Embodiment
Following examples are more of the present invention giving an example, and should not be seen as limitation of the invention.
Embodiment 1
A kind of method of preparing polypeptide active substance from enzymolysis ginkgo protein is comprised of following steps:
The first step, preparation degreasing gingko powder: with fresh ginkgo shelling, except the clothing skin, pull an oar into paste with hollander at lower than the temperature of 20 ℃, for example temperature can be chosen for 19 ℃, 18 ℃, 14 ℃, 10 ℃, 8 ℃, 6 ℃, 5 ℃, 4 ℃ ,-2 ℃ ,-6 ℃ ,-8 ℃, then with non-polar solvent or supercritical CO
2Oil and grease extracting, then at-10 ℃--50 ℃ of lyophilizes, for example lyophilize 6-50 hour, time can be 20 hours, 40 hours, and the quick moisture determination of infrared rays, the weight percentage that obtains moisture less than 5%, fragmentation, sieve, obtaining granularity is 100~200 purpose degreasing gingko powder;
second step, enzymic hydrolysis degreasing gingko powder: be mixing in 1: 5~1: 15 in mass ratio with described degreasing gingko powder and deionized water, for example can be chosen for 1: 8, 1: 9, 1: 11, 1: 14, add wherein again enzyme, the interpolation quality of enzyme is 5%~20% of described degreasing gingko powder, for example can be chosen for 7%, 9%, 13%, 15%, 16%, 18%, heating is under 30~60 ℃ mixture temperature, for example temperature can be chosen for 35 ℃, 45 ℃, 55 ℃, adjusting makes its pH value be in 7~10, stirring reaction 3~48h, for example be chosen as 4 hours, 15 hours, 20 hours, 40 hours, centrifugation after hydrolysis, collect supernatant liquor,
The 3rd step membrane sepn enzymolysis solution, molecular weight cut-off is 3000-10000 dalton polypeptide class actives, is obtaining concentrated enzymolysis solution lower than 30 ℃ of vacuum concentration; As elect-5 ℃ as, and-2 ℃, 0 ℃, 5 ℃, 8 ℃, 12 ℃, 18 ℃, 20 ℃, 25 ℃,
The purifying of the 4th step polypeptide: be 50~90% ethanol or acetone soln and described concentrated enzymolysis solution 1-10 by volume with mass percent: 1 mix and blend, for example can be chosen for: 2: 1,4: 1,7: 1,9: 1, the precipitation polysaccharose substance, centrifuging makes clear liquid, add reagent, described reagent is trichoroacetic acid(TCA), acetone, (NH again
4)
2SO
4In any or its mixture, described reagent and described clear liquid volume ratio 1-10: 1, for example can be chosen for: 2: Isosorbide-5-Nitrae: 1,8: 1,9: 1, and mix and blend, precipitation obtains polypeptide class actives;
The preparation of the 5th step gingko polypeptide powder: the described polypeptide class actives with the 4th step obtained, be precipitated with acetone, ethanol, methanol wash successively, be deposited in-10 ℃--50 ℃ of lyophilizes, for example can be chosen for-20 ℃,-40 ℃ ,-45 ℃, obtain the gingko polypeptide powder.
In technique scheme, polypeptide class actives molecular weight is 3000-10000 dalton, and the quality percentage composition of polypeptide is 50~80%, and the quality percentage composition of contained humidity lower than 5%, is for example 3%, 2%, granularity 100~200 orders; Wherein in polypeptide, lysine content is greater than 7%, and Histidine is greater than 8%, and Serine is greater than 11%, and aspartic acid is greater than 14%, and L-glutamic acid is greater than 16%.Described content is the quality percentage composition.
in technique scheme, enzyme is active papoid greater than 800,000 u/g, for example activity is 900,000 u/g, 950,000 u/g, active bacillus alkaline protease greater than 200,000 u/g, for example activity is 350,000 u/g, for example activity is 400,000 u/g, active neutral protease greater than 150,000 u/g, for example activity is 200,000 u/g, for example activity is 300,000 u/g, active cellulase greater than 1,000,000 u/g, for example activity is 1,200,000 u/g, for example activity is 1,400,000 u/g, in active polygalacturonase greater than 800,000 u/g, 900,000 u/g for example, 1,000,000 u/g, the mixture of above-mentioned one or more, if mixture, it can be arbitrary proportion.
Described non-polar solvent be in normal hexane, ether, sherwood oil, No. 6 solvent oils any, with the liquid-solid ratio of degreasing gingko powder be 1-20: 1, wherein liquid measures with milliliter, solid measures with gram.Described supercritical CO
2Extracting pressure is 10-30MPa, and extraction temperature is 25-50 ℃, and for example 35 ℃, 40 ℃, extraction time is 1-4h, for example is chosen for 2 hours, 3 hours, and CO used
2Flow is 10-30L/h, for example is chosen for 20L/h, and the functional quality percentage concentration be the 1%-15% sherwood oil as entrainment agent, for example mass concentration is 8%, 12%.
Embodiment 2 ginkgo protein enzymolysis prepare polypeptide class actives
A kind of method of efficient preparing polypeptide active substance from enzymolysis ginkgo protein comprises the following steps: the first step prepares degreasing gingko powder: fresh gingko 100g-200g is shelled, can select 100g, 120g, 150g, 180g, 200g except the clothing skin, is 5 ℃ with hollander, 10 ℃, 12 ℃, 15 ℃, 18 ℃, pull an oar into paste under 20 ℃, then with non-polar solvent or supercritical CO
2Oil and grease extracting, at-10 ℃--50 ℃ of lyophilizes, preferably-20 ℃--40 ℃, for example be chosen for-25 ℃ ,-30 ℃,-40 ℃, preparation degreasing gingko powder, the quick moisture determination of infrared rays, moisture<5% (mass percent), broken, sieve, obtain granularity 100~200 orders, protein content 11.3% (mass percent) degreasing gingko powder;
Second step enzymic hydrolysis degreasing gingko powder: be mixing in 1: 5~1: 15 by degreasing gingko powder and deionized water quality ratio, the enzyme addition is 5%~20% of degreasing gingko powder raw material, and is preferred 5%~8%, 30~60 ℃ of temperature, preferred 40 ℃, 50 ℃, pH=7~10, preferred pH=8 and pH=9, stirring reaction time 3~48h, preferred 4-8h, supernatant liquor is collected in centrifugation after hydrolysis, and degree of hydrolysis (DH%) is greater than 70%;
The 3rd step membrane sepn enzymolysis solution:
The cellulose membrane of the centrifugal enzymolysis clear liquid that obtains of second step through 0.45 μ m filtered, select molecular weight 3,000 dalton, 5,000 dalton, 10, the filtrate of 000 dalton, 30,000 daltonian polysulfone membrane fractional separation cellulose membranes, molecular weight cut-off are 3000-10000 dalton polypeptide class actives, at vacuum concentration below 30 ℃, preferred-5 ℃--40 ℃ of vacuum freezings are to 50ml-100ml;
The purifying of the 4th step polypeptide
With 50~90% ethanol or acetone and membrane sepn polypeptide concentrated solution 1-10 by volume: 1 mix and blend, the precipitation polysaccharose substance, centrifuging makes clear liquid.Select TCA (trichoroacetic acid(TCA)), acetone, (NH
4)
2SO
4Three kinds of reagent is any or with the 1-10 by volume of array mode arbitrarily: mix and blend in 1 clear liquid that adds after centrifugal, precipitation polypeptide class actives;
The preparation of the 5th step gingko polypeptide powder
With the polypeptide class actives of the 4th step precipitation, use successively acetone, ethanol, methanol wash by solid-to-liquid ratio 1: 1-30 (g/ml), throw out is at-10 ℃--and 50 ℃ of lyophilizes obtain the gingko polypeptide powder.
In the present embodiment, enzyme is selected papoid (>80 ten thousand u/g), bacillus alkaline protease (>20 ten thousand u/g), any mixture of one or more in neutral protease (>15 ten thousand u/g), cellulase (>100 ten thousand u/g), polygalacturonase (>80 ten thousand u/g).Non-polar solvent is selected in normal hexane, ether, sherwood oil, No. 6 solvent oils any, with the liquid-solid ratio (ml/g) of degreasing gingko powder be 1-20: 1.Supercritical CO
2Extracting pressure 10-30MPa, extraction temperature 25-50 ℃, extraction time 1-4h, CO
2Flow 10-30L/h, 1%-15% sherwood oil are as entrainment agent, and in preparation gingko grease, lipid acid>85% is closed in insatiable hunger.In the present embodiment, preparation polypeptide class actives molecular weight is 3000-10000 dalton, and content of peptides is 75%, moisture<5%, granularity 100~200 orders.And polypeptide contains 17 seed amino acids, and wherein aspartic acid 14.26%, L-glutamic acid 16.89%, Serine 11.32%, Histidine 8.02%, glycine 4.56%, Threonine 5.96%, arginine 6.54%, L-Ala 7.41%, tyrosine 2.25%, Gelucystine 0.21%, α-amino-isovaleric acid 4.57%, methionine(Met) 1.92%, phenylalanine 1.97%, Isoleucine 2.27%, leucine 3.15%, Methionin 7.70%, proline(Pro) 1.01%.
Embodiment 3 ginkgo protein enzymolysis prepare polypeptide class actives
With the 100g ginkgo shelling, except the clothing skin, pull an oar into paste with hollander under 15 ℃, with normal hexane equal-volume extraction 3-5 time, eliminate grease again, water layer is at-35 ℃ of lyophilize 40h, be broken into granularity 100~200 order degreasing gingko powder after drying, the infrared rays Fast Measurement, moisture 4.45%, protein content 11.8%.
Take degreasing gingko powder 20g, add deionized water 100ml, add enzyme activity 400,000 u/g neutral protease 2g, regulate pH=8, be hydrolyzed 5h under temperature 50 C, centrifugal after hydrolysis, collect supernatant liquor.Supernatant liquor is through the cellulose membrane of 0.45 μ m, and filtrate is first through the polysulfone membrane of 10,000MW, see through liquid through the polysulfone membrane of 3,000MW, trapped fluid-40 ℃ freeze concentration is to 50ml, add 250ml70% ethanol, the precipitation Crude polysaccharides, centrifugal, it is in the TCA (trichoroacetic acid(TCA))/acetone mixed solution 200ml of 1: 9 that supernatant concentration adds volume ratio to 50ml, the precipitation polypeptide, centrifugal, throw out is washed the impurity such as most moisture content again with acetone, and insolubles makes polypeptide dry powder-25 ℃ of lyophilizes.Take the tetrapeptide standard substance as check analysis, in polypeptide dry powder, content of peptides is 70%, and wherein aspartic acid 15.6%, L-glutamic acid 17.2%, Serine 11.5%, Histidine 8.7%, glycine 3.7%, Threonine 5.9%, arginine 7.4%, L-Ala 7.8%, tyrosine 1.4%, α-amino-isovaleric acid 6.4%, Isoleucine 2.8%, leucine 3.7%, Methionin 7.8%.
Embodiment 4 ginkgo protein enzymolysis prepare polypeptide class actives
With the 200g ginkgo shelling, except the clothing skin, pull an oar into paste with hollander under 15 ℃, with ether equal-volume extraction 3-5 time, eliminate grease again, water layer is at-35 ℃ of lyophilize 40h, be broken into granularity 100~200 order degreasing gingko powder after drying, the infrared rays Fast Measurement, moisture 4.45%, protein content 11.8%.
Take degreasing gingko powder 30g, add deionized water 280ml, add enzyme activity 1,200,000 u/g papoid 3g, regulate pH=9, be hydrolyzed 6h under temperature 45 C, centrifugal after hydrolysis, collect supernatant.Supernatant liquor is through the cellulose membrane of 0.45 μ m, and filtrate is first through the polysulfone membrane of 10,000MW, see through liquid through the polysulfone membrane of 3,000MW, trapped fluid-40 ℃ freeze concentration is to 50ml, add 250ml80% acetone, the precipitation Crude polysaccharides, centrifugal, supernatant concentration is to 50ml, add 10%-15%TCA (trichoroacetic acid(TCA)) precipitation polypeptide, centrifugal, throw out is washed the impurity such as most moisture content again with acetone, and insolubles makes polypeptide dry powder-35 ℃ of lyophilizes.Take the tetrapeptide standard substance as check analysis, in polypeptide dry powder, content of peptides is 58%, and wherein aspartic acid 15.6%, L-glutamic acid 17.2%, Serine 11.5%, Histidine 8.7%, glycine 3.7%, Threonine 5.9%, arginine 7.4%, L-Ala 7.8%, tyrosine 1.4%, α-amino-isovaleric acid 6.4%, Isoleucine 2.8%, leucine 3.7%, Methionin 7.8%.
Embodiment 5 ginkgo protein enzymolysis prepare polypeptide class actives
With the 200g ginkgo shelling, except the clothing skin, pull an oar into paste with hollander under 15 ℃, with sherwood oil equal-volume extraction 3-5 time, eliminate grease again, water layer is at-35 ℃ of lyophilize 40h, be broken into granularity 100~200 order gingko powder after drying, the infrared rays Fast Measurement, moisture 4.45%, protein content 11.8%.
Take degreasing gingko powder 30g, add deionized water 280ml, add enzyme activity 250,000 u/g bacillus alkaline protease 1g, 1,000,000 u/g cellulase 1g, 800,000 u/g polygalacturonase 1g regulate pH=9, are hydrolyzed 6h under temperature 45 C, and are centrifugal after hydrolysis, collect supernatant.Supernatant liquor is through the cellulose membrane of 0.45 μ m, and filtrate is first through the polysulfone membrane of 10,000MW, see through liquid through the polysulfone membrane of 3,000MW, trapped fluid-40 ℃ freeze concentration is to 50ml, add 250ml80% acetone, the precipitation Crude polysaccharides, centrifugal, supernatant concentration is to 50ml, add 70% ammoniumsulphate soln mixed precipitation polypeptide, centrifugal, throw out is washed the impurity such as most moisture content again with acetone, and insolubles makes polypeptide dry powder-35 ℃ of lyophilizes.Take the tetrapeptide standard substance as check analysis, in polypeptide dry powder, content of peptides is 65%, and wherein aspartic acid 15.6%, L-glutamic acid 17.2%, Serine 11.5%, Histidine 8.7%, glycine 3.7%, Threonine 5.9%, arginine 7.4%, L-Ala 7.8%, tyrosine 1.4%, α-amino-isovaleric acid 6.4%, Isoleucine 2.8%, leucine 3.7%, Methionin 7.8%.
The overcritical preparation degreasing of embodiment 6 gingko powder and Ginkgo oil
With the 300g ginkgo shelling, except the clothing skin, pull an oar into paste with hollander under 15 ℃, at-35 ℃ of lyophilize 40h, be broken into granularity 100 order gingko powder after drying.Weighing gingko powder 100g puts into 1L CO
2Extract extracting pressure 10-30MPa, extraction temperature 25-50 ℃, extraction time 1-4h, CO in the supercritical extraction tank
2Flow 10-30L/h,, optimum process condition is 40 ℃ of temperature, pressure 20MPa, flow velocity 15L/h, time 3h, the Ginkgo oil yield can reach 7.11%.CO
2Gingko powder after supercritical extraction is broken into granularity 100~200 order gingko powder, infrared rays Fast Measurement, moisture 4.45%, protein content 11.8% at-35 ℃ of lyophilize 20h after drying.
The GC-MS Analysis for CO
2The Ginkgo oil chemical composition of supercritical extraction shows: in Ginkgo oil, the unsaturated fatty acids total amount can reach 85.2%, and wherein 9,12-C
18:2Linolic acid accounts for 32%, 8-C
18:1Oleic acid accounts for 21%; 9-C
18:1Oleic acid accounts for 20%.Ginkgo oil has following physical features: refractive index (n
40): 1.460~1.468; Relative density (d): 0.922~0.929; Iodine number (I) is (g/100g): 95~115; Saponification value (KOH) is (mg/g): 187~196; Unsaponifiables (g/kg) :≤15.Color and luster (Lovibond cell 133.4mm)≤Huang 70 red 4.0; Moisture and volatile matter content/(%)≤0.2; Insoluble impurities/(%)≤0.2; Acid number (KOH)/(mg/g)≤4.0; Heat test (280 ℃).
The overcritical preparation degreasing of embodiment 7 gingko powder and Ginkgo oil
The present embodiment purpose is to obtain a seed amino acid to form abundant, rational polypeptide from the gingko powder, wherein polypeptide class actives molecular weight is 3000-10000 dalton, content of peptides is 50~80%, and the contained humidity mass percent is less than 5%, granularity 100~200 orders.Polypeptide contains 8 kinds of indispensable amino acids of human body, while lysine content>7%, Histidine>8%, Serine>11%, aspartic acid>14%, L-glutamic acid>16%.Especially the content of aspartic acid, L-glutamic acid, Serine and Methionin is apparently higher than the gingko raw material, for new approach is found in the deep processing of gingko.
The method that adopts is comprised of following steps:
The first step prepares degreasing gingko powder
Fresh gingko 100g-200g is shelled, except the clothing skin, pulling an oar into paste with hollander lower than 20 ℃, then with non-polar solvent or supercritical CO
2Oil and grease extracting, at-10 ℃--50 ℃ of lyophilizes, the infrared rays Fast Measurement, moisture<5%,, granularity 100~200 orders;
Second step enzymic hydrolysis degreasing gingko powder
Be mixing in 1: 5~1: 15 by degreasing gingko powder and deionized water quality ratio, the enzyme addition is 5%~20% of degreasing gingko powder raw material, 30~60 ℃ of temperature, pH=7~10, stirring reaction time 3~48h, centrifugation after hydrolysis, collect supernatant liquor, degree of hydrolysis (DH%) is greater than 70%;
The 3rd step membrane sepn enzymolysis solution
The cellulose membrane of the centrifugal enzymolysis clear liquid that obtains of second step through 0.45 μ m filtered, select molecular weight 3,000 dalton, 5,000 dalton, 10,000 dalton, 30, the filtrate of 000 daltonian polysulfone membrane fractional separation cellulose membrane, molecular weight cut-off are 3000-10000 dalton polypeptide class actives, at vacuum concentration below 30 ℃ to 50ml-100ml;
The purifying of the 4th step polypeptide
With 50~90% ethanol or acetone and membrane sepn polypeptide concentrated solution 1-10 by volume: 1 mix and blend, the precipitation polysaccharose substance, centrifuging makes clear liquid.Select TCA (trichoroacetic acid(TCA)), acetone, (NH
4)
2SO
4Three kinds of reagent is any or with the 1-10 by volume of array mode arbitrarily: mix and blend in 1 clear liquid that adds after centrifugal, precipitation polypeptide class actives;
The preparation of the 5th step gingko polypeptide powder
With the polypeptide class actives of the 4th step precipitation, use successively acetone, ethanol, methanol wash by solid-to-liquid ratio 1: 1-30 (g/ml), throw out obtains the gingko polypeptide powder-10 ℃ of-50 ℃ of lyophilizes.
The present invention does not degrade for the quality that guarantees gingko and protein and polypeptide, with water content in the shelling of the fresh gingko of 50%-65% with except after the clothing skin, employing is lower than 20 ℃ of making beating and cryodesiccated method, at-10 ℃ extremely--50 ℃ of lyophilizes, freezing time 6-50 hour, preferably-15 ℃--40 ℃, freezing time 10-40 hour, water content<6%.If do not adopt the art of this patent to process, higher than 50%, issue the metabolism of biochemical composition due to the water content of fresh gingko in the effect of self enzyme, enzyme liberating, easy moldy metamorphism, the nutritive ingredients such as protein and grease are destroyed.The easy enzymolysis of ginkgo protein produces peculiar smell, also easily goes mouldy, and has influence on the local flavor of gingko.If adopt heated drying or Exposure to Sunlight to process, the original volatile component of gingko easily loses, and grease easily produces and becomes sour simultaneously.If 190 ℃ of-220 ℃ of lower spraying dryings, obtain water content at the powder of 10% left and right; Due to through pyroprocessing, ginkgo protein, polypeptide, the sex change of enzyme isoreactivity thing can not keep the structure of original proteins and peptides.And experiment shows protein content 11.9% in lyophilize gingko powder, greater than protein content 9.7% in spraying drying gingko powder.
Contain the fat-soluble ginkgotoxins such as hydrocyanic acid, ginkgolic acid, Hydroginkgolic acid and bilobol due to gingko, easily cause untoward reaction, affect people's neural system, Digestive tract, integumentary system and hemopoietic system.Therefore, the people of some responsive physique easily occurs irritated to ginkgotoxin, occur various types of dermatitis fash after food; After the edible gingko of somebody, the gastrointestinal discomfort symptom can appear, as situations such as nauseating, vomiting, stomachache, diarrhoea.In addition, gingko contains the grease of 4%-10%, is mainly wherein unsaturated fatty acids, and to cause that easily oyster loses rotten due to unsaturated fatty acids, and the existence of grease simultaneously affects proteolysis and becomes polypeptide.In order to remove the gingko grease, this patent adopted with non-polar solvent or supercritical CO before preparation gingko powder
2Then oil and grease extracting becomes degreasing gingko powder-10 ℃ to-50 ℃ lyophilizes.
This patent non-polar solvent is selected in normal hexane, ether, sherwood oil, No. 6 solvent oils any, with the liquid-solid ratio (ml/g) of degreasing gingko powder be 1-20: 1, preferred 10-15: 1, continuous extraction 3-6 time, residuum becomes degreasing gingko powder-10 ℃ to-50 ℃ lyophilizes.This patent adopts supercritical CO simultaneously
2Oil and grease extracting, supercritical CO
2Extracting pressure 10-30MPa, extraction temperature 25-50 ℃, extraction time 1-4h, CO
2Flow 10-30L/h, 1%-15% sherwood oil be as entrainment agent, and preparation gingko grease unsaturated fatty acid content is brought up to more than 85%, and wherein 9,12-C
18:2Linolic acid accounts for 31.876%, 8-C
18:1Oleic acid accounts for 20.360%; 9-C
18:1Oleic acid accounts for 19.345%.As table 1.
The chemical constitution of table 1 supercritical extraction gingko powder
The present embodiment discloses the method that enzymolysis ginkgo protein matter prepares the gingko polypeptide, enzyme is selected papoid (>80 ten thousand u/g), bacillus alkaline protease (>20 ten thousand u/g), neutral protease (>15 ten thousand u/g), cellulase (>100 ten thousand u/g)), one or more any mixture in polygalacturonase (>80 ten thousand u/g), preferred neutral protease, papoid and Sumizyme MP.Degreasing gingko powder and deionized water quality ratio are mixing in 1: 5~1: 15, and the enzyme addition is 5%~20% of degreasing gingko powder raw material, 30~60 ℃ of temperature, pH=7~10, stirring reaction time 3~48h, centrifugation after hydrolysis, collect supernatant liquor, degree of hydrolysis (DH%) is greater than 65%.By investigating the impact of the factors such as hydrolysis temperature, pH value, enzyme substrates concentration, enzyme dosage, time, by Design expert experimental analysis software design, adopt Box-Behnken model (seeing Table 2), design 3 factor 3 level optimization experiments (seeing Table 3), the Surface Method that meets with a response analytical model R
2=0.8521, F value is 4.48, P value 0.0303, the model highly significant, and the optimum enzymolysis condition that obtains on this model basis is: time 4-10h, enzyme dosage 2g, pH are 9, and hydrolysis temperature is 45-50 ℃, and concentration of substrate is 2.17%.The present invention adopts formol titration to measure its amino acid nitrogen content, the degree of hydrolysis (DH%) of protein when calculating enzymolysis according to formula (1), and degree of hydrolysis (DH%) is greater than 70% with this understanding.
V
1-alkali-titration initial value; V
2-alkali-titration end point values; TN---total nitrogen content, mg;
Table 2Box-Behnken experimental design factor and level code value
Adopt the Design Expert 7.1.3.1 of statistical software to carry out the multiple regression match to experimental data, model analysis shows that secondary model (Quadratic) is best fit model, and the results of analysis of variance of this model sees Table 4.
Regression diagnostics shows, coefficient of determination R-Squared=81.01%, and signal to noise ratio (Adeq-Precision)=7.498>4 illustrates that the degree of fitting of equation and confidence level are all very high, can be used for the degree of hydrolysis of pre-measured reaction.The tolerance range of C.V.% (variation coefficient of Y) expression experiment, the C.V.% value is higher, and the reliability of experiment is lower, and C.V.=14.65% in this patent contrived experiment is lower, and the illustrative experiment operation is credible.Therefore regression equation provides a model to the enzymolysis process of protein.
DH%=599.51725-229.33925 * A+16.46370 * B-12.04075 * C+2.509 * A * B+6.05250 * A * C-0.594 * B * C+7.12425 * A2-0.40213 * B2-3.35575 * C2, A-pH wherein, B-temperature, C-concentration of substrate.
Table 3 experimental design and result
The enzymolysis clear liquid filters through the cellulose membrane of 0.45 μ m, this patent adopts the centrifugal enzymolysis clear liquid that obtains of polysulfone membrane stage treatment of different molecular weight, select molecular weight 3,000 dalton, 5,000 dalton, 10,000 dalton, 30,000 daltonian polysulfone membrane, molecular weight cut-off are 3000-10000 dalton polypeptide class actives, at vacuum concentration below 30 ℃, preferably-5 ℃--40 ℃ of vacuum freezings can keep the activity of polypeptide better to 50ml-100ml.Select 50~90% ethanol or acetone, preferred 70~80% ethanol or acetone, and membrane sepn polypeptide concentrated solution 1-10 by volume: 1 mix and blend, preferred 3-6: 1, the precipitation polysaccharose substance, centrifuging makes clear liquid.Select again TCA (trichoroacetic acid(TCA)), acetone, (NH
4)
2SO
4Three kinds of reagent is any or with the 1-10 by volume of array mode arbitrarily: mix and blend in 1 clear liquid that adds after centrifugal, precipitation polypeptide class actives; Use successively acetone, ethanol, methanol wash by solid-to-liquid ratio 1: 1-30 (g/ml), preferred solid-to-liquid ratio 1: 1-10 (g/ml) throw out is at-10 ℃--50 ℃ of lyophilizes, obtain the gingko polypeptide powder, wherein polypeptide class actives molecular weight is 3000-10000 dalton, content of peptides is 50~80%, moisture<5%, granularity 100~200 orders.
The present embodiment adopts biuret method, measures content of peptides with the drafting of tetrapeptide typical curve.Draw respectively tetrapeptide standard substance (4mg/ml) 0.0ml, 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, 3.0ml, 3.5ml, 4.0ml 4.5ml is in the 10ml volumetric flask, label adds respectively 2ml biuret reagent A, sway evenly, then add biuret reagent B, sway evenly, adding distil water is settled to scale, shake up, it was fully developed the color in standing 3 minutes, centrifugal, get supernatant, measure optical density value (OD) in the 540nm place, blank reagent (No. 1 volumetric flask) is reference.Take OD as ordinate zou, tetrapeptide standard substance concentration is X-coordinate, and the drawing standard curve carries out linear regression analysis with method of least squares, tries to achieve standard regressive method Y=aX+b.Y is absorbancy (A); X is concentration (C); A is slope; B is intercept.During sample determination, with diluted sample to proper concn,
And add biuret reagent, and processing mode is the same, and after shaking up, in 540nm place mensuration light absorption value (A), namely the y value, bring standard regressive method into, tries to achieve x, i.e. sample concentration (C).Adopt biuret method to survey its content of peptides, obtain typical curve equation Y=0.2475X+0.0004, R
2=0.9992.
The present embodiment is set up the HPLC method and is measured polypeptide amino acid, HPLC adopts 17 seed amino acid standard substance to do the external standard contrast, sets up HPLC reversed-phase system analysis of amino acid and forms, concrete operation method: get polypeptide sample 29.14mg, add 0.8ml5.7NHCl to put into the acid treatment of hydrolysis pipe, be filled with N
2Rear tube sealing is hydrolyzed 22h under 110 ℃.Open pipe is settled to 2ml after neutralization, get supernatant after centrifugal and put into volumetric flask and treat that HPLC analyzes.Analysis condition: o-phthalaldehyde(OPA) column front derivation, stationary phase ODS C
18(4.6 * 50mm, 5 μ), mobile phase A: sodium acetate soln (including 3 ‰ tetrahydrofuran (THF)s); Mobile phase B: water: methyl alcohol: acetonitrile=200: 450: 350, carry out linear gradient elution, flow velocity: 0.5ml/min, ultraviolet detection 338.1nm.HPLC collection of illustrative plates such as Fig. 4 of 17 seed amino acid standard substance, HPLC collection of illustrative plates such as Fig. 5 of polypeptide amino acid.
Table 4 secondary model analysis of variance table
The degreasing gingko polypeptide of the present embodiment preparation, wherein polypeptide class actives molecular weight is 3000-10000 dalton, and content of peptides is 50~80%, moisture<5%, granularity 100~200 orders, and polypeptide contains 17 seed amino acids, comprises the amino acid of 8 kinds of needed by human, simultaneously lysine content>7%, Histidine>8%, Serine>11%, aspartic acid>14%, L-glutamic acid>16%.Especially the content of aspartic acid, L-glutamic acid, Serine and Methionin amino acid in the ginkgo protein powder, therefore, this patent utilizes biological enzyme degrading activity albumen, the biologically active substances such as preparation active polypeptide and amino acid, after eating human body, more easily absorbed by the body, showing as molecular weight is the daltonian small molecules of 3000-10000, and amino acid whose composition is comprehensive, and ratio is reasonable, and nutrition is abundanter.As table 5.
In table 5 gingko polypeptide and ginkgo protein matter, amino acid forms contrast