CN107586320A - A kind of brown croaker air bladder reducing blood lipid oligopeptides and its application - Google Patents
A kind of brown croaker air bladder reducing blood lipid oligopeptides and its application Download PDFInfo
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Abstract
The invention discloses a kind of brown croaker air bladder reducing blood lipid oligopeptides and its application, is specially：Using brown croaker air bladder as raw material, enzymolysis liquid is digested to obtain by degreasing, alkali protease, enzymolysis liquid isolates and purifies to obtain reducing blood lipid oligopeptides Phe Ser Gly Leu Arg (FSGLR) using ultrafiltration, anion exchange resin chromatography, gel filtration chromatography and RPLC, and ESI MS measure molecular weight is 831.98Da.Have the beneficial effect that：Reducing blood lipid oligopeptides of the present invention can reduce the HepG2 lipid within endothelial cells accumulation of OA inductions；It is in dose dependent decomposition to T-CHOL, triglycerides；Expression of the aliphatic radical because of SREBP 1c, ACC, SREBP 2, HMGR and FAS can be tuned into down, suppresses into fat factors A CC protein expression level, activates LXR α, can be used for preparing reducing blood lipid medicine, health product or food additives.
Description
Technical field
The present invention relates to a kind of reducing blood lipid oligopeptides, more particularly, to a kind of brown croaker air bladder reducing blood lipid oligopeptides and its application.
Background technology
Blood fat is the general name of contained lipoids in blood, mainly comprising cholesterol, triglyceride, cholesterol ester, β-fat egg
In vain, the composition such as phosphatide, not esterified resin acid.Hyperlipidemia refers to that blood lipid level is too high, so as to directly cause some serious harms
The disease of health, such as atherosclerosis, coronary heart disease, pancreatitis.Hyperlipidemia is a kind of common disease, particularly 50
The common disease of more than year the elderly health, has the characteristics of high illness rate, high disability rate and high mortality.Therefore, hyperlipemia
The seriousness of disease definitely can not be ignored, and it is to treat a kind of important way of hyperlipidemia to take health products auxiliary treatment, for
The quality of life for improving people is significant.
Brown croaker（Miichthys miiuy）, also known as rice fish, slate cod croaker fish, belong to Perciformes Sciaenidae.Brown croaker air bladder fat content
Low, main component is large biological molecule collagenic protein isoreactivity goods and materials, is easy to be digested and utilizes, so as to improve
Histotrophic nutrition situation and acceleration metabolism, influence some particular organization's physiological functions, and enhancing development strengthens resistance against diseases,
Play anti-aging and resist the efficiency of cancer.Based on the application of fish spiral shell mainly culinary art and dried product are processed currently on the market,
The fish spiral shell added value of drying production is low, and has obvious localized feature, is concentrated mainly on coastal area, and rare food interiorly
With constraining the sale of air bladder to a certain extent, economic value is limited, therefore works out a kind of feasible air bladder deep processing skill
Art turns into the task of top priority.And using brown croaker air bladder as raw material, the technique that brown croaker air bladder reducing blood lipid oligopeptides is prepared using zymolysis technique is ground
Study carefully and be in blank stage, and it is even more to have no to prepare high activity reducing blood lipid oligopeptides and its application as material using brown croaker air bladder enzymolysis product
Report.
Prior art such as Authorization Notice No. is the B of CN 102363800 Chinese invention patent, discloses a kind of ultrasonic wave
The method that solution air bladder prepares anti-oxidant oligopeptides, it is air bladder powder first to crush air bladder；It is 6.0 to add pH value into air bladder powder again
Phosphate buffer and trypsase, stir, obtain air bladder solution；Then air bladder solution is digested in a water bath, then
Air bladder solution is carried out into supersound process to be digested, continues to digest in water bath with thermostatic control after supersound process；After enzymolysis terminates, go out
Enzyme, cool down, be filtrated to get supernatant；By supernatant 10000Da milipore filter ultrafiltration, the solution after ultrafiltration is freeze-dried, obtains
To the anti-oxidant oligopeptides of air bladder.The anti-oxidant oligopeptides of above-mentioned preparation has good scavenging action to the free radical of human body, can play anti-
Only aging, inflammation, and artery sclerosis and the cardiovascular and cerebrovascular disease triggered, a kind of nourishing additive agent can also be used as to be applied to food
In product.But the preparation method increases the extraction difficulty of anti-oxidation peptide, the obtained anti-oxidation peptide is pure in addition without defatting step
Spend it is relatively low, and be not suitable for extraction reducing blood lipid oligopeptides.
The content of the invention
It is an object of the invention to provide a kind of brown croaker air bladder reducing blood lipid oligopeptides with effect for reducing blood fat, the reducing blood lipid are few
Peptide is as reducing blood lipid medicine, health product and food additives.
The present invention is directed to the problem of being mentioned in above-mentioned technology, and the technical scheme taken is：A kind of brown croaker air bladder reducing blood lipid is few
Peptide, the amino acid sequence of above-mentioned reducing blood lipid oligopeptides is Phe-Ser-Gly-Leu-Arg (FSGLR), molecular weight 578.67Da.
A kind of preparation method of brown croaker air bladder reducing blood lipid oligopeptides, specifically includes following step：
Brown croaker air bladder pre-processes：Brown croaker air bladder is cleaned, after homogenate to pasty state, by solid-to-liquid ratio 1g:10~15mL adds 0.08-
0.12M NaOH solution, 12~15h is soaked at 0~5 DEG C, filtering, neutrality is washed to distillation, by solid-to-liquid ratio 1g:5~8mL
8-12% aqueous isopropanol is added, 36~48h of degreasing, filtering, abandons filtrate, solids is rinsed well isopropanol with distilled water,
Dry, obtain degreasing brown croaker air bladder, the step can thoroughly remove the adipose tissue degreasing of air bladder, and the extraction for reducing fish maw protein is difficult
Degree, no reagent residual, moreover it is possible to remove the foreign protein of non-collagen, improve the purity of gained reducing blood lipid oligopeptides, and the processing side
Method has no effect to the other compositions in bucket air bladder and effect；
The enzymolysis of brown croaker air bladder：Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5~8mL addition distilled water, regulation pH value to 9~
10, add the alkali protease of air bladder weight 2~3%, 3~4h digested at 50~60 DEG C, then 90~100 DEG C of enzyme deactivations 10~
15min, room temperature is cooled to, 15-25min is centrifuged under 10000~15000rmp, collect supernatant, produce brown croaker air bladder enzymolysis
Liquid, enzyme activity >=2.0 × 105U/g of above-mentioned alkali protease, the activity that high activity can be effectively extracted from air bladder are more
Peptide, improve the conversion ratio of protein so that the yield of active peptides is high, and reaction condition is gentle, and cost is relatively low, safe, no
The composition and activity of active peptides will not be only destroyed, and the quality of active peptides can be improved；
The preparation of brown croaker air bladder reducing blood lipid oligopeptides：Ultrafiltration by brown croaker air bladder enzymolysis liquid through molecular cut off for 3kDa and 10kDa
Film is classified, collect molecular cut off be less than 3kDa components, obtain ultrafiltration enzymolysis liquid, then by ultrafiltration enzymolysis liquid successively through from
Sub-exchange resin chromatography, gel filtration chromatography and RPLC（RP-HPLC）Purifying, produce brown croaker air bladder reducing blood lipid widow
The higher brown croaker air bladder reducing blood lipid oligopeptides of purity can be made in peptide, the step, and the reducing blood lipid oligopeptides is to DPPH free radicals, hydroxyl free
Base and ultra-oxygen anion free radical have good scavenging action, and have good lipid oxidation resistance, while have good
Good antioxidation activity, is easy to digest and assimilate, safe without toxic side effect.
Preferably, brown croaker is Miichthys miiuy.
Preferably, the detailed process of ion exchange chromatography, gel filtration chromatography and RP-HPLC purifying is：
Ion exchange chromatography：By above-mentioned ultrafiltration enzymolysis liquid with distilled water be made into concentration be 45~55mg/mL solution, 4 DEG C,
12000r/min centrifuges 20min, removes insoluble impurities, and supernatant is added to DEAE-52 anion exchange resin chromatographic columns, according to
It is secondary with distilled water, 0.1,0.5 and 1mol/L NaCl solution elute, collect 0.5mol/L NaCl elution fractions, as ion hand over
Chromatography zymolyte is changed, this method has advantage reproducible, simple to operate, that Elution range is wide, can carried out according to actual conditions
Amplification condition realizes industrialized production；
Gel filtration chromatography：It is the molten of 45~55 mg/mL that above-mentioned ion-exchange chromatography zymolyte, which is dissolved in distilled water to be made into concentration,
Liquid, 20min is centrifuged under 4 DEG C, 12000r/min, remove insoluble impurities, supernatant passes through sephadex SephadexG-
25 column chromatography for separation, are eluted with ultra-pure water, and elution fraction is collected according to the absorbance curve under 280nm, wherein, reducing blood lipid
The most strong component of activity is gel chromatography zymolyte, and this method is different according to the translational speed in chromatographic column, the component of macromolecular
First it is eluted, is eluted after the component of small molecule, it is easy to operate, do not influence so as to reach the purpose isolated and purified
Target components chemical property；
RP-HPLC is purified：Above-mentioned gel chromatography zymolyte is made into 80~100 μ g/mL solution with distilled water, utilizes RP-
HPLC is purified, and reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR), this method are obtained according to hypolipidemic activity
High sensitivity, good separating effect, can fast separating and purifying target product.
Further preferably, RP-HPLC purification conditions are：Sample size is 10~15 μ L；Chromatographic column is Kromasil C18（250mm×
4.6mm, 5 μm）；Mobile phase is 40% acetonitrile；Elution speed is 0.8~1.0mL/min；The nm of ultraviolet detection wavelength 280.
A kind of brown croaker air bladder reducing blood lipid oligopeptides, can reduce oleic acid（OA）The HepG2 lipid within endothelial cells accumulation of induction, 1~
To T-CHOL under 10 μm of ol/L concentration（TC）, triglycerides（TG）In dose dependent decomposition, Phe-Ser-Gly-
Leu-Arg (FSGLR) can be tuned into expression of the aliphatic radical because of SREBP-1c, ACC, SREBP-2, HMGR and FAS down, suppress into fat because
Sub- ACC protein expression level, activation LXR α.Therefore, Phe-Ser-Gly-Leu-Arg (FSGLR) can lower most of into fat
Gene, lipid accumulation is reduced, play effect for reducing blood fat, can be developed into reducing blood lipid medicine, health product and food additives.
Compared with prior art, the advantage of the invention is that：Brown croaker air bladder reducing blood lipid oligopeptides of the present invention can reduce oleic acid
（OA）The HepG2 lipid within endothelial cells accumulation of induction, can be tuned into aliphatic radical down because of SREBP-1c, ACC, SREBP-2, HMGR and FAS
Expression, suppress into fat factors A CC protein expression level, activate LXR α, suppress absorption fatty in enteron aisle, play reducing blood lipid and make
With；The reducing blood lipid oligopeptides can effectively reduce blood lipids level, have and be easy to digest and assimilate and the characteristics of safe without toxic side effect,
It can be developed into reducing blood lipid medicine, health product and food additives；Air bladder reducing blood lipid oligopeptides of the present invention is made by enzyme solution,
Enzymolysis process is easy to control, and reducing blood lipid oligopeptides can be made to discharge to greatest extent, improve reducing blood lipid oligopeptides yield, and pass through through
Ion exchange chromatography, gel filtration chromatography and RPLC purifying, obtained reducing blood lipid oligopeptides purity are high；This hair
It is bright to use brown croaker air bladder to produce reducing blood lipid oligopeptides for raw material, industry blank is compensate for, one kind is provided for the deep processing of air bladder and cuts
Real feasible technology.
Brief description of the drawings
Fig. 1 is the DEAE-52 anion exchange resin tomographic maps of the embodiment of the present invention 2；
Fig. 2 is the sephadex Sephadex G-25 tomographic maps of the embodiment of the present invention 2；
Fig. 3 is the RP-HPLC analyses that the sephadex Sephadex G-25 of the embodiment of the present invention 2 prepare zymolyte；
Fig. 4 is the mass spectrogram of reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention；
Fig. 5 is the influence that reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention is bred to HepG2 cells；
Fig. 6 is the shadow that reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention is accumulated to HepG2 lipid within endothelial cells
Ring；
Fig. 7 is the mirror that reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention is accumulated to HepG2 lipid within endothelial cells
Lower observation result；
Fig. 8 is reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention to HepG2 intracellular triglycerides（TG）
The influence of content；
Fig. 9 is reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention to the intracellular T-CHOLs of HepG2（TC）
The influence of content；
Figure 10 is various concentrations reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention to HepG2 cell lactones
The influence of matter synthesis related gene expression；
Figure 11 is that reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention aoxidizes phase to HepG2 lipid within endothelial cells
The influence of correlation gene expression；
Figure 12 be reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) SREBP-1c intracellular to HepG2 of the present invention and
ACC genes influence in the expression of protein level；
Figure 13 is reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) of the present invention to the intracellular liver receptor-inducibles of HepG2
The influence of expression.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of brown croaker air bladder reducing blood lipid oligopeptides, its preparation method specifically include following step：
1）Brown croaker air bladder pre-processes：Brown croaker（Miichthys miiuy）Air bladder is cleaned, after homogenate to pasty state, by solid-to-liquid ratio 1g:
15mL adds 0.08M NaOH solution, and 12h is soaked at 5 DEG C, filters, neutrality is washed to distillation, by solid-to-liquid ratio 1g:8mL adds
Enter 10% aqueous isopropanol, degreasing 36h, filtering, abandon filtrate, solids is rinsed well isopropanol with distilled water, is dried, is obtained
Degreasing brown croaker air bladder；
2）The enzymolysis of brown croaker air bladder：Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:8mL adds distilled water, regulation pH value to 9, adds
The alkali protease of air bladder weight 2%, digests 4h at 60 DEG C, then in 90 DEG C of enzyme deactivation 15min, room temperature is cooled to, in 10000rmp
Lower centrifugation 25min, supernatant is collected, produces brown croaker air bladder enzymolysis liquid, enzyme activity >=2.0 × 105U/ of above-mentioned alkali protease
g；
3）The preparation of brown croaker air bladder reducing blood lipid oligopeptides：Through molecular cut off it is the super of 3kDa and 10kDa by brown croaker air bladder enzymolysis liquid
Filter membrane is classified, and is collected molecular cut off and is less than 3kDa components, obtains ultrafiltration enzymolysis liquid, then pass through ultrafiltration enzymolysis liquid successively
Ion exchange chromatography, gel filtration chromatography and RPLC（RP-HPLC）Purifying, produces brown croaker air bladder reducing blood lipid
Oligopeptides.
Above-mentioned ion exchange chromatography, gel filtration chromatography and RP-HPLC purifying detailed process be：
Ion exchange chromatography：Above-mentioned ultrafiltration enzymolysis liquid is made into the solution that concentration is 55mg/mL with distilled water, 4 DEG C,
12000r/min centrifuges 20min, removes insoluble impurities, and supernatant is added to DEAE-52 anion exchange resin chromatographic columns, according to
It is secondary with distilled water, 0.1,0.5 and 1mol/L NaCl solution elute, collect 0.5mol/L NaCl elution fractions, as ion hand over
Change chromatography zymolyte；
Gel filtration chromatography：Above-mentioned ion-exchange chromatography zymolyte is dissolved in distilled water and is made into the solution that concentration is 55mg/mL, 4
DEG C, 20min is centrifuged under 12000r/min, remove insoluble impurities, supernatant passes through sephadex SephadexG-25 post layers
Analysis separation, is eluted with ultra-pure water, and elution fraction is collected according to the absorbance curve under 280nm, wherein, hypolipidemic activity is most
Strong component is gel chromatography zymolyte, and this method is different according to the translational speed in chromatographic column, and the component of macromolecular is first washed
Take off, be eluted after the component of small molecule, it is easy to operate, do not influence target group so as to reach the purpose isolated and purified
Divide chemical property；
RP-HPLC is purified：Above-mentioned gel chromatography zymolyte is made into 80 μ g/mL solution with distilled water, carried out using RP-HPLC
Purifying, hypolipidemic activity oligopeptides is prepared according to hypolipidemic activity, this method high sensitivity, good separating effect, can quick separating it is pure
Change target product.
Above-mentioned RP-HPLC purification conditions are：Sample size is 10 μ L；Chromatographic column is Kromasil C18（250mm × 4.6mm, 5
μm）；Mobile phase is 40% acetonitrile；Elution speed is 1.0mL/min；Ultraviolet detection wavelength 280nm.
Embodiment 2：
A kind of brown croaker air bladder reducing blood lipid oligopeptides, its preparation method specifically include following step：
1）Brown croaker air bladder pre-processes：Brown croaker（Miichthys miiuy）Air bladder is after over cleaning, homogenate to pasty state, according to 1 g:
10mL adds 0.1MNaOH and 15h is soaked in 4 DEG C, filtering, neutrality is washed to distillation, according to 1g:5mL adds 10% isopropanol
Solution, degreasing 48h, filtering, supernatant is abandoned, is rinsed well isopropanol with distilled water, dried, obtain degreasing brown croaker air bladder；
2）The enzymolysis of brown croaker air bladder：Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5mL adds distilled water.PH value is adjusted to 9~10,
Add the alkali protease of air bladder weight 2.5%（Enzyme activity >=2.0 × 105U/g）, 55 DEG C of enzymolysis 3.5h, gone out in 95 DEG C, 10min
Enzyme, normal temperature is cooled to, 12000rmp centrifugation 20min, collects supernatant, i.e. brown croaker air bladder enzymolysis liquid；
3）The preparation of brown croaker air bladder reducing blood lipid oligopeptides：Through molecular cut off it is the super of 3kDa and 10kDa by brown croaker air bladder enzymolysis liquid
Filter membrane is classified, and is collected molecular cut off and is less than 3kDa components, obtains ultrafiltration enzymolysis liquid, ultrafiltration enzymolysis liquid is handed over through ion successively
Change resin chromatography, gel filtration chromatography and RPLC（RP-HPLC）Purifying, obtains brown croaker air bladder reducing blood lipid oligopeptides,
Utilize amino acid sequence analysis instrument and mass spectroscopy its structure.
Above-mentioned ion exchange chromatography, gel filtration chromatography and RP-HPLC purifying detailed process be：
Ion exchange chromatography：Above-mentioned ultrafiltration enzymolysis liquid is made into the solution that concentration is 50mg/mL with distilled water, 4 DEG C,
12000r/min centrifuges 20min, removes insoluble impurities, and supernatant is added to DEAE-52 anion exchange resin chromatographic columns, according to
It is secondary with distilled water, 0.1,0.5 and 1mol/L NaCl solution elute, collect 0.5mol/L NaCl elution fractions（DMS-Ⅲ）, i.e.,
It is as shown in Figure 1 for ion-exchange chromatography zymolyte, DEAE-52 anion exchange resin tomographic maps；
Gel filtration chromatography：DMS- III is dissolved in distilled water and is made into the solution that concentration is 50 mg/mL, 4 DEG C, 12000r/min centrifugations
20min, removes insoluble impurities, and supernatant passes through sephadex Sephadex G-25 column chromatography for separation, carried out with ultra-pure water
Elution, elution fraction is collected according to the absorbance curve under 280nm（GS-III）, as gel chromatography zymolyte, glucan coagulate
Glue Sephadex G-25 tomographic maps are as shown in Figure 2；
RP-HPLC is purified and structure detection：GS-III is made into 100 μ g/mL solution with distilled water, utilizes RP-HPLC（Sample introduction
Measure 10 μ L；Chromatographic column Kromasil C18（250mm × 4.6mm, 5 μm）；Mobile phase：40% acetonitrile；Elution speed 1.0mL/min；
Ultraviolet detection wavelength 280nm）Purified, hypolipidemic activity oligopeptides, the RP-HPLC figures of zymolyte are prepared according to hypolipidemic activity
As shown in figure 3, being detected as simple spike through RP-HPLC, it is Phe- to determine amino acid sequence using protein/polypeptide sequenator
Ser-Gly-Leu-Arg (FSGLR), ESI-MS measure molecular weight are 578.67Da, mass spectrogram such as Fig. 4 institutes of reducing blood lipid oligopeptides
Show.
Obtained brown croaker air bladder reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) is subjected to activity rating, as a result
As is shown in figures 5-12, the results showed that：Phe-Ser-Gly-Leu-Arg (FSGLR) can reduce oleic acid（OA）The HepG2 cells of induction
Inner lipid is accumulated；To T-CHOL under 1~10 μm of ol/L concentration（TC）, triglycerides（TG）Decompose and make in dose dependent
With；Phe-Ser-Gly-Leu-Arg (FSGLR) can be tuned into table of the aliphatic radical because of SREBP-1c, ACC, SREBP-2, HMGR and FAS down
Reach, suppress into fat factors A CC protein expression level, activation LXR α.Therefore, under Phe-Ser-Gly-Leu-Arg (FSGLR) energy
Majority is tuned up into aliphatic radical because reducing lipid accumulation.
A kind of brown croaker air bladder reducing blood lipid oligopeptides, the reducing blood lipid oligopeptides can be as the additives of medicine, health food and food.
Embodiment 3：
A kind of brown croaker air bladder reducing blood lipid oligopeptides, its preparation method specifically include following step：
1）Brown croaker air bladder pre-processes：Brown croaker（Miichthys miiuy）Air bladder is after over cleaning, homogenate to pasty state, according to 1g:
10mL adds 0.1MNaOH and 15h is soaked in 4 DEG C, filtering, neutrality is washed to distillation, according to 1g:5mL adds 10% isopropanol
Solution, the neopelex and carvacrol of air bladder weight 0.022% and 0.004% are added, degreasing 48h, filtering, abandons filter
Liquid, solids are rinsed well isopropanol with distilled water, are dried, are obtained degreasing brown croaker air bladder, above-mentioned neopelex and
Carvacrol can play the effect mutually cooperateed with isopropanol, destroy adipocyte plasma membrane so that and intracellular fat oozes out, while
Increase the solvability of isopropanol, different types of fatty acid glyceryl ester can be promoted quickly to be dissolved in solution, and then remove,
Accelerate the debinding rate and effect of air bladder, the step can thoroughly remove the adipose tissue degreasing of air bladder, reduce carrying for fish maw protein
Take difficulty, no reagent residual, moreover it is possible to the foreign protein of non-collagen is removed, improves the purity of gained reducing blood lipid oligopeptides, and at this
Reason method has no effect to the other compositions in bucket air bladder and effect；
2）The enzymolysis of brown croaker air bladder：Extracting degreasing brown croaker air bladder, by solid-liquid ratio 1g:5mL adds distilled water.PH value is adjusted to 9~10,
Add the alkali protease of air bladder weight 2.5%（Enzyme activity >=2.0 × 105U/g）, 55 DEG C of enzymolysis 3.5h, gone out in 95 DEG C, 10min
Enzyme, normal temperature is cooled to, 12000rmp centrifugation 20min, collects supernatant, i.e. brown croaker air bladder enzymolysis liquid；
3）The preparation of brown croaker air bladder reducing blood lipid oligopeptides：Through molecular cut off it is the super of 3kDa and 10kDa by brown croaker air bladder enzymolysis liquid
Filter membrane is classified, and is collected molecular cut off and is less than 3kDa components, obtains ultrafiltration enzymolysis liquid, ultrafiltration enzymolysis liquid is handed over through ion successively
Resin chromatography, gel filtration chromatography and RP-HPLC purifying are changed, obtains brown croaker air bladder reducing blood lipid oligopeptides.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
Sequence table
<110>Zhejiang Ocean university
<120>A kind of brown croaker air bladder anti-oxidation peptide and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213>Brown croaker (Miichthysmiiuy)
<400> 1
Phe Ser Gly Leu Arg
1 5
Claims (10)
- A kind of 1. brown croaker air bladder reducing blood lipid oligopeptides, it is characterised in that：The amino acid sequence of the reducing blood lipid oligopeptides is Phe-Ser- Gly-Leu-Arg (FSGLR)。
- A kind of 2. brown croaker air bladder reducing blood lipid oligopeptides according to claim 1, it is characterised in that：The reducing blood lipid oligopeptides ESI-MS measure molecular weight is 578.67Da.
- A kind of 3. brown croaker air bladder reducing blood lipid oligopeptides according to claim 1, it is characterised in that：The system of the reducing blood lipid oligopeptides Preparation Method comprises the steps：1）Brown croaker air bladder pre-processes：Brown croaker air bladder is cleaned, after homogenate to pasty state, adds NaOH solution, soaks, filtering, with steaming Distilled water is washed till neutrality, adds aqueous isopropanol, degreasing, filtering, abandons filtrate, and solids is rinsed well isopropanol with distilled water, Dry, obtain degreasing brown croaker air bladder；2）The enzymolysis of brown croaker air bladder：Extracting degreasing brown croaker air bladder, distilled water is added, adjust pH value, added alkali protease enzymolysis, go out Enzyme, room temperature is cooled to, centrifuged, collected supernatant, produce brown croaker air bladder enzymolysis liquid；3）The preparation of brown croaker air bladder reducing blood lipid oligopeptides：Brown croaker air bladder enzymolysis liquid is classified to obtain ultrafiltration enzymolysis through milipore filter Liquid, then by ultrafiltration enzymolysis liquid successively through ion exchange chromatography, gel filtration chromatography and RPLC（RP- HPLC）Purifying, produces brown croaker air bladder reducing blood lipid oligopeptides.
- A kind of 4. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step The concentration of aqueous isopropanol is 8~12% in 1, the solid-to-liquid ratio 1g of air bladder and aqueous isopropanol:5~8mL, 36~48h of degreasing.
- A kind of 5. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step Enzyme activity >=2.0 × 105U/g of 2 neutral and alkali protease, alkali protease are air bladder weight 2~3%, and hydrolysis temperature is 50~60 DEG C, pH value be 9~10, the time be 3~4h.
- A kind of 6. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step Centrifugal rotational speed is 10000~15000rmp in 2, and the time is 15~25min.
- A kind of 7. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step The molecular cut off of milipore filter is 3kDa and 10kDa in 3.
- A kind of 8. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step 3 ion exchange resins chromatography, gel filtration chromatography and RP-HPLC purifying detailed process be：1）Ion exchange chromatography：Aforementioned polypeptides mixture is dissolved in distilled water and is made into the solution that concentration is 45~55mg/mL, 4 DEG C, 12000r/min centrifugation 20min, remove insoluble impurities, and supernatant is added to DEAE-52 anion exchange resin chromatography Post, successively with distilled water, 0.1,0.5 and 1mol/L NaCl solution elute, collect 0.5mol/L NaCl elution fractions, be Ion-exchange chromatography zymolyte；2）Gel filtration chromatography：It is the molten of 45~55mg/mL that above-mentioned ion-exchange chromatography zymolyte, which is dissolved in distilled water to be made into concentration, Liquid, 4 DEG C, 12000r/min centrifugation 20min, removes insoluble impurities, supernatant passes through sephadex SephadexG-25 posts Chromatography, eluted with ultra-pure water, elution fraction is collected according to the absorbance curve under 280nm, wherein, hypolipidemic activity Most strong component is gel chromatography zymolyte；3）RP-HPLC is purified：Above-mentioned gel chromatography zymolyte is made into 80~100 μ g/mL solution with distilled water, utilizes RP- HPLC is purified, and reducing blood lipid oligopeptides Phe-Ser-Gly-Leu-Arg (FSGLR) is obtained according to hypolipidemic activity.
- A kind of 9. preparation method of brown croaker air bladder reducing blood lipid oligopeptides according to claim 3, it is characterised in that：The step RP-HPLC purification conditions are in 3：Sample size is 10~15 μ L；Chromatographic column is Kromasil C18（250mm × 4.6mm, 5 μm）； Mobile phase is 40% acetonitrile；Elution speed is 0.8~1.0mL/min；The nm of ultraviolet detection wavelength 280.
- A kind of 10. application of brown croaker air bladder reducing blood lipid oligopeptides, it is characterised in that：The air bladder reducing blood lipid oligopeptides, which is applied to prepare, to drop The additive of hypolipidemic medicine, health products and food.
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| CN110201140A (en) * | 2019-07-01 | 2019-09-06 | 南通大学 | The application of air bladder polypeptide |
| CN114044807A (en) * | 2021-11-19 | 2022-02-15 | 浙江海洋大学 | Mussel blood fat reducing oligopeptide for treating hyperlipidemia |
| CN116003578A (en) * | 2023-02-27 | 2023-04-25 | 湖北省农业科学院农产品加工与核农技术研究所 | Sturgeon swim bladder protein peptide and application thereof in antioxidation |
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| CN110201140A (en) * | 2019-07-01 | 2019-09-06 | 南通大学 | The application of air bladder polypeptide |
| CN114044807A (en) * | 2021-11-19 | 2022-02-15 | 浙江海洋大学 | Mussel blood fat reducing oligopeptide for treating hyperlipidemia |
| CN114044807B (en) * | 2021-11-19 | 2023-08-22 | 浙江海洋大学 | Mussel hypolipidemic oligopeptide for treating hyperlipidemia |
| CN116003578A (en) * | 2023-02-27 | 2023-04-25 | 湖北省农业科学院农产品加工与核农技术研究所 | Sturgeon swim bladder protein peptide and application thereof in antioxidation |
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