CN104593337A - Orgotein extracted from beer yeast - Google Patents
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- CN104593337A CN104593337A CN201410854458.3A CN201410854458A CN104593337A CN 104593337 A CN104593337 A CN 104593337A CN 201410854458 A CN201410854458 A CN 201410854458A CN 104593337 A CN104593337 A CN 104593337A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention relates to orgotein extracted from beer yeast; the beer yeast is the key raw material in the beer production process; therefore, the beer yeast is widely used in the industrial field; and furthermore, the orgotein in the beer yeast or a waste beer yeast is abundant. The orgotein disclosed by the invention is prepared by the following steps of: homogenizing the beer yeast serving as the raw material at a high pressure by taking phosphate buffer solution with the pH of 6.5-6.7 and the concentration of 0.04-0.06mol/L as a homogenizing solvent so as to obtain crude extraction solution rich in orgotein; and eluting and purifying the orgotein by sequentially using phosphate buffer solution with the pH of 6.5-6.7 and the concentration of 0.02-0.03mol/L and citric acid buffer solution with the pH of 4.5-6.0 and the concentration of 0.02-0.03mol/L in the further treatment process of orgotein, concentrating and freeze-drying so as to obtain orgotein. The invention further relates to application of orgotein. The orgotein provided by the invention has high purity, excellent antioxidant activity and good application prospect in the field of cosmetics.
Description
Technical field
The present invention relates to the extraction of activated protein, be specifically related to a kind of Proteins, orgoteins extracted from cereuisiae fermentum.
Background technology
Proteins, orgoteins (Orgotein) is organized as raw material separation and Extraction and obtained a kind of water soluble protein by the liver of pig, ox, sheep and red corpuscle etc., is a kind of peptide chain macromole metalloenzyme.In field of biology, there is the effects such as anti-inflammatory, at present, be just applied to cosmetic field more and more.Proteins, orgoteins is present in organism more, and synthetic has great difficulty, therefore, select applicable extraction raw material and corresponding extracting method, obtain Proteins, orgoteins, thus solve Proteins, orgoteins carry out source problem to adapt to the demand a large amount of to it, be this area need at present solve problem.
The critical materials in beer process produced by cereuisiae fermentum, wherein containing a large amount of Proteins, orgoteins compositions.And, a large amount of waste beer yeast can be produced in the production process of beer, have a lot of manufacturing enterprise to recycle the waste beer yeast produced in beer production at present, beer waste beer yeast carried out purify, rejuvenation, after enlarged culturing, as raw material for extracting various active substance.In sum, Proteins, orgoteins rich content in cereuisiae fermentum or waste beer yeast, and there is a large amount of resources, be used to the desirable feedstock extracting Proteins, orgoteins.
Patent documentation CN1800377A discloses a kind of method that yeast saccharomyces cerevisiae thalline extraction purification obtains Cu/Zn superoxide dismutase.Described by the production process of yeast saccharomyces cerevisiae thalline extraction purification Cu/Zn superoxide dismutase is: yeast saccharomyces cerevisiae thalline is carried enzyme through broken wall, acetone precipitation, DEAE-32 cellulose chromatography obtains the enzyme of purification refine.Through comparing and studying known, what prior art provided extracts in the technique of Proteins, orgoteins from yeast, and acetone precipitation is still key link wherein.As known to those skilled in the art, as the acetone of organic solvent, have certain toxicity, do not meet current environmental protection trend, and acetone also can cause protein active to lose to a certain extent.
Summary of the invention
The present invention is intended to overcome the defect that prior art exists, and provides a kind of Proteins, orgoteins with excellent anti-oxidant activity extracted from cereuisiae fermentum, the abundant raw material source of described Proteins, orgoteins, extraction process is efficient, safety, environmental protection, using value is extensive.
The invention provides a kind of Proteins, orgoteins extracted from cereuisiae fermentum, described Proteins, orgoteins is prepared from by the method comprised the following steps:
1) be dissolved in phosphate buffered saline buffer by cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, and concentration is 0.04 ~ 0.06mol/L; High-pressure homogeneous under 1000 ~ 2000bar condition, circulate 5 ~ 10 times, centrifugal, get supernatant liquor; Add that ammonium sulfate is standing, centrifugal, collecting precipitation;
2) by step 1) gained is precipitated and dissolved in phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, concentration is 0.02 ~ 0.03mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) gained concentrated solution adds the affine capital end of the metal-chelating balanced, and carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 4.5 ~ 6.0, and concentration is 0.02 ~ 0.03mol/L; Collect the elutriant containing protein, concentrated, lyophilize, obtains Proteins, orgoteins.
Step 1 of the present invention) described in cereuisiae fermentum as extracting the raw material of Proteins, orgoteins, there is wide material sources, feature that biological safety is high; And Proteins, orgoteins content is higher in cereuisiae fermentum, be suitable for large-scale production Proteins, orgoteins.Described cereuisiae fermentum can be fresh cereuisiae fermentum, also can the waste beer yeast produced in beer production be recycled, conventionally beer waste beer yeast is carried out purifying, rejuvenation, after enlarged culturing, as raw material for extracting Proteins, orgoteins.
Step 1 of the present invention) in: described high-pressure homogeneous pressure is 1000 ~ 2000bar, and be preferably 1500bar, high-pressure homogeneous should circulation is carried out repeatedly, and to ensure extraction yield, cycle index is 5 ~ 10 times, is preferably 8 times; Described phosphate buffered saline buffer is potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, and the concentration of this damping fluid is preferably 0.05mol/L, and pH value is preferably 6.6; The mass volume ratio of cereuisiae fermentum and phosphate buffered saline buffer is 1:2 ~ 8, and be preferably 1:4, the mass unit of cereuisiae fermentum is g herein, and the volume unit of phosphate buffered saline buffer is ml; Described centrifugal temperature is 2 ~ 4 DEG C, and be preferably 3 DEG C, centrifugal speed is 1000 ~ 5000rpm, and be preferably 3000rpm, centrifugation time is 5 ~ 15min, is preferably 10min.
This step precipitates albumen after adding ammonium sulfate after obtaining supernatant liquor; Concrete employing twice classification ammonium sulfate precipitation, step is: be dissolved in supernatant liquor by ammonium sulfate with mass volume ratio 0.3 ~ 0.5:1, leaves standstill 30 ~ 60min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.7 ~ 0.9:1, leaves standstill 15 ~ 45min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000 conditions, collecting precipitation; The precipitation of twice being collected gained merges; The mass unit of ammonium sulfate is g herein, and the volume unit of crude extract is ml.
Step 2 of the present invention) adopt gel chromatography removing ammonium sulfate, the protein solution of not liquid containing ammonium sulfate can be obtained fast, the lengthy and tedious process avoiding conventional dialysis desalination method and the target product loss that may cause.Concrete, described phosphate buffered saline buffer is potassium primary phosphate-dipotassium hydrogen phosphate damping fluid; The concentration of damping fluid is preferably 0.025mol/L, and pH value is preferably 6.6; Step 3) gained precipitation be dissolved in phosphate buffered saline buffer with mass volume ratio 1:1 ~ 5, the mass unit herein precipitated is g, and the unit of phosphate buffered saline buffer volume is ml; Sephadex column selects Sephadex G-25, and the balance of gel column and the wash-out of moving phase are routine operation; Described concentrated preferably polyoxyethylene glycol method of enrichment, described polyoxyethylene glycol is preferably PEG4000, and enrichment step is routine operation.
Step 2 of the present invention) adopt ammonium sulfate in Nessler's reagent colorimetry qualification elutriant; The method is specially: get 1 elutriant, drops in white colorimetric disk hole, and add 1 Nessler's reagent mixing, if occur, brown color precipitates, and shows in elutriant containing ammonium sulfate.According to above-mentioned standard, step 2) after the target elutriant collected carries out this detection, should not occur that brown color precipitates.
Step 2 of the present invention) and step 3) in, adopt the protein in sulfosalisylic acid colorimetric method qualification elutriant; The method is specially: get 1 elutriant, drops in black colorimetric disk, adds 1 20% sulphosalicylic acid mixing, if there is white flock precipitate, shows in elutriant containing protein.According to above-mentioned standard, step 2) after the target elutriant collected carries out this detection, should white flock precipitate be there is.
Step 3 of the present invention) carry out protein purification by metal affinity chromatography method, the Proteins, orgoteins after purifying can be obtained fast, the loss of activity of Proteins, orgoteins can be avoided, and security is higher, more environmental protection; Specifically, in order to improve purification efficiency, in step 2) in gained concentrated solution, add 0.5 ~ 0.8mol/L NaCl, then add the affine capital end of the metal-chelating balanced; In this step, the metal ion of metal-chelating affine post institute chelating is selected from chelating metal ion conventional in the metal chelate affinity chromatographies such as copper, zinc, iron, nickel ion; The concentration of citrate buffer solution is preferably 0.025mol/L, and pH value is preferably 5.2; Described concentrated preferably polyoxyethylene glycol method of enrichment, described polyoxyethylene glycol is preferably PEG4000, and enrichment step is routine operation.
As most preferably scheme of the present invention, described Proteins, orgoteins is prepared from by the method comprised the following steps:
1) be dissolved in phosphate buffered saline buffer by cereuisiae fermentum with mass volume ratio 1:4, the pH value of described phosphate buffered saline buffer is 6.6, and concentration is 0.05mol/L; High-pressure homogeneous under 1500bar condition, circulate 8 times, at 3 DEG C, centrifugal 10min under 3000rpm condition, get supernatant liquor; Ammonium sulfate is dissolved in supernatant liquor with mass volume ratio 0.4:1, leaves standstill 45min, at 3 DEG C, centrifugal 20min under 8500rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.8:1, leaves standstill 30min, at 3 DEG C, centrifugal 20min under 8500 conditions, collecting precipitation; The precipitation of twice being collected gained merges;
2) by step 1) gained is precipitated and dissolved in phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.6, concentration is 0.025mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carries out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) add 0.6mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 5.2, and concentration is 0.025mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains Proteins, orgoteins.
Phosphate buffered saline buffer of the present invention is potassium primary phosphate-dipotassium hydrogen phosphate damping fluid.
In order to ensure the activity of the Proteins, orgoteins prepared, the service temperature of each step of the present invention is all preferably 0 ~ 4 DEG C.
Proteins, orgoteins provided by the invention is in blue or blue-greenish colour.
The present invention protects the described Proteins, orgoteins extracted from cereuisiae fermentum preparing the application in makeup further.Because Proteins, orgoteins provided by the invention has excellent anti-oxidant activity, therefore can as the desirable feedstock preparing makeup.
Compared with prior art, the Proteins, orgoteins productive rate that invention provides is high, purity is high, activity good, may be used for preparing makeup, has a good application prospect.And preparation technology's safety of this Proteins, orgoteins, environmental protection, abundant raw material source, is suitable for industrialization scale operation.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In various embodiments of the present invention, in elutriant, the discrimination method of ammonium sulfate is specially: get 1 elutriant, drops in white colorimetric disk hole, and add 1 Nessler's reagent mixing, if occur, brown color precipitates, and shows in elutriant containing ammonium sulfate; In elutriant, the discrimination method of protein is specially: get 1 elutriant, drops in black colorimetric disk, adds 1 20% sulphosalicylic acid mixing, if there is white flock precipitate, shows in elutriant containing protein.
Embodiment 1
Proteins, orgoteins is prepared from by following steps:
1) be dissolved in 400ml phosphate buffered saline buffer by 100g cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.6, and concentration is 0.05mol/L; High-pressure homogeneous under 1500bar condition, circulate 8 times, at 3 DEG C, centrifugal 10min under 3000rpm condition, get supernatant liquor; In supernatant liquor, add 187.6g ammonium sulfate, leave standstill 45min, at 3 DEG C, centrifugal 20min under 8500rpm condition, separation of supernatant, collecting precipitation; In supernatant liquor, add 300g ammonium sulfate, leave standstill 30min, at 3 DEG C, centrifugal 20min under 8500 conditions, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 5.17g;
2) by step 1) gained is precipitated and dissolved in 15ml phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.6, concentration is 0.025mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution 17ml;
3) in step 2) add 0.6mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 5.2, and concentration is 0.025mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains cyan powders 18.62mg.
Detect through proteins gel electrophoresis SDS-PAGE, products obtained therefrom molecular weight is identical with commercially available Proteins, orgoteins standard substance.
Embodiment 2
Proteins, orgoteins is prepared from by following steps:
1) be dissolved in 800ml phosphate buffered saline buffer by 100g cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.7, and concentration is 0.06mol/L; High-pressure homogeneous under 2000bar condition, circulate 10 times, at 4 DEG C, centrifugal 15min under 5000rpm condition, get supernatant liquor; In supernatant liquor, add 300g ammonium sulfate, leave standstill 60min, at 4 DEG C, centrifugal 30min under 9000rpm condition, separation of supernatant, collecting precipitation; In supernatant liquor, add 405g ammonium sulfate, leave standstill 45min, at 4 DEG C, centrifugal 30min under 9000 conditions, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 4.76g;
2) by step 1) gained is precipitated and dissolved in 23ml phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.7, concentration is 0.03mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution 25ml;
3) in step 2) add 0.8mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 6.0, and concentration is 0.03mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains cyan powders 15.22mg.
Embodiment 3
Proteins, orgoteins is prepared from by following steps:
1) be dissolved in 200ml phosphate buffered saline buffer by 100g cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.5, and concentration is 0.04mol/L; High-pressure homogeneous under 1000bar condition, circulate 5 times, at 2 DEG C, centrifugal 5min under 1000rpm condition, get supernatant liquor; In supernatant liquor, add 105g ammonium sulfate, leave standstill 30min, at 2 DEG C, centrifugal 15min under 8000rpm condition, separation of supernatant, collecting precipitation; In supernatant liquor, add 175g ammonium sulfate, leave standstill 15min, at 2 DEG C, centrifugal 15min under 8000 conditions, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 4.49g;
2) by step 1) gained is precipitated and dissolved in 8ml phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.5, concentration is 0.02mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution 5ml;
3) in step 2) add 0.5mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 4.5, and concentration is 0.02mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains cyan powders 13.87mg.
Comparative example 1
Compared with embodiment 1, difference is only, described step 2) replace in order to lower operation: by step 3) gained is precipitated and dissolved in pH6.6,0.025mol/L phosphate buffer 1 5ml, be placed in dialysis tubing, conventionally ammonium sulfate is removed in dialysis, changes a dialysis buffer liquid every 5 hours, after 50 hours, not containing ammonium sulfate in solution, concentrate with PEG4000, obtain concentrated solution.
Products therefrom is cyan powders 8.67mg.
Comparative example 2
Compared with embodiment 1, difference is only, step 3) described citrate buffer solution pH6.6,0.025mol/L phosphate buffered saline buffer replacement.
Products therefrom is white powder 15.98mg.
Experimental example: Proteins, orgoteins detects
1, sample is detected: embodiment 1 ~ 3 and comparative example 1,2 products therefrom, commercially available Proteins, orgoteins standard substance.
2, purity detecting: with C18 post for stationary phase, with the acetonitrile-water containing 0.3% trifluoroacetic acid for moving phase, adopts reversed-phased high performace liquid chromatographic, conveniently operate, reference standard product go out peak situation, determine the target absorption peak of sample, adopt peak area method counting yield purity.
3, Activity determination: because Proteins, orgoteins can remove superoxide anion, superoxide anion is produced by xanthine and XOD reactive system, nitroblue tetrazolium(NBT) is reduced to blue formazan, the latter has strong absorption at 560nm place, and Proteins, orgoteins can suppress the formation of formazan; Reaction solution blueness is darker, illustrate that the activity of Proteins, orgoteins is lower, otherwise then activity is higher, just can calculate the activity level of Proteins, orgoteins accordingly by colorimetric analysis.Concrete operation step, refers to the specification sheets that superoxide-dismutase test kit detects (total number born activity detection kit (NBT method), the green skies, Haimen City biotechnology research institute, production code member S0109) this test kit.
4, the purity of Proteins, orgoteins and the detected result of enzymic activity are in table 1; The activity of Proteins, orgoteins represents by specific activity (U/mg) herein.
Table 1: Proteins, orgoteins detected result
| Sample | Purity (%) | Specific activity (U/mg) |
| Embodiment 1 | 90.36 | 5013.8 |
| Embodiment 2 | 88.76 | 4937.5 |
| Embodiment 3 | 89.03 | 4956.8 |
| Comparative example 1 | 85.15 | 4683.5 |
| Comparative example 2 | 61.3 | 4068.9 |
| Standard substance | -- | 4967.4 |
From above detected result, the purity of the Proteins, orgoteins that method provided by the invention prepares all is greater than 88%, and be significantly better than comparative example 2, although comparative example 1 is on the low side close to product yield (mass ratio of product and raw material cereuisiae fermentum) more each embodiment with each embodiment purity; Proteins, orgoteins specific activity provided by the invention is all greater than 4900U/mg, and is not less than the specific activity of standard substance, is better than comparative example 1, and is significantly better than comparative example 2.In each embodiment, the bulk properties of embodiment 1 supplying method products obtained therefrom is best.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1. the Proteins, orgoteins extracted from cereuisiae fermentum, is characterized in that, described Proteins, orgoteins is prepared from by the method comprised the following steps:
1) be dissolved in phosphate buffered saline buffer by cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, and concentration is 0.04 ~ 0.06mol/L; High-pressure homogeneous under 1000 ~ 2000bar condition, circulate 5 ~ 10 times, centrifugal, get supernatant liquor; Add that ammonium sulfate is standing, centrifugal, collecting precipitation;
2) by step 1) gained is precipitated and dissolved in phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, concentration is 0.02 ~ 0.03mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) gained concentrated solution adds the affine capital end of the metal-chelating balanced, and carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 4.5 ~ 6.0, and concentration is 0.02 ~ 0.03mol/L; Collect the elutriant containing protein, concentrated, lyophilize, obtains Proteins, orgoteins.
2. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, is characterized in that, step 1) concentration of described phosphate buffered saline buffer is 0.05mol/L, pH value is 6.6.
3. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, it is characterized in that, step 1) described in add that ammonium sulfate is standing, centrifugal, collecting precipitation is specially: ammonium sulfate is dissolved in supernatant liquor with mass volume ratio 0.3 ~ 0.5:1, leave standstill 30 ~ 60min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.7 ~ 0.9:1, leaves standstill 15 ~ 45min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000 conditions, collecting precipitation; The precipitation of twice being collected gained merges.
4. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, is characterized in that, step 2) concentration of described phosphate buffered saline buffer is 0.025mol/L, pH value is 6.6.
5. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, is characterized in that, step 3) concentration of described citrate buffer solution is 0.025mol/L, pH value is 5.2.
6. the Proteins, orgoteins extracted from cereuisiae fermentum according to Claims 1 to 5 any one, is characterized in that, step 3) further comprising the steps of: in step 2) in gained concentrated solution, add 0.5 ~ 0.8mol/L NaCl.
7. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, is characterized in that, described Proteins, orgoteins is prepared from by the method comprised the following steps:
1) be dissolved in phosphate buffered saline buffer by cereuisiae fermentum, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, and concentration is 0.04 ~ 0.06mol/L; High-pressure homogeneous under 1000 ~ 2000bar condition, circulate 5 ~ 10 times, at 2 ~ 4 DEG C, centrifugal 5 ~ 15min under 1000 ~ 5000rpm condition, get supernatant liquor; Ammonium sulfate is dissolved in supernatant liquor with mass volume ratio 0.3 ~ 0.5:1, leaves standstill 30 ~ 60min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.7 ~ 0.9:1, leaves standstill 15 ~ 45min, at 2 ~ 4 DEG C, centrifugal 15 ~ 30min under 8000 ~ 9000 conditions, collecting precipitation; The precipitation of twice being collected gained merges;
2) by step 1) gained is precipitated and dissolved in phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.5 ~ 6.7, concentration is 0.02 ~ 0.03mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carry out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) add 0.5 ~ 0.8mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 4.5 ~ 6.0, and concentration is 0.02 ~ 0.03mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains Proteins, orgoteins.
8. the Proteins, orgoteins extracted from cereuisiae fermentum according to claim 1, is characterized in that, described Proteins, orgoteins is prepared from by the method comprised the following steps:
1) be dissolved in phosphate buffered saline buffer by cereuisiae fermentum with mass volume ratio 1:4, the pH value of described phosphate buffered saline buffer is 6.6, and concentration is 0.05mol/L; High-pressure homogeneous under 1500bar condition, circulate 8 times, at 3 DEG C, centrifugal 10min under 3000rpm condition, get supernatant liquor; Ammonium sulfate is dissolved in supernatant liquor with mass volume ratio 0.4:1, leaves standstill 45min, at 3 DEG C, centrifugal 20min under 8500rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.8:1, leaves standstill 30min, at 3 DEG C, centrifugal 20min under 8500 conditions, collecting precipitation; The precipitation of twice being collected gained merges;
2) by step 1) gained is precipitated and dissolved in phosphate buffered saline buffer, the pH value of described phosphate buffered saline buffer is 6.6, concentration is 0.025mol/L, gained solution is added the sephadex column Sephadex G-25 top balanced, carries out wash-out with phosphate buffered saline buffer described in this step; Collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) add 0.6mol/L NaCl in gained concentrated solution and dissolve, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with citrate buffer solution, the pH value of described citrate buffer solution is 5.2, and concentration is 0.025mol/L; Collect the elutriant containing protein, concentrate with PEG4000, lyophilize, obtains Proteins, orgoteins.
9. the Proteins, orgoteins extracted from cereuisiae fermentum described in claim 1 ~ 8 any one, is preparing the application in makeup.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471084A (en) * | 2019-01-23 | 2020-07-31 | 中国科学院青岛生物能源与过程研究所 | Device for protein precipitation and method for separating and purifying target protein from protein mixed solution |
| CN117683102A (en) * | 2024-02-02 | 2024-03-12 | 广州华淼生物科技研究院有限公司 | Yeast protein and extraction method and application thereof |
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