A kind of tree peony seed blood sugar reducing peptide and purification process thereof and application
Technical field
The invention belongs to technical field of food biotechnology, it is specifically related to a kind of tree peony seed blood sugar reducing peptide and purification process thereof and application.
Background technology
Diabetes are the diseases of a kind of serious threat human health. Along with the develop rapidly of economic level, the diets tend of people is good, and amount of exercise reduces increasingly, causes the sickness rate of diabetes to increase year by year. Traditional hypoglycemic agents comprises glucosidase inhibitor, euglycemic agent, Insulin secretagogues, Chinese patent medicine, Regular Insulin, biguanides, sulfonephthalein carbamide type medicine etc., and owing to pharmacological agent exists side effect, life-time service patient is difficult to accept. In recent years, reached the favor that the method controlling blood sugar is more and more subject to people by dietotherapy, wherein develop the striving direction that new alpha-glucosidase inhibitor is numerous scholar.
Having scholar recently taking albumen such as mottle clam, silk, egg white and wheys as raw material, enzyme-squash techniqued has the bioactive peptide of Inhibiting ��-glucosidase. But the activity of these polypeptide is not high, such as two tripeptides isolated in the blood sugar lowing polypeptide of silk source, half suppresses dosage to be respectively 2.7mg/mL and 1.5mg/mL. Therefore, the blood sugar reducing peptide researching and developing novel high-activity is the difficult problem that industry emphasis to be solved.
Since peony seed oil is become Novel edible oil by Ministry of Health's official approval, China's oil cultivated area cumulative year after year of tree peony, the tree peony seed dregs of rice output after oil expression also constantly increases, and wherein crude protein accounts for 28.34% (W/W) of the tree peony seed dregs of rice. Tree peony seed albumen contains 18 kinds of natural amino acids, and wherein 8 kinds of human bodies must account for 30.44% by amino acid, and nutritive props is reasonable, has great DEVELOPMENT PROSPECT.
Summary of the invention
In order to solve the problem, the present invention provides a kind of tree peony seed blood sugar reducing peptide and purification process thereof, adopts ultrasound-assisted enzymolysis tree peony seed albumen, carries out separation and purification by ultrafiltration, ion exchange resin, dextrane gel, obtain the blood sugar reducing peptide of high reactivity. The comprehensive utilization not being only tree peony seed provides novel method, and even more important is the blood sugar reducing peptide obtaining a kind of novel, high reactivity, for numerous diabetic subjects bring Gospel.
Technical scheme: for realizing above-mentioned technical purpose, the tree peony seed blood sugar reducing peptide of the present invention prepares by the following method:
(1) the tree peony seed spent meal obtained after the oil expression of tree peony seed is mixed according to solid-liquid ratio 1: 8��12 with water, regulate pH to 9��9.5, at 40��60 DEG C, ultrasonic agitation extracts albumen, then the centrifugal 10��15min of 3500��3800r/min, get supernatant liquid, the precipitation of gained repeats extraction twice as stated above, merge the supernatant liquid of gained, 1mol/LHCl is used to regulate pH to 4��4.5, centrifugal 10��the 15min of 3500��3800r/min, get precipitation and carry out lyophilize, obtain tree peony seed albumen;
(2) 1��3% tree peony seed protein solution is prepared, use trypsin digestion, according to adding trypsinase 5000��125000U/g, pH7.5��8.5, temperature 35��40 DEG C, time 15��the 25min of ultrasonic wave added enzymolysis, ultrasonic frequency 22��40kHz, power 50��200W/L, reaction terminates, boiling water bath 10��15min carries out going out enzyme, can obtain tree peony seed blood sugar reducing peptide.
In a preferred embodiment, tree peony seed blood sugar reducing peptide solution is prepared by the following method:
A. raw material and distilled water are mixed even, the NaOH of 1mol/L is used to regulate pH value of solution to 9.5,50 DEG C of water bath sonicator intermittent stirrings extract albumen 2h, the centrifugal 15min of 3800r/min, gained precipitation extracts twice again, and gained supernatant liquid uses 1mol/LHCl to regulate the centrifugal 15min of pH to 4.5,3800r/min, get precipitation and carry out lyophilize, tree peony seed albumen can be obtained.
B. 1��3% tree peony seed protein solution is prepared, use trypsinase according to adding trypsinase 5000��125000U/g, pH7.5��8.5, temperature 35��40 DEG C, the time 15��25min of ultrasonic wave added enzymolysis, ultrasonic frequency 22��40kHz, power 50��200W/L, reaction terminates, and boiling water bath 10min carries out going out enzyme, can obtain tree peony seed polypeptide solution.
Present invention further proposes the purification process of above-mentioned tree peony seed blood sugar reducing peptide, it is characterised in that, comprise the steps:
(1) by the tree peony seed centrifugal 15��20min of blood sugar reducing peptide 8000��10000r/min, get middle level clarified liq, first use 0.46 ��m or 0.22 ��m of membrane filtration, ultrafiltration is carried out after filtration, the ultra-filtration membrane of 10000Da is first used to carry out initial filter, obtaining the first filtrate of molecular weight at below 10000Da, then use the filter membrane of 5000Da to carry out second ultrafiltration initial filter solution, the final best molecular weight of inhibiting rate that obtains is below 5000Da filtrate;
(2) adopt resin to carry out ion-exchange absorption, more than Static Adsorption 1h, collect eluate and carry out lyophilize, obtain one-level lyophilize polypeptide;
(3) the lyophilize polypeptide that step (2) obtains is mixed with the aqueous solution, adopts SephadexG-15 to carry out column chromatography, collect the sample lyophilize that obtain, obtain two grades of lyophilize polypeptide;
(4) the two grades of lyophilize polypeptide obtained are used the separation of C-18 reverse-phase chromatographic column, obtain the tree peony seed blood sugar reducing peptide after purifying.
Preferably, in step (2), described resin is any one in 732 type resin cation (R.C.)s, D151 large hole cation exchanger resin, 717 type anionite-exchange resin. More preferably, adopting 732 type adsorption rates the highest, effect is best.
Specifically, use 732 types absorption preferred steps be: will handle well 732 sodium form resin cation (R.C.)s load chromatography columns, distilled water is used to balance with flow velocity 2%CV/min, use UV-detector to detect at 220nm in downstream, until baseline is steady, add 5000kD ultrafiltration flow of solution through resin bed, until also becoming red below resin, distilled water is used to rinse resin with flow velocity 2%CV/min, 220nm baseline is steadily, use 0.5��2% (v/v) ammoniacal liquor instead and carry out wash-out according to 2%CV/min, 220nm detects, collect the 2nd elution peak and carry out lyophilize, wherein, the post that CV represents actual holds volume.
Preferably, in step (3), the step adopting SephadexG-15 to carry out column chromatography is: the SephadexG-15 handled well is loaded chromatography column, blade diameter length ratio 1: 30��1: 45, use distilled water to balance according to 2%CV/min flow velocity, use in downstream UV-detector to detect at 220nm; The polypeptide solution loading that will prepare, volume is the 5��10% of column volume, treats that sample enters gel column bed completely, it may also be useful to distilled water rinses according to 2%CV/min, and 220nm detects and collects the 2nd elution peak, and the post that CV represents actual holds volume.
Preferably, the step of the C-18 reverse-phase chromatographic column separation in step (4) is: preparation moving phase, wherein, mobile phase A is according to volume ratio ultrapure water: TFA=99.95: 0.05, and Mobile phase B is according to volume ratio acetonitrile: TFA=99.95: 0.05, and mobile phase A, B all use 0.22 ��m of membrane filtration, according to volume ratio mobile phase A: Mobile phase B=85: 15, flow velocity 12%CV/min, determined wavelength 220nm carry out detecting and collect, and the post that CV represents actual holds volume.
Closer, the present invention proposes the tree peony seed blood sugar reducing peptide after above-mentioned tree peony seed blood sugar reducing peptide or the purified purifying obtained for the preparation of the application in hypoglycemic medicine or healthcare products.
Useful effect: compared with prior art, tool of the present invention has the following advantages:
(1) the present invention carries out ultrasonic wave complex enzyme hydrolysis by the tree peony seed dregs of rice after being extracted oil by tree peony seed, and carries out separation and purification, prepares tree peony seed blood sugar lowing polypeptide novel, high reactivity. The long reaction time that tradition enzymolysis protein matter needs, and inefficiency, waste time and energy. The present invention uses ultrasound-assisted enzymolysis, is possible not only to realize Reaction time shorten, and can increase efficiency of pcr product.
(2) membrane separation technique of ultra-filtration technique to be a kind of taking pressure be impellent, according to big young pathbreaker's macromole of molecular weight of material and the separating substances of small molecules, simple and convenient, with low cost, do not increase any chemical reagent, do not cause the deactivation of macromolecular substance, and there is pack density height in unit container, the advantages such as floor space is little. The application adopts ultra-filtration technique, ensures the activity after polypeptide initial gross separation to greatest extent.
It is big that (3) 732 type Zeo-karbs have processing power, can use by repeated regeneration, long working life, the advantages such as working cost is lower, it is applicable to industrial production demand, the advantages such as D151 large hole cation exchanger resin has aperture and specific surface area is big, loading capacity is big, selectivity is good, rate of adsorption is fast, desorption condition is gentle, manipulation of regeneration is convenient, life cycle length, meet industrial production needs. 717 type anionite-exchange resin have regeneration efficiency height, alkali water consumption is low, exchange capacity is big, anti-Organic pollutants and advantage, the applicable industrial production requirement such as resistance of oxidation is strong, physical strength is good. Sephadex series is the three-dimensional netted gel particle being cross-linked to form by epoxy chloropropane by dextran, and wherein the molecular weight of G-15 separate substance is less than 1500Da, is applicable to being separated of desalination, peptide and other small molecules in this molecular weight ranges. Advantage is that SephadexG-15 belongs to inert support, not band electric charge, and adsorptive power is weak, and operational condition is very gentle, and physico-chemical property, the activity of separation composition is had good maintenance. Mainly utilize its mild condition to carry out being separated according to molecular size range and the active advantage kept herein.
(4) the present invention uses the method that high-efficient liquid is separated, it is possible to prepare the target product polypeptide that purity is higher, have application prospect. Current China is entering aging society, and diabetes are high morbidity and the regular incidences of elderly population, and have the trend strided forward to becoming younger, therefore very urgent for the research and development reducing blood sugar based food. Biological industry flourish, the popularization for tree peony seed blood sugar reducing peptide has good promoter action, and following process makes capsule, tablet or the product such as beverage, drink class.
Accompanying drawing explanation
Fig. 1 is preparation and the purifying schematic flow sheet of tree peony seed blood sugar reducing peptide of the present invention;
Fig. 2 is the ultraviolet detection schematic diagram of the polypeptide solution after 732 sodium form cationic resin column layers are analysed;
Fig. 3 is the ultraviolet detection schematic diagram of the polypeptide solution obtained after SephadexG-15 column chromatography;
Fig. 4 is the HPLC schematic diagram of the polypeptide solution obtained after C-18 reverse-phase chromatographic column is separated.
Embodiment
As shown in Figure 1, the present invention provides a kind of tree peony seed blood sugar reducing peptide and purification process thereof, comprise the preparation of tree peony seed albumen, the preparation of polypeptide enzymolysis solution, and the polypeptide being less than 5000 by ultrafiltration collection molecular weight further, then spent ion exchange resin separation successively, dextrane gel separation and liquid phase separation, obtain the tree peony seed blood sugar reducing peptide of purifying.
Below by specific embodiment, the present invention is described in detail. Wherein, in following embodiment, the post that CV represents actual holds volume; Ammonia concn is volume fraction (V/V).
Embodiment 1
Raw material and distilled water are carried out proportioning according to solid-liquid ratio 1: 10, stirring 15min makes it fully mixed even, pH value of solution to 9.5 is regulated with 1mol/LNaOH, 50 DEG C of water bath sonicator 2h, the centrifugal 15��20min of 3800r/min, gained precipitation is extracted twice again, merging gained supernatant liquid uses 1mol/LHCl to regulate pH to 4.5, centrifugal 15��the 20min of 3800r/min, gets precipitation and carries out lyophilize, can obtain tree peony seed albumen.
Embodiment 2��13
Reaction conditions is: compound concentration tree peony seed protein solution 1��3%, enzyme concentration 5000��12500U/g, pH7.5��8.5, temperature 35��40 DEG C, time 15��25min, ultrasonic frequency 28kHz, power 100W/L, enzyme digestion reaction terminates rear boiling water bath 10min, can obtain tree peony seed polypeptide enzymolysis solution.
External hypoglycemic measuring method:
(1) mouse alpha-glucosidase preparation
Mouse is put to death in dislocation, washes impurity elimination thing, liquid nitrogen grinding with 0 DEG C of physiological saline. Being mixed with lapping powder by PBS, according to volume mass than 10: 1 proportionings, extraction of ocean eddies 60s, 4 DEG C of centrifugal 15min of 8000r/min, get supernatant.
(2) alpha-glucosaccharase enzyme inhibition rate detection method
Water-bath 37 DEG C reaction 15min, 405nm place detection light absorption value;
Inhibiting rate method of calculation are as follows:
(3) reagent needed for
PBS:0.05mol/LpH6.8 phosphate buffered saline buffer
PNPG solution: the pNPG (p-NP-��-D-glucopyranoside) accurately taking 50mg, is dissolved in 10mLPBS.
Enzyme solution: extract mouse enzyme solution in 1.6mLPBS+0.2mLpNPG+0.2mL enzyme solution reaction system, water-bath 37 DEG C reaction 15min, OD405Between 0.4��0.45.
Detected result is as shown in table 1:
Blood sugar reducing peptide under the different preparation condition of table 1 is active
Best by the inhibition of upper table 1 known embodiment 13 gained enzymolysis polypeptide liquid, therefore select embodiment 13 to carry out further separation and purification.
Embodiment 14
Embodiment 13 is obtained the polypeptidase solution centrifugal 15min of liquid 8000r/min, get middle level clarified liq, 0.46 ��m of membrane filtration, ultrafiltration is carried out at use ultrafiltration apparatus after membrane filtration, the ultra-filtration membrane of 10000Da is first used to carry out initial filter, use the filter membrane of 5000Da to carry out second ultrafiltration initial filter solution again, finally choose best below the 5000Da filtrate of inhibiting rate and preserve stand-by;
Embodiment 15��17
The 732 type resin cation (R.C.)s handled well, D151 large hole cation exchanger resin, 717 type anionite-exchange resin are carried out Static Adsorption respectively, after absorption 1h, measures adsorption rate respectively, as shown in table 2.
The Static Adsorption rate of the dissimilar resin of table 2
Embodiment 18��21
Take the 732 type resin cation (R.C.)s loading chromatography columns that 1g handles well, distilled water balance is used to rinse 3CV, use UV-detector 220nm to detect in downstream, again 5000kD ultrafiltration solution is flowed through resin column according to 2%CV/min, until outlet also becomes red below resin column, it is replaced with distilled water, rinse, resin is rinsed according to 2%CV/min, till 220nm ultraviolet detection numerical value no longer fluctuates, using 0.5,1.0,1.5,2.0% concentration ammoniacal liquor instead and carry out wash-out as elutriant, result is as shown in table 3:
The eluting rate of table 3 different concns ammoniacal liquor
Embodiment 22
The 732 type resin cation (R.C.)s handled well load chromatography column, distilled water is used to rinse according to flow velocity 2%CV/min flow velocity, downstream uses UV-detector to detect at 220nm, the molecular weight of preparation in embodiment 14 is less than 5000kD ultrafiltrated and flows through resin column according to flow velocity 0.2%CV/min, along with polypeptide is adsorbed, resin column turns into redness, until outlet resin also turns red below post, using distilled water to rinse resin according to flow velocity 0.2%CV/min, after rinsing 1CV, 220nm ultraviolet detection numerical value no longer fluctuates; Using 2% concentration ammoniacal liquor to be adsorbed polypeptide according to flow velocity 0.2%CV/min wash-out, 220nm detects, and collects and is occurred that lyophilize, obtains lyophilized products such as the 2nd elution peak in Fig. 2.
Embodiment 23
Polypeptide sample preparation lyophilize in embodiment 22 obtained becomes 50mg/mL solution; The SephadexG-15 handled well is loaded chromatography column, and blade diameter length ratio is 1: 40, it may also be useful to distilled water balances according to 0.2%CV/min flow velocity, uses UV-detector to detect at 220nm in downstream; The polypeptide solution loading that will prepare, volume is column volume 7.5%, it may also be useful to distilled water rinses according to flow velocity 0.2CV/min, and 220nm detects and collects such as the 2nd elution peak in Fig. 3, and lyophilize, obtains lyophilized products.
Embodiment 24
Embodiment 23 lyophilized products is configured to 25mg/mL solution for standby; Moving phase is configured according to the ratio of the volume of ultrapure water, TFA (trifluoroacetic acid), acetonitrile, mobile phase A ultrapure water: TFA=99.95: 0.05, Mobile phase B acetonitrile: TFA=99.95: 0.05, mobile phase A, B all use 0.22 ��m of membrane filtration, ratio mobile phase A according to volume: Mobile phase B=85: 15, flow velocity 12%CV/min, determined wavelength 220nm, post temperature 30 DEG C, C-18 reverse-phase chromatographic column is separated; Separating resulting as shown in Figure 4, collect No. 2 peaks material, lyophilize namely obtain us required for blood sugar lowing polypeptide, IC50It is 47.256 �� g/mL.
The present invention utilizes ultrasonic assistant biological enzyme degraded tree peony seed albumen, is cut into multiple different molecular weight polypeptide, then uses ultrafiltration, ion-exchange, dextrane gel and high performance liquid chromatography to carry out separation and purification. It is possible not only to carry out efficiently endonuclease reaction, obtains a large amount of tree peony seed blood sugar reducing peptides, and polypeptide active can be kept not lose in purge process, it is possible to well Commercial cultivation. Meanwhile, the present invention prepares tree peony seed blood sugar reducing peptide by ultrasonic assistant tree peony seed proteolysis, is possible not only to the comprehensive utilization substantially increasing tree peony seed, increases its economic worth, and provides a kind of novel blood sugar lowing peptide with high activity.