, it is significantly higher than gamma-Linolenic acid in loading sample and accounts for total fatty acids and crude extract total fatty acids
Ratio (loading sample：Gamma-Linolenic acid/total fatty acids=25.58%, total fatty acids/thick fat extract=42.18%), on
Sample amount and column chromatography silica gel amount ratio 1:The spirulina glycolipid MGDG rich in gamma-Linolenic acid can be equally obtained when 100 (g/g).
The sample of table 2 is 1 with column chromatography silica gel amount ratio：Total fatty acids and total GLA content yield when 100
Note：Sample number into spectrum is consistent with being numbered in Fig. 3, and 1,7,8,9 and 13-17 collected silicagel column sample for order；[please be true
Recognize numbering]
Wherein 7+8+9 is that sample 7,8 and 9 collection liquids merge measure.
Embodiment 3
The spirulina glycolipid rich in gamma-Linolenic acid, other concrete operations are further isolated and purified using silica gel column chromatography
Step such as embodiment 1.Applied sample amount and column chromatography silica gel amount ratio 1:50(g/g).
As a result as shown in Fig. 4 and table 3, after thick fat extract sample is by silicagel column, it is possible to achieve chlorophyll and MGDG,
DGDG's and SQDG is efficiently separated, and gamma-Linolenic acid is primarily present in MGDG, and wherein gamma-Linolenic acid highest can in total fatty acids
Up to 46.87%, up to 70.49%, it is significantly higher than gamma-Linolenic acid in loading sample and accounts for total fatty acids and crude extract total fatty acids
Ratio (loading sample：Gamma-Linolenic acid/total fatty acids=25.58%, total fatty acids/thick fat extract=42.18%), on
Sample amount and column chromatography silica gel amount ratio 1:The spirulina glycolipid MGDG rich in gamma-Linolenic acid can be equally obtained when 50 (g/g).
The sample of table 3 is 1 with column chromatography silica gel amount ratio：Total fatty acids and total GLA content yield when 50
Note：Sample number into spectrum is consistent with being numbered in Fig. 4, and 4-12 collected silicagel column sample for order, and 1-3 is free of or containing a small amount of
Object, not as the sample objects of analysis；
Wherein 5+6 is that the collection liquid of sample 5 and 6 merges measure.
Embodiment 4
The spirulina glycolipid rich in gamma-Linolenic acid is further isolated and purified using silica gel column chromatography, concrete operation step is such as
Embodiment 1.Applied sample amount and column chromatography silica gel amount ratio 1:30(g/g).
As a result as shown in Fig. 5 and table 4, after thick fat extract sample is by silicagel column, it is possible to achieve chlorophyll and MGDG,
DGDG's and SQDG is efficiently separated, and gamma-Linolenic acid is primarily present in MGDG, and wherein gamma-Linolenic acid highest can in total fatty acids
Up to 48.35%, up to 67.45%, it is significantly higher than gamma-Linolenic acid in loading sample and accounts for total fatty acids and crude extract total fatty acids
Ratio (loading sample：Gamma-Linolenic acid/total fatty acids=25.58%, total fatty acids/thick fat extract=42.18%), on
Sample amount and column chromatography silica gel amount ratio 1:The spirulina glycolipid MGDG rich in gamma-Linolenic acid can be equally obtained when 30 (g/g).
The sample of table 4 is 1 with column chromatography silica gel amount ratio：Total fatty acids and total GLA content yield when 30
Note：Sample number into spectrum is consistent with being numbered in Fig. 5, and 4-6,8-12 collected silicagel column sample, numbering 1-3 and 7 for order
Be free of or containing a small amount of object, not as the sample objects of analysis.
Embodiment 5
Spirulina lipid extracts remove the pigments such as lutein, chlorophyll, concrete operations by macroporous absorbent resin first：
2.5 column volumes of sample concentration 2mg/mL loadings, 70% column volume of ethanol elution 2, then with 95% ethanol elution, 4 cylinders
Product, collect eluent.Eluent produces the spiral of removing pigment using removing organic solvent is concentrated in vacuo after vacuum freeze drying
The thick fat extract of algae；
The spirulina glycolipid rich in gamma-Linolenic acid is further isolated and purified using silica gel column chromatography, concrete operation step is such as
Embodiment 1, applied sample amount and column chromatography silica gel amount ratio 1:200(g/g).
As a result as shown in Fig. 6 and table 5, thick fat extract sample by macroporous absorbent resin (after macroporous absorbent resin,
Pigment removal rate is up to more than 90%) and silicagel column after, lead to the same conclusion, it is possible to achieve chlorophyll and MGDG, DGDG and
SQDG's is efficiently separated, and gamma-Linolenic acid is primarily present in MGDG, and wherein gamma-Linolenic acid reaches as high as in total fatty acids
51.23%, total fatty acids 57.48%, it is significantly higher than gamma-Linolenic acid in loading sample and accounts for total fatty acids and the ratio of crude extract
(loading sample：Gamma-Linolenic acid/total fatty acids=28.05%, thick fat=45.40% of total fatty acids/depigmentation), remove pigment
Carry out silica gel column chromatography again afterwards further to isolate and purify, can equally obtain the spirulina glycolipid MGDG rich in gamma-Linolenic acid.And
Compare and find through absorbance detection, by pigment removal rate after macroporous absorbent resin up to more than 90%, further increase extraction
The condition of thing.
Table 5 removes silica gel column chromatography separating purification sample total fatty acids and total GLA content after pigment
Note：Sample number into spectrum is consistent with being numbered in Fig. 6, and 2-20 collected silicagel column sample for order, and numbering 1 is free of target
Thing, not as the sample objects of analysis.
Embodiment 6
The spirulina glycolipid rich in gamma-Linolenic acid is further isolated and purified using silica gel column chromatography, concrete operation step is such as
Embodiment 1, applied sample amount and column chromatography silica gel amount ratio 1:200 (g/g), eluent system are chloroform：Methanol：Water (4:1:0.1v/v/
v)。
As a result shown in table 6 and table 7, table 5 is that ethanol extraction carries with Bligh-Dyer methods extraction total fatty acids and gamma-Linolenic acid
The comparison of efficiency is taken, using in-situ transesterification methyl esterization as reference, the extraction efficiencies of total fatty acids is less than Bligh-Dyer methods, but γ-
Leukotrienes extraction efficiency is 88.5%, higher than Bligh-Dyer methods, is illustrated for the spirulina glycolipid rich in gamma-Linolenic acid, second
Alcohol extracting is more selective.Table 7 is using chloroform：Methanol：Water (4:1:0.1v/v/v) silica gel column chromatography knot during eluent system
Fruit, gamma-Linolenic acid are primarily present in MGDG, and gamma-Linolenic acid reaches as high as 46.78% wherein in total fatty acids, in crude extract
Total fatty acids 59.58%, it is significantly higher than, and gamma-Linolenic acid in loading sample accounts for total fatty acids and the ratio of crude extract is (upper all
Product：Gamma-Linolenic acid/total fatty acids=25.58%, total fatty acids/thick fat extract=42.18%).Although using Bligh-
Dyer methods are extracted, chloroform：Methanol：Water elution system isolates and purifies, and can equally obtain the spirulina glycolipid rich in gamma-Linolenic acid
MGDG, but selected reagent is respectively provided with stronger toxicity and can not be applied in food industry, and use range is limited.
The other method of table 6 and the comparison of the total fatty acids and gamma-Linolenic acid yield of the inventive method extraction
The chloroform of table 7：Methanol：Water elution system isolates and purifies total fatty acids and total GLA content in sample
Note：Sample number into spectrum 1-17 collected silicagel column sample for order.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (12)
1. a kind of spirulina glycolipid preparation method rich in gamma-Linolenic acid, this method comprise the following steps：
S1：Alcohol steep, spirulina powder and ethanol are pressed into mass volume ratio 1:6~10 ratio is well mixed, and constant temperature stirring carries
Take, Extracting temperature is room temperature~60 DEG C, and extraction time is 30~120min；
S2：The thick fat of spirulina obtains, and the crude extract after ethanol extracts, be centrifuged at a high speed algae-residue and supernatant, supernatant profit
Organic solvent is removed with being concentrated in vacuo, the thick fat extract of spirulina is produced after vacuum freeze drying；
S3：Column chromatographic isolation and purification, the thick fat extract of spirulina that column chromatographic isolation and purification step S2 is obtained, applied sample amount and post layer
Analyse silica gel amount in mass ratio 1:30~200 loadings, column chromatography mobile phase are ethanol and n-hexane mixed solvent.
2. the method as described in claim 1, it is characterised in that Extracting temperature is 35-50 DEG C in S1, spirulina powder and ethanol
Ratio is 1：6~8.
3. the method as described in claim 1, it is characterised in that described spirulina be blunt top spirulina, spirulina maxim with
And any one in Spirulina subaalsa.
4. the method as described in claim 1, it is characterised in that the described filler that isolates and purifies is 200-400 mesh column chromatography silicon
Glue.
5. the method as described in claim 1, it is characterised in that described extracting and developing purification solvent ethanol and n-hexane are
Food-grade ethanol and n-hexane.
6. method as claimed in claim 5, it is characterised in that ethanol and n-hexane the in the mixed solvent ethanol and n-hexane
Ratio is 1:1~10 (v/v).
7. the method as described in claim 1, it is characterised in that the applied sample amount presses 1 with column chromatography silica gel amount:50~200 (g/
g)。
8. method as claimed in claim 7, it is characterised in that the applied sample amount presses 1 with column chromatography silica gel amount:200(g/g).
9. the method as described in claim 1~7 any one, it is characterised in that also include macroporous absorption tree between S2 and S3
Fat removes step.
10. method as claimed in claim 8, it is characterised in that the macroporous absorbent resin removes the thick fat extract of spirulina
In pigment, 1.25~2.50mg/mL of sample concentration, 2~3 column volumes of loading, eluant, eluent select 70~100% ethanol, wash
2~6 column volumes are taken off, pigment spirulina crude extract must be removed.
11. method as claimed in claim 10, it is characterised in that the pigment includes lutein and chlorophyll；The macropore
Polymeric adsorbent is one kind in D101, HP20 and HP2MGL.
12. the method as described in claim 1~7 any one, it is characterised in that also including gas Chromatographic Determination total fatty acids
And the step of gamma -linolenic acid content, it is specially：
S4：The eluent that S3 is collected carries out thin-layered chromatography analysis, iodine vapor dyeing, determines glycolipid MGDG in eluent
Position；
S5:The eluent that S4 is collected, which is concentrated in vacuo, removes organic solvent, after vacuum freeze drying, produces rich in gamma-Linolenic acid
Spirulina glycolipid, then carry out directly turning esterification reaction of organic acid, so by total fatty acids in GC-MS measure eluents and γ-
Linolenic content.
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Application publication date: 20180406 |