CN107173815A - A kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland - Google Patents
A kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland Download PDFInfo
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- CN107173815A CN107173815A CN201710160726.5A CN201710160726A CN107173815A CN 107173815 A CN107173815 A CN 107173815A CN 201710160726 A CN201710160726 A CN 201710160726A CN 107173815 A CN107173815 A CN 107173815A
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- oxidant
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
It is used for the purposes for preparing antioxidant functional food and food additives the invention discloses a kind of anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland.The present invention is purified by ultrafiltration, HPLC and the anti-oxidant enzymolysis oligopeptide of North Pacific's squid spawn tangled gland is made using North Pacific's squid spawn tangled gland as raw material, and the amino acid sequence of the oligopeptides is Ala Tyr Ala Ser Ser, and molecular weight is 497.50Da.The present invention has also correspondingly provided a kind of anti-oxidation preparation.
Description
Technical field
The present invention relates to a kind of purposes of marine biomaterial, and in particular to a kind of North Pacific squid spawn tangled gland resists
Aoxidize the purposes of enzymolysis oligopeptide.
Background technology
The intermediate product that body is produced in metabolic processes such as superoxide anion（O2-）、H2O2, peroxidating from
By base（ROO）And hydroxy radical（-OH）With very strong oxidation.Under normal physiological conditions, internal antioxidase and non-
The enzyme factor can effectively remove free radical, so as to maintain the poised state of free radical.But in pathological conditions, can be rapid
The peroxidating of lipid is caused to cause the damage of cell membrane, DNA and protein active, so as to cause a variety of human diseases related.Therefore,
It is an important channel for defending organism disease to occur to the removing of interior free yl.Because the antioxidant majority of synthesis is present
Potential safety hazard, therefore, screening is efficient, low toxicity antioxidant turns into the new trend of biology, medical science and food scientific research.
There are some researches show the active peptide from multiple protein all has antioxidation activity, and enzyme solution is that biology is obtained from albumen
One of effective ways of active peptide, have been widely used as a kind of method for improving protein active function and nutritive value.
Protein among diet is mainly absorbed in enteron aisle in the form of polypeptide and amino acid, and polypeptide is in body antioxidation process
Play an important role.In recent years to oceanic biological active peptides, the research of particularly antioxidation active peptides has made great progress.
But marine active peptide not yet forms industrial scale, therefore, the enzyme engineering technology of land active peptide exploitation is used for reference, with marine organisms egg
Bai Ziyuan is raw material, passes through the comprehensive study to techniques such as enzymolysis, preparations, you can develop land protein sources and chemical synthesis institute
Series natural, the biologically active peptide efficiently, novel that can not be produced.
Squid is ocean cephalopod important in the world, and its growth cycle is short, fertility is strong and resource recovery is fast
Speed, is a kind of sustainable marine fishery resources.In recent years, with the development of domestic and international deep sea fishing technology, squid has turned into
The main marine fishing of China and aquatic products processing kind.At present, the annual squid processing capacity of China is up to 40 ~ 500,000 t, occupies the world
One, processing variety is mainly North Pacific squid, Argentinian squid and Peru squid.Squid is because of the fine and smooth nutrition of its meat, wind
Delicious, a variety of necessary amino acid with high protein and low fat, rich in needed by human body, enjoys consumers.In squid
Typically its trunk is processed in process, the accessory substances, wherein gonad such as 50% or so internal organ is produced（Spawn tangled gland,
Spermary, ovary etc.）The 20% of internal organ is accounted for, the accessory substance such as these internal organ is usually processed into feed, is used as the bait of fish；Squid liver
Dirty be extracted after Squid Oil is buried；Obtained from squid spawn tangled gland generally only to make culinary art edible, biological added value it is low and not and
When processing can certain pollution be caused to environment.Extraction on squid spawn tangled gland enzymolysis polypeptide has not yet to see report.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of North Pacific's squid spawn tangled gland with anti-oxidation efficacy
Anti-oxidant enzymolysis oligopeptide.
The present invention is that the technical scheme that solution above-mentioned technical problem is taken is：
A kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland, the amino acid sequence of the enzymolysis oligopeptide is Ala-
Tyr-Ala-Ser-Ser, molecular mass is that the molecular structural formula of 497.50 Da, the i.e. enzymolysis oligopeptide is：
,
Enzymolysis oligopeptide is used for the purposes for preparing antioxidant functional food or food additives.Contain North Pacific's squid spawn tangled gland
Anti-oxidant enzymolysis oligopeptide 0.01-0.5 parts, 1-3 parts of barrenwort, 1-5 parts of the Radix Astragali, 1-10 parts of Ramulus Taxilli, 10-20 parts of the red sage root, radish
Sub- 1-10 parts, 1-10 parts of Radix Angelicae Sinensis, the preparation of 3-20 parts of the root of kudzu vine, the purposes for food additives.
Compared with prior art, the use for a kind of anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland that the present invention is provided
On the way.Meanwhile, North Pacific's anti-oxidant enzymolysis oligopeptide of squid spawn tangled gland prepared by the present invention is that natural material is made through enzymolysis, peace
Entirely, have no toxic side effect, significantly, North Pacific's anti-oxidant enzymolysis oligopeptide of squid spawn tangled gland can be used as antioxidant drug to antioxidation activity
Product, functional food are further researched and developed.
Brief description of the drawings
Fig. 1 is the anti-oxidant enzymolysis oligopeptide amino acid molecular structure formula of the embodiment of the present invention 1；
Fig. 2 is absorbance of the ultrafiltration component 3-5 KD molecules section of the embodiment of the present invention 1 at 280 nm（Absorbance-pipe number）；
Fig. 3 is the DPPH clearance rates at different peaks in Fig. 2 of the present invention；
Fig. 4 is the superoxide anion clearance rate of the enzymolysis polypeptide different molecular section of the embodiment of the present invention 1；
Fig. 5 is clearance rate of the enzymolysis polypeptide different molecular section of the embodiment of the present invention 1 to DPPH；
Fig. 6 is the reducing power of the enzymolysis polypeptide different molecular section of the embodiment of the present invention 1；
Fig. 7 is the Scavenging action to hydroxyl free radical of the enzymolysis polypeptide different molecular section of the embodiment of the present invention 1；
Fig. 8 is the peak I of the embodiment of the present invention 1 high-efficient liquid phase chromatogram at 280 nm.
Embodiment
The present invention is described in further detail with reference to embodiments.
Embodiment 1：
A kind of anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland, the molecular structure of the enzymolysis oligopeptide is：
,
I.e. the amino acid sequence of the enzymolysis oligopeptide is Ala-Tyr-Ala-Ser-Ser, and molecular mass is 497.50 Da.
The preparation method of the anti-oxidant enzymolysis oligopeptide of North Pacific's squid spawn tangled gland, with following steps：
1）North Pacific's squid spawn tangled gland is taken to smash homogenate to pieces, with organized enzyme at peak enzymolysis-ability temperature and pH value condition, plus albumen
Enzyme, insulation are digested；Then inactivate, 0 ~ 4 oC, centrifuge under conditions of 6000 ~ 12000 r/min, take supernatant；
2）Supernatant is taken, ultrafiltration molecular cut off is that the ultrafiltration component of 3-5 KD molecules section is stand-by；
3）Take described ultrafiltration component to carry out agarose gel chromatography separation, collect first occurred at 280 nm in three peaks
The component at peak；
4）The component at first described peak is taken to cross the HPLC further isolated anti-oxidant enzymolysis of North Pacific squid spawn tangled gland
Oligopeptides, its molecular structure is：
。
Above-mentioned organized enzyme is alkali protease, it is preferred that hydrolysis temperature be located at 40~50 DEG C between, hydrolysis time 5~
Between 7h, pH between 8.0~10.0, enzyme concentration be located at 2500~3500U/g, solid-liquid ratio be located at 1：3~1：5.
It is preferred that the condition of enzymolysis be：Temperature 50 C, the h of enzymolysis time 7, U/g, pH value 9.0 of enzyme concentration 3500, material
Liquor ratio is 1：6 be optimum enzymolysis condition
Step 3）The condition of described elution is：Column dimension：10×300-310 mm；Post material：Agarose；Post material granular size：
10 ± 2 µm；Applied sample amount：500 µL；Mobile phase：Ultra-pure water；Eluent flow rate：0.5 mL/min；Detection wavelength：280nm；
Automatic collected volume：3.2 mL/ are managed.
A kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland, the amino acid sequence of the enzymolysis oligopeptide is
Ala-Tyr-Ala-Ser-Ser, molecular mass is that the molecular structural formula of 497.50 Da, the i.e. enzymolysis oligopeptide is：
,
Enzymolysis oligopeptide is used for the purposes for preparing antioxidant functional food or food additives.Contain North Pacific's squid spawn tangled gland
Anti-oxidant enzymolysis oligopeptide 0.01-0.5 parts, 1-3 parts of barrenwort, 1-5 parts of the Radix Astragali, 1-10 parts of Ramulus Taxilli, 10-20 parts of the red sage root, radish
Sub- 1-10 parts, 1-10 parts of Radix Angelicae Sinensis, the preparation of 3-20 parts of the root of kudzu vine, the purposes for food additives.
Embodiment 2：
Materials and methods
Material to be tested
North Pacific's squid spawn tangled gland is purchased in local aquatic product enterprise；Alkali protease is purchased in Asia-Pacific perseverance letter biotechnology（North
Capital）Co., Ltd；DPPH is purchased in West Asia reagent；Luxuriant and rich with fragrance Lip river piperazine, Phen, ferric trichloride, disodium hydrogen phosphate are purchased in me
Fourth Reagent Company；Remaining reagent is that analysis is pure, is purchased in Chemical Reagent Co., Ltd., Sinopharm Group.
Key instrument
SSW-420-2S thermostat water baths, Shanghai Min Yi Electronics Co., Ltd.s；BSA124S type electronic balances, Germany
SartoriusAG companies；DS-1 tissue mashing machines, Shanghai Sample Model Factory；PHS-250PH is counted, Lida Instrument Factory, Shanghai；
The type high speed low temperature centrifugal machines of CF16RX II, HIT；The UV-R type ultrapure water systems of Direct-Q 5, the U.S.
Millipore companies；Cogent u Scale type ultrafiltration systems, Millipore companies of the U.S.；ALPHA 1-4/LDplus types are cold
Lyophilizer, Beijing development in science and technology Co., Ltd of Song Yuan Huaxing；Ultraviolet specrophotometer（752FC）, Shanghai spectral instrument is limited
Company；Agilent-1260 type high performance liquid chromatographs, Agilent company of the U.S..
Method
Experiment of single factor
Proteolysis assay is carried out from alkali protease, it is considered to hydrolysis temperature, enzymolysis time, enzyme concentration, pH value of solution and solid-liquid ratio.
Hydrolysis process is as follows:
Squid spawn tangled gland is smashed → is homogenized → 1 mol/L sodium hydroxides to pieces and adjusts pH value → insulation → being hydrolyzed after enzyme-added → to go out enzyme (90
DEG C 15 min of heating) → centrifugation (10000 r/min, 15 min) → supernatant constant volume → amino nitrogen content (AAN) determines.
Between temperature is located at 40~50 DEG C, hydrolysis time between 5~7h, pH is between 8.0~10.0, enzyme concentration
It is located at 1 positioned at 2500~3500U/g, solid-liquid ratio：3~1：Preferably, obtained free ANN amounts at most, are hydrolyzed hydrolysis effect when 5
Degree is higher.However, temperature 50 C, the h of enzymolysis time 7, U/g, pH value 9.0 of enzyme concentration 3500, solid-liquid ratio are 1：6 is most
Good enzymatic hydrolysis condition.
Sephadex G-25 isolate and purify result
Take the lyophilized sample of 3-5 KD molecules section to carry out Ago-Gel Sephadex G-25 chromatographies, occur three at 280 nm
Peak, i.e. peak I, peak II and peak III, detect that it, to DPPH clearance rates, obtains appearance I after three peak components of collection are freeze-dried respectively
There is preferable Scavenging activity, see Fig. 3, take the molecule section of peak I to carry out HPLC purifying.
HPLC separation, purity detecting and subject peptide sequence measurement result
Peak I prepares result as shown in figure 8, when retention time is about 18min, there is simple spike, peak height is through HPLC chromatogram post
161.2, through amino acid sequence instrument N-terminal Sequence Detection, its amino acid sequence is Ala-Tyr-Ala-Ser-Ser（AYASS）, relatively
Molecular mass is 497.50 Da, and its structural formula is as follows：
After being chromatographed through Ago-Gel G-25, three peaks are drawn, according to the measure to DPPH clearance rates, the effect of appearance I are obtained obvious
Really, DPPH clearance rate is isolated and purified by HPLC up to 61.8% and through amino acid sequence instrument N-terminal Sequence Detection, obtains target
The amino acid sequence of peptide is Ala-Tyr-Ala-Ser-Ser.North Pacific's squid spawn tangled gland can obtain anti-oxidant work by enzymolysis
Property preferable oligopeptides, can develop the intensive processing of squid byproduct, improve biological added value and have great importance.
Finally, in addition it is also necessary to it is noted that listed above is only a specific embodiment of the invention.Obviously, it is of the invention
Above example is not limited to, there can also be many deformations.One of ordinary skill in the art can be straight from present disclosure
Export or all deformations associated are connect, protection scope of the present invention is considered as.
SEQUENCE LISTING
<110>Zhejiang Ocean university
<120>A kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland
<130> zjou-yzs-20161203
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213>It is artificial synthesized
<400> 1
Ala Tyr Ala Ser Ser
1 5
Claims (2)
1. a kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland, it is characterised in that：The amino of the enzymolysis oligopeptide
Acid sequence is Ala-Tyr-Ala-Ser-Ser, and molecular mass is 497.50 Da；Described enzymolysis oligopeptide is anti-oxidant for preparing
The purposes of health food or food additives.
2. a kind of purposes of the anti-oxidant enzymolysis oligopeptide of North Pacific squid spawn tangled gland, it is characterised in that：The molecule of the enzymolysis oligopeptide
Structural formula is, molecular mass is 497.50 Da；Contain the North Pacific
Anti-oxidant enzymolysis oligopeptide 0.01-0.5 parts of squid spawn tangled gland, 1-3 parts of barrenwort, 1-5 parts of the Radix Astragali, 1-10 parts of Ramulus Taxilli, red sage root 10-
20 parts, 1-10 parts of radish seed, 1-10 parts of Radix Angelicae Sinensis, the preparation of 3-20 parts of the root of kudzu vine, the purposes for food additives.
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| CN201710160726.5A CN107173815B (en) | 2017-03-17 | 2017-03-17 | Application of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710160726.5A CN107173815B (en) | 2017-03-17 | 2017-03-17 | Application of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
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| CN107173815A true CN107173815A (en) | 2017-09-19 |
| CN107173815B CN107173815B (en) | 2020-10-30 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109748951A (en) * | 2019-01-09 | 2019-05-14 | 中南大学湘雅医院 | A kind of Radix Angelicae Sinensis antioxidation polypeptide and the preparation method and application thereof |
| CN110759969A (en) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451257A (en) * | 2013-06-17 | 2013-12-18 | 浙江海洋学院 | Method for preparing polypeptide with high antioxidant activity through enzymolysis of internal organs of squids |
| CN104531663A (en) * | 2014-12-17 | 2015-04-22 | 浙江海洋学院 | Immobilized enzyme hydrolysis method for preparing high F value oligopeptide by using minced squid meat |
-
2017
- 2017-03-17 CN CN201710160726.5A patent/CN107173815B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451257A (en) * | 2013-06-17 | 2013-12-18 | 浙江海洋学院 | Method for preparing polypeptide with high antioxidant activity through enzymolysis of internal organs of squids |
| CN104531663A (en) * | 2014-12-17 | 2015-04-22 | 浙江海洋学院 | Immobilized enzyme hydrolysis method for preparing high F value oligopeptide by using minced squid meat |
Non-Patent Citations (1)
| Title |
|---|
| 刘倩茹 等: "北太平洋鱿鱼缠卵腺抗氧化酶解寡肽的制备", 《湖北农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109748951A (en) * | 2019-01-09 | 2019-05-14 | 中南大学湘雅医院 | A kind of Radix Angelicae Sinensis antioxidation polypeptide and the preparation method and application thereof |
| CN109748951B (en) * | 2019-01-09 | 2021-12-03 | 中南大学湘雅医院 | Angelica sinensis antioxidant polypeptide and preparation method and application thereof |
| CN110759969A (en) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
| CN110759969B (en) * | 2019-10-14 | 2021-10-22 | 浙江海洋大学 | Preparation method of antioxidant enzymolysis oligopeptide from peripherical glands of northern pacific squid |
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| CN107173815B (en) | 2020-10-30 |
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