CN109400678A - A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source - Google Patents
A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source Download PDFInfo
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- CN109400678A CN109400678A CN201811214652.XA CN201811214652A CN109400678A CN 109400678 A CN109400678 A CN 109400678A CN 201811214652 A CN201811214652 A CN 201811214652A CN 109400678 A CN109400678 A CN 109400678A
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Abstract
The invention belongs to marine organisms small active peptides fields, and in particular to stichopus japonicus digests the anti-oxidant and DPP-IV inhibitory activity peptide in source, amino acid sequence Ser-Arg-Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser.Fresh stichopus japonicus is added water homogenate to be placed in enzymatic vessel by the present invention, it is digested using compound protease and obtains enzymolysis liquid, enzymolysis liquid obtains micromolecule active polypeptide crude product by membrane separation technique, the crude product is separated through sephadex Sephadex LH-20, small active peptides are obtained, its purity are measured using RP-HPLC, purity is greater than 95%, liquid chromatograph mass spectrography measures its amino acid composition, finally determines the amino acid sequence of the small-molecular peptides.The active small molecular peptide in the stichopus japonicus source, molecular weight is small, purification procedures are simple, it is easily prepared, purity is high, and there is preferable anti-oxidant and DPP-IV inhibitory activity, have the characteristics that natural, safe and efficient, it can be applied to the prevention and treatment of the related diseases such as diabetes as antioxidant and DPP-IV inhibitor, have broad application prospects in food, health care product and field of medicaments.
Description
Technical field
The invention belongs to marine organisms small active peptides fields, and in particular to the anti-oxidant and DPP- in stichopus japonicus enzymatic hydrolysis source
IV inhibitory activity peptide.
Background technique
Polypeptide drugs have the characteristics that bioactivity is high, specific compared with strong, less immunogenic, stability are good, so that more
Peptide medicine becomes the emphasis of new drug development, and more and more polypeptide drugs are developed and are applied to clinic, at present peptide drug owner
To derive from endogenous polypeptide or some natural polypeptides, but these endogenous or natural polypeptides exist biological in-vivo content compared with
It is low, extract it is difficult, be difficult to the problems such as obtaining, along with being constantly progressive for biological enzymolysis technology, digest the active peptides in source at
For the effective way for obtaining active peptides.
The enzymolysis activity polypeptide researched and developed at present is mainly derived from terrestrial organism, such as soybean peptide, corn peptide, gelatine
Former protein peptides etc., and the active peptide of marine source is not developed and utilized well.China's sea area resources are abundant, gather around
There are numerous good protein resources, the polypeptide in marine protein source is that developmental research ocean polypeptide drugs and functional health are eaten
The raw material treasure-house of product isolates anti-oxidant and DPP-IV inhibitory activity peptide from the protein enzymatic hydrolyzate of different marine sources at present
Deng report, such as shellfish, oyster, jellyfish, giant salamander etc..
Sea cucumber belongs to one of big treasure in the world eight, is not only precious food, also the expensive medicinal material of generic name, especially distant stichopus japonicus,
Excellent tonic product even more in sea cucumber, protein content is high, and nutritive value is abundant, has nourishing yin and nourishing blood, antifatigue, adjustment immunity of organism
Numerous effects such as power, anti-aging.Utilize the peptide fragment that can get various active after different protease hydrolyzed sea cucumber protein.At present
About the separation of active peptide in sea cucumber, the only isolated 2 antioxidation active peptides (NO:CN 104402972 of patent report
104356201 A of A, CN), but do not have the report of DPP-IV inhibitory activity peptide.
Dipeptidyl peptidase-IV (DPP-IV) belongs to the serine protease in prolyl oligopeptidase family, is distributed widely in
Human body, when the 2nd amino acids of N-terminal are proline or alanine, DPP-IV can rive albumen or more at the position
Peptide makes its inactivation.Incretin (incretin) is that a kind of polypeptide with promoting insulin secretion generated in enteron aisle swashs
Element, main component glucagon-like-peptide-1 (GLP-1) and glucose-dependent-insulinotropic peptide (GIP), they are in body
It is interior to degrade vulnerable to DPP-IV, so that half-life period is very short to play the role of promoting insulin secretion, therefore, DPP-IV
Inhibitor becomes the drug of new class treatment diabetes B, and the DPP-IV inhibitor listed at present has Xi Gelieting
(sitagliptin), vildagliptin (vildagliptin) etc. achieves significant effect, and DPP-IV inhibitor will
The new therapeutic agent of diabetes B as great prospect has the research increased and Development volue.
Summary of the invention
There are problem, status and development prospects for above-mentioned by the present invention, isolate and purify from stichopus japonicus oligopeptide and obtain one kind
Anti-oxidant and DPP-IV peptide for inhibiting, the bioactive peptide molecule amount is small, is easily isolated purifying, and preparation process is simple, purity is high, activity
By force, the fields such as food, health care product, daily chemical product and medicine bioengineering be can be applied to.
A kind of technical solution that the present invention is taken to achieve the above object are as follows: anti-oxidant and DPP-IV suppression in stichopus japonicus source
Active peptide processed, which is characterized in that its amino acid sequence of the active peptide is Ser-Arg-Pro-Gln-Tyr-Pro-Gln-Tyr-
Pro-Ser。
Using the bioactive peptides sequence as core, any corresponding adjustment or modification that it is carried out.
The active peptide has anti-oxidant and DPP-IV inhibitory activity, can be applied to prepare hypoglycemic or antioxidant food,
In terms of health care product and drug.
The isolation and purification method of the active peptide, comprising the following steps:
The preparation of S1, stichopus japonicus enzymolysis product
The water that 2~3 times of quality volumes are added in fresh stichopus japonicus body wall is made homogenate and is placed in enzymatic vessel, and stichopus japonicus matter is then added
2~5 ‰ compound protease of amount, digests 4 hours at 40~50 DEG C, and the control of enzyme reaction pH value 8.0~9.0, tie by enzymatic hydrolysis
It is warming up to 90 DEG C of enzyme deactivations 10 minutes after beam, obtains stichopus japonicus protein enzymatic hydrolyzate；
The Ultra filtration membrane of S2, stichopus japonicus protein enzymatic hydrolyzate
The stichopus japonicus protein enzymatic hydrolyzate of above-mentioned acquisition is centrifuged 10 minutes at 8000 revs/min, removes particulate material, is then used and is cut
It stays the ultrafiltration membrane that molecular weight is 3000 Da to carry out molecular weight separation, obtains UF membrane sample, sample vacuum at a temperature of 60 DEG C
It is concentrated under reduced pressure into certain volume, is freeze-dried to obtain small active peptides crude product；
S3, small active peptides isolate and purify
Small active peptides crude product in S2 is dissolved in water, the solution that concentration is 100 mg/mL is configured to, it is solidifying using glucan
Glue Sephadex LH-20 column chromatography is isolated and purified, and mobile phase is 30% methanol, and flow velocity is 0.3~0.5 mL/min,
280 nm of eluent measures absorbance, and each peak needed for being collected according to absorbance value, freeze-drying obtains small active peptides；
S4, the purity analysis of small active peptides and amino acid sequence test
Measure purity using high performance liquid chromatography, chromatographic condition is as follows: C18 chromatographic column, mobile phase A are containing percentage by volume
0.05%~0.1% trifluoroacetic acid water, Mobile phase B are acetonitrile, condition of gradient elution are as follows: 0~15min, 3%B, 15~20min, 3%
~10%B, 20~30min, 10%B~20%B, 30~40min, 20%B~35%B, flow velocity are 1.0 mL/min, and Detection wavelength is
220 or 280 nm, the retention time of small-molecular peptides are 31min, 95% or more purity；
Determined amino acid sequence is carried out using high performance liquid chromatography-tandem mass, determines its amino acid sequence are as follows: Ser-Arg-
Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser。
The quality proportioning of compound protease in the step S1 are as follows: neutral proteinase: papain: flavor protease
=(2 ~ 4): (3 ~ 5): (3 ~ 5).
Sephadex Sephadex LH-20 column internal diameter in the step S3 is 3.0 cm, 100 cm of column length.
The C18 chromatography column internal diameter be 4.6 mm, 250 mm of column length, 5 μm of granularity.
Beneficial effects of the present invention:
(1) the distant stichopus japonicus for selecting high nutritive value is digested using the specific compound protein enzyme preparation that multiple screening obtains,
Efficient sea cucumber oligopeptide is obtained after after further Ultra filtration membrane, for the product based on small-molecular peptides, relative molecular weight is small
It is greater than 95% in the protolysate proportion of 1000 Da.
(2) it is separated using gel column chromatography Sephadex LH-20, finally obtains the active peptide of a high-purity, it is high
Effect liquid phase chromatogram analysis shows that, purity be greater than 95%, through Mass Spectrometric Identification amino acid sequence are as follows: Ser-Arg-Pro-Gln-Tyr-
Pro-Gln-Tyr-Pro-Ser is retrieved through online database BIOPEP and EROP-Moscow, and the sequence is new small molecule
Active peptide.Molecular weight is small, and purification procedures are simple, is easily obtained, which has stronger antioxidant activity and DPP-
IV inhibitory activity belongs to food-borne anti-oxidation peptide, is expected to replace the anti-oxidation peptide of synthesis, while having potential reduction blood glucose
Effect can be used for diabetes relevant food, health care product exploitation or as lead compound.
(3) problem lower present invention also improves sea cucumber absorbing proteins utilization efficiency is the further essence of Holothurian Resources
Deep processing provides reliable basis.
Detailed description of the invention
Fig. 1 is that stichopus japonicus activity peptide analysis of the present invention purifies and analyze and identify flow chart.
Fig. 2 is that gel column chromatography Sephadex LH-20 separates curve graph.
Fig. 3 is the high-efficient liquid phase chromatogram of stichopus japonicus active peptide.
Fig. 4 is the DPPH free radical scavenging activity figure of stichopus japonicus peptide and its separation component.
Fig. 5 is the DPP-IV inhibitory activity figure of stichopus japonicus peptide and its separation component.
Specific embodiment
The present invention is further described with embodiment with reference to the accompanying drawing, but the present invention is not limited to specific embodiment.
Embodiment 1
A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source, its amino acid sequence of the active peptide are Ser-Arg-
Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser。
Using the bioactive peptides sequence as core, any corresponding adjustment or modification that it is carried out.
The active peptide has anti-oxidant and DPP-IV inhibitory activity, can be applied to prepare hypoglycemic or antioxidant food,
In terms of health care product and drug.
Embodiment 2
As shown in Figure 1, the isolation and purification method of the anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source described in embodiment 1,
The following steps are included:
The preparation of S1, stichopus japonicus enzymolysis product
The water that 2 times of quality volumes are added in fresh stichopus japonicus body wall is made homogenate and is placed in enzymatic vessel, and stichopus japonicus quality is then added
2 ‰ compound protease, the quality proportioning of compound protease are as follows: neutral proteinase: papain: flavor protease=2:3:
3, it is digested 4 hours at 40 DEG C, the control of enzyme reaction pH value is warming up to 90 DEG C of enzyme deactivations 10 minutes after 8.0, enzymatic hydrolysis, obtains
Stichopus japonicus protein enzymatic hydrolyzate.
The Ultra filtration membrane of S2, stichopus japonicus protein enzymatic hydrolyzate
The stichopus japonicus protein enzymatic hydrolyzate of above-mentioned acquisition is centrifuged 10 minutes at 8000 revs/min, removes particulate material, is then used and is cut
It stays the ultrafiltration membrane that molecular weight is 3000 Da to carry out molecular weight separation, obtains UF membrane sample, sample vacuum at a temperature of 60 DEG C
It is concentrated under reduced pressure into certain volume, is freeze-dried to obtain small active peptides crude product.
S3, small active peptides isolate and purify
Small active peptides crude product in S2 is dissolved in water, the solution that concentration is 100 mg/mL is configured to, it is solidifying using glucan
Glue Sephadex LH-20 column chromatography (3.0 × 100cm) is isolated and purified (as shown in Figure 2), and mobile phase is 30% methanol,
Flow velocity is 0.3 mL/min, and 280 nm of eluent measures absorbance, and detached peaks needed for being collected according to absorbance value are freeze-dried
To small active peptides.
The Purity and determined amino acid sequence of S4, small active peptides
As shown in figure 3, measuring purity using high performance liquid chromatography, chromatographic condition is as follows: Yi Lite C18 chromatographic column (4.6 mm ×
250 mm, 5 μm), mobile phase A is 0.05% trifluoroacetic acid water (V/V), and Mobile phase B is acetonitrile, condition of gradient elution are as follows: 0~15
Min, 3%B, 15~20 min, 3%~10%B, 20~30 min, 10%B~20%B, 30~40 min, 20%B~35%B, flow velocity
For 1.0 mL/min, Detection wavelength is 280 nm, and the retention time of small-molecular peptides is 31 min, 95% or more purity.
Determined amino acid sequence is carried out using high performance liquid chromatography-tandem mass, determines its amino acid sequence are as follows: Ser-
Arg-Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser。
The active testing of small active peptides:
The measurement of DPPH free radical scavenging ability:
The solution of small active peptides distilled water 1mg/mL concentration after precise freeze-drying.0.1mmol/ is prepared in advance
L DPPH solution, is kept in dark place.
A1: taking the 100 μ L of sample solution of various concentration, each that 100 μ L 0.1mmol/lDPPH are added, and stands 30 after shaking up
Min measures absorbance value with microplate reader under 517nm wavelength, the sample of each concentration do three groups it is parallel.
A2: taking the 100 μ L of sample solution of various concentration, each that 100 μ L dehydrated alcohols are added, and stands 30min after shaking up,
Absorbance value is measured under 517nm wavelength with microplate reader.
A0: taking 100 μ L DPPH, and 100 μ L dehydrated alcohols are added, 30min are stood after shaking up, with microplate reader in 517nm
Absorbance value is measured under wavelength.
Calculation formula are as follows: DPPH clearance rate=[(A0- (A1-A2))/A0] × 100%, as a result as shown in Figure 4.
The test of DPP-IV inhibitory activity:
Small active peptides after precise freeze-drying are configured to 5 mg/mL with 0.1mol/L Tris-HCl (pH=8.0)
Stock solution.Substrate Gly-Pro-pNA is configured to the molten of 50 mmol/L concentration with 0.1mol/L Tris-HCl (pH=8.0)
Liquid.DPP-IV is configured to the solution of 1U/L with 0.1mol/L Tris-HCl (pH=8.0).
Small active peptides stock solution addition DPP-IV solution is made into the solution of various concentration, small molecule is replaced with pure water
Active peptide solution is as negative control, after ice bath 30 minutes, takes 100 μ L to be added in 96 orifice plates, it is molten that 100 μ L substrates are then added
Liquid, at the same using 0.1mol/L Tris-HCl (pH=8.0) replace substrate solution as blank control, 37 DEG C incubation 60 minutes, 405
Absorbance is measured at nm, calculates inhibiting rate.
Inhibiting rate=[(A negative control-A negative control blank)-(A inhibits drug-A to inhibit drug blank)]/(A feminine gender is right
According to-A negative control blank) × 100%, as a result as shown in Figure 5.
The small-molecular peptides have preferable antioxidant activity and DPP-IV inhibitory activity to active testing as the result is shown, can be used as food
The antioxidant of source property can also be applied to food, health care product and drug of the related diseases such as diabetes etc..
Embodiment 3
Each step of the isolation and purification method of the anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source described in the present embodiment
It is in the same manner as in Example 2, difference are as follows:
(1) water that 2.5 times of quality volumes are added in fresh stichopus japonicus body wall in step S1 is made homogenate and is placed in enzymatic vessel, then plus
Enter 3.5 ‰ compound protease of stichopus japonicus quality, the quality proportioning of compound protease are as follows: neutral proteinase: papain: wind
Taste protease=3:4:4 digests 4 hours at 45 DEG C, and enzyme reaction pH value is controlled 8.5；
(2) mobile phase is 30% methanol in step S3, and flow velocity is 0.4 mL/min；
(3) mobile phase A is 0.75% trifluoroacetic acid water (V/V) in step S4；
(4) Detection wavelength is 220nm in step S4.
Embodiment 4
Each step of the isolation and purification method of the anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source described in the present embodiment
It is in the same manner as in Example 2, difference are as follows:
(1) water that 3 times of quality volumes are added in fresh stichopus japonicus body wall in step S1 is made homogenate and is placed in enzymatic vessel, then plus
Enter 5 ‰ compound protease of stichopus japonicus quality, the quality proportioning of compound protease are as follows: neutral proteinase: papain: flavor
Protease=4:5:5 digests 4 hours at 50 DEG C, and enzyme reaction pH value is controlled 9.0；
(2) mobile phase is 30% methanol in step S3, and flow velocity is 0.5 mL/min；
(3) mobile phase A is 0.1% trifluoroacetic acid water (V/V) in step S4.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
SEQUENCE LISTING
<110>the dark blue peptide science and technology in Dalian researches and develops Co., Ltd
<120>the anti-oxidant and DPP-IV peptide for inhibiting in a kind of stichopus japonicus source
<130> 0002S
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Apostichopus japonicus
<400> 1
Ser Arg Pro Gln Tyr Pro Gln Tyr Pro Ser
1 5 10
SEQUENCE LISTING
<110>the dark blue peptide science and technology in Dalian researches and develops Co., Ltd
<120>the anti-oxidant and DPP-IV peptide for inhibiting in a kind of stichopus japonicus source
<130> 0002S
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Apostichopus japonicus
<400> 1
Ser Arg Pro Gln Tyr Pro Gln Tyr Pro Ser
1 5 10
Claims (7)
1. a kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source, which is characterized in that its amino acid sequence of the active peptide
It is classified as Ser-Arg-Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser.
2. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 1, which is characterized in that with described
Bioactive peptides sequence is core, any corresponding adjustment or modification carried out to it.
3. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 1, which is characterized in that the work
Property peptide have anti-oxidant and DPP-IV inhibitory activity, can be applied to prepare hypoglycemic or antioxidant food, health care product and drug side
Face.
4. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 1, which is characterized in that the work
The isolation and purification method of property peptide, comprising the following steps:
The preparation of S1, stichopus japonicus enzymolysis product
The water that 2~3 times of quality volumes are added in fresh stichopus japonicus body wall is made homogenate and is placed in enzymatic vessel, and stichopus japonicus matter is then added
2~5 ‰ compound protease of amount, digests 4 hours at 40~50 DEG C, and the control of enzyme reaction pH value 8.0~9.0, tie by enzymatic hydrolysis
It is warming up to 90 DEG C of enzyme deactivations 10 minutes after beam, obtains stichopus japonicus protein enzymatic hydrolyzate；
The Ultra filtration membrane of S2, stichopus japonicus protein enzymatic hydrolyzate
The stichopus japonicus protein enzymatic hydrolyzate of above-mentioned acquisition is centrifuged 10 minutes at 8000 revs/min, removes particulate material, is then used and is cut
It stays the ultrafiltration membrane that molecular weight is 3000 Da to carry out molecular weight separation, obtains UF membrane sample, sample vacuum at a temperature of 60 DEG C
It is concentrated under reduced pressure into certain volume, is freeze-dried to obtain small active peptides crude product；
S3, small active peptides isolate and purify
Small active peptides crude product in S2 is dissolved in water, the solution that concentration is 100 mg/mL is configured to, it is solidifying using glucan
Glue Sephadex LH-20 column chromatography is isolated and purified, and mobile phase is 30% methanol, and flow velocity is 0.3~0.5 mL/min,
280 nm of eluent measures absorbance, and each peak needed for being collected according to absorbance value, freeze-drying obtains small active peptides；
S4, the purity analysis of small active peptides and amino acid sequence test
Measure purity using high performance liquid chromatography, chromatographic condition is as follows: C18 chromatographic column, mobile phase A are containing percentage by volume
0.05%~0.1% trifluoroacetic acid water, Mobile phase B are acetonitrile, condition of gradient elution are as follows: 0~15min, 3%B, 15~20min, 3%
~10%B, 20~30min, 10%B~20%B, 30~40min, 20%B~35%B, flow velocity are 1.0 mL/min, and Detection wavelength is
220 or 280 nm, the retention time of small-molecular peptides are 31min, 95% or more purity；
Determined amino acid sequence is carried out using high performance liquid chromatography-tandem mass, determines its amino acid sequence are as follows: Ser-Arg-
Pro-Gln-Tyr-Pro-Gln-Tyr-Pro-Ser。
5. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 4, which is characterized in that the step
The quality proportioning of compound protease in rapid S1 are as follows: neutral proteinase: papain: flavor protease=(2 ~ 4): (3 ~ 5):
(3 ~ 5).
6. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 4, which is characterized in that the step
Sephadex Sephadex LH-20 column internal diameter in rapid S3 is 3.0 cm, 100 cm of column length.
7. a kind of anti-oxidant and DPP-IV peptide for inhibiting in stichopus japonicus source according to claim 4, which is characterized in that described
C18 chromatography column internal diameter be 4.6 mm, 250 mm of column length, 5 μm of granularity.
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| CN110845578A (en) * | 2019-12-10 | 2020-02-28 | 扬州大学 | Novel intestinal mucosa antioxidant active peptide and preparation method thereof |
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| CN111825745A (en) * | 2020-07-29 | 2020-10-27 | 中国科学院华南植物园 | Immunoregulatory active heptapeptide and preparation method and application thereof |
| CN111978374A (en) * | 2020-07-29 | 2020-11-24 | 中国科学院华南植物园 | Immunoregulation active hexapeptide and preparation method and application thereof |
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| CN113151388A (en) * | 2021-04-19 | 2021-07-23 | 国肽生物工程（常德）有限公司 | Sea cucumber peptide with antioxidant and DPP-IV (dipeptidyl peptidase-IV) inhibiting functions and preparation method thereof |
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| CN114163500A (en) * | 2021-11-12 | 2022-03-11 | 广东海洋大学 | Oyster protein source anti-skin photoaging active peptide and preparation method and application thereof |
| CN114686551A (en) * | 2022-03-16 | 2022-07-01 | 江南大学 | Pea protein source DPP-IV inhibitory peptide and screening method thereof |
| CN114805479A (en) * | 2022-04-13 | 2022-07-29 | 锡林郭勒职业学院 | Bioactive peptide with dipeptidyl peptidase IV inhibitory activity |
| CN115028706A (en) * | 2022-01-05 | 2022-09-09 | 昆明理工大学 | Application of fetoprotein small molecule peptide in preparation of dipeptidyl peptidase IV inhibitor |
| CN117925765A (en) * | 2024-03-21 | 2024-04-26 | 山东助邦生物科技有限公司 | Biological processing method of food sea cucumber |
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