CN108703252A - A kind of preparation method of green onion albumen - Google Patents
A kind of preparation method of green onion albumen Download PDFInfo
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- CN108703252A CN108703252A CN201810547145.1A CN201810547145A CN108703252A CN 108703252 A CN108703252 A CN 108703252A CN 201810547145 A CN201810547145 A CN 201810547145A CN 108703252 A CN108703252 A CN 108703252A
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- green onion
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- 241000234282 Allium Species 0.000 title claims abstract description 69
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000010992 reflux Methods 0.000 claims abstract description 10
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000008055 phosphate buffer solution Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
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- 239000002904 solvent Substances 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 5
- 235000020679 tap water Nutrition 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
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- 108090000623 proteins and genes Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 150000001408 amides Chemical class 0.000 description 7
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000003078 antioxidant effect Effects 0.000 description 4
- 238000005238 degreasing Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000005452 bending Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000000751 protein extraction Methods 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 230000002000 scavenging effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 1
- 235000010167 Allium cepa var aggregatum Nutrition 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- -1 DPPH free radical Chemical class 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000004438 eyesight Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000000848 glutamatergic effect Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000001696 ion exchange chromatographie Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012913 prioritisation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000003257 protein preparation method Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/185—Vegetable proteins
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Biochemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to vegetable protein extractive technique field more particularly to a kind of preparation methods of green onion albumen, include the following steps：(1) it pre-processes；(2) refluxing extraction；(3) it saltouts；(4) it dialyses；Easy to operate using the preparation method in the present invention, the green onion albumen of gained has antioxidation activity in vitro.
Description
Technical field
The present invention relates to vegetable protein extractive technique field more particularly to a kind of preparation methods of green onion albumen.
Background technology
Shallot is Liliaceae allium herbaceos perennial, as one of medicinal and edible plants, has antibacterial, anticancer, resists
The bioactivity such as oxidation.Green onion is the dry mature seed of green onion, and warm-natured, acrid flavour has the effect of bowl spares strengthening the essence, improving eyesight relieve heat,
It can be used for kidney deficiency, dizzy, the illnesss such as anemofrigid cold.
Many diseases such as cancer, diabetes, atherosclerosis, senile dementia, parkinsonism occur and body
Interior free radical is related.Based on this, antioxidant research and development have become the important directions of field of medicaments concern.Vegetable protein has antioxygen
The functions such as change, hypoglycemic, antitumor, immunological regulation.
Due to containing abundant protein in green onion, in order to comprehensively utilize green onion child resource, the present invention is built by raw material of green onion
A kind of green onion protein preparation method has been found, and has carried out characterization and antioxidation in vitro evaluation to it.
Invention content
The technical problem to be solved by the present invention is to：In view of the deficiencies of the prior art, it provides a kind of with preferably anti-oxidant
The preparation method of active green onion albumen.
In order to solve the above technical problems, the technical scheme is that：
A kind of preparation method of green onion albumen, the preparation method comprises the following steps：
(1) it pre-processes：Dry green onion is crushed, is filtered after ungrease treatment, collects filter residue；
(2) refluxing extraction：Filter residue in step (1) is taken, Extraction solvent heating and refluxing extraction, filtering, the extraction of collection is added
Liquid obtains supernatant by centrifugation；
(3) it saltouts：Supernatant in step (2) is taken, ammonium sulfate is added, is centrifuged after saltouing overnight and obtains albumen precipitation；
(4) it dialyses：Albumen precipitation in step (3) is taken, dissolves, is handled by bag filter, the trapped fluid of collection is by freezing
Dry is green onion albumen crude product.
As an improvement technical solution, according to solid-liquid ratio 1 in the step (1):The ratio of 6-8 be added petroleum ether into
Row ungrease treatment, skimming temp are 40 DEG C, time 2h.
As an improvement technical solution, the solid-liquid ratio of filter residue and Extraction solvent is 1 in the step (2):25-1:
35, Extracting temperature is 40-60 DEG C, extraction time 90-150min.
As an improvement technical solution, Extraction solvent is 0.01-0.03mol/L in the step (2), pH8-9's
Phosphate buffer solution.
As a kind of perferred technical scheme, Extraction solvent is that the phosphate of 0.02mol/L, pH8 are slow in the step (2)
Solution is rushed, Extracting temperature is 60 DEG C, extraction time 150min, and filter residue and the solid-liquid ratio of phosphate buffer solution are 1:35.
As a kind of perferred technical scheme, the temperature in the step (2) when centrifugation is 4 DEG C, rotating speed 5000-6000r/
Min, centrifugation time 25-40min.
As a kind of perferred technical scheme, the saturation degree of ammonium sulfate is 95% in the step (3), is placed at 4 DEG C
Overnight.
As a kind of perferred technical scheme, the molecular cut off of bag filter is 3500Da- in the step (4)
5000Da, and first being dialysed 24-48h with tap water when dialysing, then dialysed 24-48h with distilled water.
After using above-mentioned technical proposal, the beneficial effects of the invention are as follows：
(1) present invention is using green onion as raw material, after petroleum ether degreasing is handled, carries out reflux with phosphate buffer solution and carries
It takes, then by the supernatant being collected by centrifugation after ammonium sulfate precipitation is handled, protein component precipitates in supernatant, and it is heavy to be collected by centrifugation
It forms sediment, then is dissolved albumen precipitation with phosphate buffer solution, dialyse by bag filter, the trapped fluid of collection is by freeze-drying
For green onion albumen crude product.Preparation method is simple, and raw material is easy to get, and is to investigate with the recovery rate of green onion albumen by orthogonal test
Index has obtained the best preparation process of green onion albumen, and pH value 8 extracts 150min, solid-liquid ratio 1:35,60 DEG C of Extracting temperature,
The recovery rate of green onion albumen is 6.29% under this process conditions.
(2) present invention passes through green onion proteolysis chromatogram, it can be seen that amino acid contained type is complete；By removing
DPPH free radical activities are tested and hydroxy radical (OH) clearance test, it was confirmed that in the present invention green onion albumen for preparing have compared with
Good antioxidant activity, is expected to obtain a kind of potential natural.
Description of the drawings
Fig. 1 is the infrared spectrogram of green onion albumen；
Fig. 2 is amino acid standard chromatogram；
Fig. 3 is green onion protein hydrolytic amino acid chromatogram；
Fig. 4 is green onion protein SDS-PAGE electrophoretogram.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
It takes freeze-drying green onion protein sample appropriate, rear tabletting is fully ground with dry KBr powder, in 4000cm-1-
400cm-1Wave band scans.In infared spectrum as shown in Figure 1, the absorption peak of N-H stretching vibrations is in 3400~3440cm-1Range
It is interior, but after same hydrogen bond forms association body, peak will be subjected to displacement to lower wave number.As seen from the figure, the amide A of green onion albumen takes out of
Present 3299.11cm-1Place shows that its N-H is strong and forms association body with hydrogen bond.2961.23cm-1The weak absorbing at place is amide B bands
Characteristic absorption peak caused by C-N is flexible；In 1655.10cm-1There is strong absworption peak in place, is the C=O stretching vibration peaks of amide Ⅰ,
It is commonly used for Secondary structure analysis.1480~1600cm-1For the feature of II band of amide, the predominantly N- of green onion albumen
The characteristic absorption of the reflection of H bendings, II band of green onion albumen amide is 1539.53cm-1The strong absorption ownership at place, belongs to the C=of His
The C=N stretching vibrations of C and Arg make the intensity at peak increase in this overlapping；1200~1360cm-1The assignments at place are in amide III
Band is caused by C-N is flexible and N-H bendings；Belong to the CH of Gly skeletons and Pro side chains2Rocking vibration peak is also herein
It is embodied in region；1241.97cm-1Place is the characteristic absorption of Amide Ⅲ band caused by the N-H bendings of green onion albumen.
With SDS-PAGE electrophoresis technique determining protein relative molecular weights.Deposition condition is：5% concentration glue, 15% separation gel,
Using constant pressure concentration glue 120V, separation gel 150V, time 2.5h, it is determined that green onion protein relative molecular weight.As shown in Figure 4：
Protein molecule has the deeper band of color between 55kDa~40kDa, illustrates that protein molecular weight is concentrated mainly on this range
It is interior；The band that 15kDa and 10kDa nearby also has color shallower.
Embodiment 1
A kind of preparation method of green onion albumen, includes the following steps：
(1) it pre-processes：Dry green onion is crushed, according to solid-liquid ratio 1:6 ratio is added petroleum ether, at 40 DEG C, degreasing 2h,
Filter residue is collected in filtering；
(2) refluxing extraction：Filter residue in step (1) is taken, is 1 according to solid-liquid ratio:25 ratio is added 0.01mol/L, pH8's
Phosphate buffer solution, Extracting temperature are 40 DEG C, extract 90min, filtering, the extracting solution of collection in 4 DEG C, rotating speed 5000r/min,
Supernatant is collected after centrifugation 25min；
(3) it saltouts：Take supernatant in step (2), ammonium sulfate (saturation degree 95%) be added, saltouts, 4 DEG C overnight after centrifuge
Obtain albumen precipitation；
(4) it dialyses：Albumen precipitation in step (3) is taken, is dissolved with the phosphate buffer solution of pH8, by molecular cut off
For the processing of 3500Da bag filters, first for 24 hours with tap water dialysis, then for 24 hours with distilled water dialysis, the trapped fluid of collection is dry by freezing
Dry is green onion albumen crude product.
Embodiment 2
A kind of preparation method of green onion albumen, includes the following steps：
(1) it pre-processes：Dry green onion is crushed, according to solid-liquid ratio 1:7 ratio is added petroleum ether, at 40 DEG C, degreasing 2h,
Filter residue is collected in filtering；
(2) refluxing extraction：Filter residue in step (1) is taken, is 1 according to solid-liquid ratio:0.03mol/L, pH8.5 is added in 30 ratio
Phosphate buffer solution, Extracting temperature be 50 DEG C, extract 120min, filtering, the extracting solution of collection is in 4 DEG C, rotating speed 5500r/
Min collects supernatant after centrifuging 35min；
(3) it saltouts：Take supernatant in step (2), ammonium sulfate (saturation degree 95%) be added, saltouts, 4 DEG C overnight after centrifuge
Obtain albumen precipitation；
(4) it dialyses：Albumen precipitation in step (3) is taken, is dissolved with the phosphate buffer solution of pH8.5, by retaining molecule
Amount is that 5000Da bag filters are handled, and is first dialysed 36h with tap water, then is dialysed 36h with distilled water, and the trapped fluid of collection is by freezing
Dry is green onion albumen crude product.
Embodiment 3
A kind of preparation method of green onion albumen, includes the following steps：
(1) it pre-processes：Dry green onion is crushed, according to solid-liquid ratio 1:8 ratio is added petroleum ether, at 40 DEG C, degreasing 2h,
Filter residue is collected in filtering；
(2) refluxing extraction：Filter residue in step (1) is taken, is 1 according to solid-liquid ratio:35 ratio is added 0.02mol/L, pH9's
Phosphate buffer solution, Extracting temperature are 60 DEG C, extract 150min, filtering, the extracting solution of collection is in 4 DEG C, rotating speed 6000r/
Min collects supernatant after centrifuging 40min；
(3) it saltouts：Take supernatant in step (2), ammonium sulfate (saturation degree 95%) be added, saltouts, 4 DEG C overnight after centrifuge
Obtain albumen precipitation；
(4) it dialyses：Albumen precipitation in step (3) is taken, the phosphate buffer solution dissolving of pH9 is added, by retaining molecule
Amount is that 3500Da bag filters are handled, and is first dialysed 48h with tap water, then is dialysed 48h with distilled water, and the trapped fluid of collection is by freezing
Dry is green onion albumen crude product.
According to experiment of single factor as a result, it is investigation factor to select pH value, extraction time, Extracting temperature, liquid ratio, with L9
(34) orthogonal arrage arrangement experiment, it determines green onion protein extraction technique, sees Tables 1 and 2.
1 green onion Protein Extraction technological factor water-glass of table
2 orthogonal experiments of table
From Tables 1 and 2：Using green onion extraction rate of protein as index, green onion Protein Extraction prioritization scheme is
A1B3C3D3, i.e. pH value is 8, extracts 150min, solid-liquid ratio 1:35,60 DEG C of Extracting temperature.
4 green onion Amino Acids in Proteins composition analysis of embodiment
Protein pre-treatment
Sample 0.02796g is taken to be hydrolyzed in pipe in 20mL, addition 6M hydrochloric acid 15mL vacuum outgas 30min, nitrogen charging, tube sealing,
22h is hydrolyzed in 110 DEG C of baking ovens；Open pipe after cooling is transferred in 50mL volumetric flasks, ultra-pure water constant volume and mixing；Take 1mL hydrolyzates
In 5mL bottles, depickling drying in 50 DEG C of vacuum drying chambers, after draining plus 1mL ultra-pure waters are drained again.Accurately add 0.02M hydrochloric acid
1mL redissolves, and 0.22 μm of water film filtering is spare.
Chromatographiccondition
2622#PH ion-exchange chromatographies, specification 4.5*60mm；Sample size is 20 μ L；In addition to proline is 440nm,
His amino acid Detection wavelength is 570nm；Ninhydrin, buffer flow rate 0.35mL/min, flow velocity 0.4mL/min；It is derivative anti-
It is 57 DEG C to answer device temperature；This experiment is single point correction, and the concentration of each standard amino acid is 2nmol/20 μ L, the results are shown in Table 3.
3 green onion protein hydrolysising amino acid content result of table
Note：T total amino acid contents；E must amino acid；N nonessential amino acids；
As known from Table 3, the amino acid contained type of green onion is completely (except tryptophan because hydrolysis is almost destroyed entirely), gross mass point
Number 81.59% is 15.27% wherein the highest of mass fraction containing glutamic acid, and Glutamatergic, which is combined in human body with blood ammonia, to be formed pair
The harmless glutamine of body releases the ammonia toxic action generated during tissue metabolism, and participates in brain tissue metabolism, makes brain work(
It can be active；E/T is 33.5%, E/N 50.5%, is proposed with the World Health Organization (WHO) and FAO (Food and Agriculture Organization of the United Nation) (FAO)
E/T is about that 40%, E/N is about 60% to be close.
Embodiment 5
The measurement of green onion albumen free radical scavenging
A series of green onion protein sample solution and Vc solution (positive control) are configured according to table 4, are configured to absolute ethyl alcohol
The DPPH solution of 0.04mg/mL.The sample solution of 2mL various concentrations is taken respectively, 2mL DPPH solution is added, and is uniformly mixed, room
After temperature places 30min, 5000r/min centrifuges 10min.Supernatant is taken to survey light absorption value at 517nm.Use Vc as positive control.
Sample calculates the clearance rate of DPPH free radicals with following formula：
A0The light absorption value of -2mL absolute ethyl alcohol+2mL DPPH solution；
A1The light absorption value of -2mL sample solution+2mL DPPH solution；
A2The light absorption value of -2mL absolute ethyl alcohol+2mL sample solutions.
The clearance rate of 4 green onion albumen of table and Vc to DPPH free radicals
The experimental results showed that in 0.05-5mg/ml concentration ranges, green onion albumen to hydroxy radical there are scavenging effect,
And as the increase scavenging effect of concentration enhances.
Embodiment 6
Measurement of the green onion albumen to hydroxy radical (OH) clearance rate
A series of green onion protein sample solution and Vc solution (control group), every part of sample solution are prepared according to 5 concentration of table
Take 1mL in test tube, addition 1mL sodium phosphate buffers (150mmol/L, pH=7.4), 1ml safron T (360 μ g/mL),
0.5mL EDTANa2-Fe(Ⅱ)-H2O2, 1mL 3%H2O2Solution, swirl mixing device mixing, whole system are anti-under 37 DEG C of constant temperature
After answering 30min, OD values at its 520nm are detected.Blank group：Isometric prepare liquid, control group are replaced with 1mL distilled water：It is steamed with 1mL
Distilled water replaces H2O2.Calculation formula：Clearance rate (%)=(ASample-ABlank)/(AControl-ABlank) × 100%
The clearance rate of 5 green onion albumen of table and VC to hydroxy radical (OH)
The experimental results showed that under various concentration, there is scavenging effect in green onion albumen and Vc to hydroxy radical (OH),
In 0.05-5mg/ml concentration ranges, with the increase green onion albumen of concentration to the clearance rate of hydroxy radical (OH) also therewith
Increase, in a concentration of 5mg/mL, concentration may be up to 90% or more, and compared with Vc, remote to hydroxy radical (OH) clearance rate
Higher than Vc.
In conclusion the preparation method of the present invention using green onion as raw material, by ungrease treatment, refluxing extraction, saltouts, dialyses
A kind of green onion albumen with antioxidant activity has been obtained afterwards.
Claims (8)
1. a kind of preparation method of green onion albumen, it is characterised in that the preparation method comprises the following steps：
(1) it pre-processes：Dry green onion is crushed, is filtered after ungrease treatment, collects filter residue；
(2) refluxing extraction：Filter residue in step (1) is taken, Extraction solvent heating and refluxing extraction, filtering, the extracting solution warp of collection is added
It crosses centrifugation and obtains supernatant；
(3) it saltouts：Supernatant in step (2) is taken, ammonium sulfate is added, is centrifuged after saltouing overnight and obtains albumen precipitation；
(4) it dialyses：Albumen precipitation in step (3) is taken, dissolves, is handled by bag filter, the trapped fluid of collection is by freeze-drying
As green onion albumen crude product.
2. a kind of preparation method of green onion albumen according to claim 1, it is characterised in that：In the step (1) according to
Solid-liquid ratio 1:The ratio of 6-8 is added petroleum ether and carries out ungrease treatment, and skimming temp is 40 DEG C, time 2h.
3. a kind of preparation method of green onion albumen according to claim 1, it is characterised in that：Filter residue in the step (2)
Solid-liquid ratio with Extraction solvent is 1:25-1:35, Extracting temperature is 40-60 DEG C, extraction time 90-150min.
4. a kind of preparation method of green onion albumen according to claim 1 or 3, it is characterised in that：It is carried in the step (2)
It is 0.01-0.03mol/L, the phosphate buffer solution of pH8-9 to take solvent.
5. a kind of preparation method of green onion albumen according to claim 4, it is characterised in that：Extraction in the step (2)
Solvent be 0.02mol/L, pH8 phosphate buffer solution, Extracting temperature be 60 DEG C, extraction time 150min, and filter residue and
The solid-liquid ratio of phosphate buffer solution is 1:35.
6. a kind of preparation method of green onion albumen according to claim 1, it is characterised in that：Centrifugation in the step (2)
When temperature be 4 DEG C, rotating speed 5000-6000r/min, centrifugation time 25-40min.
7. a kind of preparation method of green onion albumen according to claim 1, it is characterised in that：Sulfuric acid in the step (3)
The saturation degree of ammonium is 95%, is stood overnight at 4 DEG C.
8. a kind of preparation method of green onion albumen according to claim 1, it is characterised in that：Dialysis in the step (4)
The molecular cut off of bag is 3500Da-5000Da, and is first dialysed 24-48h with tap water when dialysing, then is dialysed with distilled water
24-48h。
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| CN107602663A (en) * | 2015-03-17 | 2018-01-19 | 上海应用技术大学 | A kind of tartary buckwheat bioactive peptide and its application |
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| CN101181581A (en) * | 2007-12-04 | 2008-05-21 | 中国人民解放军第二军医大学 | Shallot extract as well as preparation method and usage thereof |
| CN102697826A (en) * | 2012-06-18 | 2012-10-03 | 江苏省中国科学院植物研究所 | Gynura divaricata protein extract and preparation method and application thereof |
| CN107602663A (en) * | 2015-03-17 | 2018-01-19 | 上海应用技术大学 | A kind of tartary buckwheat bioactive peptide and its application |
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Application publication date: 20181026 |