CN104479001B - The preparation and application of κ-casein derived biologically active peptide - Google Patents
The preparation and application of κ-casein derived biologically active peptide Download PDFInfo
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- CN104479001B CN104479001B CN201410809413.4A CN201410809413A CN104479001B CN 104479001 B CN104479001 B CN 104479001B CN 201410809413 A CN201410809413 A CN 201410809413A CN 104479001 B CN104479001 B CN 104479001B
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- biologically active
- ntvpa
- active polypeptide
- polypeptide
- milk
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to albumen field, and in particular to multiple milk-derived biologically active polypeptide NTVPA, VVTIL, PKKNQ from κ caseins.Promote experiment by antioxidation in vitro experiment and ion vitro immunization function, it was found that biologically active polypeptide of the invention has different antioxidation biology activity and promotes the activity of cellular immunity；Result of study shows that three kinds of milk-derived biologically active peptides of the present invention have the function of potentially to slow down aging as small molecule material.In addition, digestion experiment is simulated the result shows that three biologically active polypeptides found are stablized under the conditions of common animal body is digested, will not further it be degraded, it can directly be absorbed by animal body and play its bioactive functions having, be of great significance to developing with anti-oxidation function, the dairy products of enhancing immune function and anti-aging, health products and medicine tool.
Description
Technical field
The present invention relates to albumen field, and in particular to a variety of biologically active polypeptides from κ-casein and its prepare and
Using.
Background technology
Since biologically active peptide is as sanatory bioactive ingredients, has and transmit physiologic information, adjust physiology work(
The effect of energy, for the dimension of the normal physiological activity of the systems such as the nerve of human body, digestion, reproduction, growth, movement, metabolism, circulation
Hold extremely important.They not only have the effect such as antiviral, bacterium, anti-hypertension, norcholesterol, but also are used as immunomodulator
Have the function of to adjust immune response, antitumor etc.；Free radical, which can be removed, to be had effects that to slow down aging, and is current international food
The function factor of the most popular research topic in boundary and great development prospect.Various active peptides are used frequently as functional food adding ingredient
Produced in the practice of food, particularly have been achieved with good result in the application aspect of milk beverage, food additives.Milk-derived is given birth to
The function of thing active peptide biology and the positive attention of healthy influence to consumer.
In recent years, more and more scientific investigations showed that much the small peptides from bioprotein are lived with various biological
Property, such as hormonal action, immunological regulation, antithrombotic, anti-hypertension, norcholesterol, antibacterial, antiviral, antitumaous effect.Meanwhile
Research finds that small peptide preferably can be absorbed and utilized by human body, and amino acid can only be degraded to just by changing traditional protein
The viewpoint that can be absorbed and used.In numerous bioproteins various cow's milk proteins be very important biologically active polypeptide source it
One, they are decomposed by lactic acid bacteria and utilize, and there occurs a series of biochemical reactions, protein is changed into polypeptide or free
Amino acid, is finally digested or is directly entered by the absorption and transport of intestinal epithelial cell the blood circulation of human body,
Play its biological action.
Therefore, these bioactive micro peptides not only become the natural resources treasure-house of screening medicine, and functional food work
The primary raw material component of industry.At present, biologically active peptide preparation and its application and development have become the heat of research in world wide
Point.
Immune-active peptides are to obtain and prove that one kind biology of its physiological activity is living from breast first after opioid peptides discovery
Property peptide.1981, Jolles et al. had found first, and using trypsin hydrolysis people lactoprotein, from hydrolysate obtaining one kind exempts from
Epidemic disease active peptide segment, its amino acid sequence are Val-Glu-Pro-Ile-Pro-Tyr, which is proved to can in testing in vitro
Strengthen phagocytosis of the Turnover of Mouse Peritoneal Macrophages to sheep erythrocyte, intravenous injection, then can strengthen mouse to pneumonia kirschner
The resistivity of bar infection.Elitsur et al. obtains Arg-Tyr-Leu-Gly-Tyr-Leu- through pepsin casein
Glu and Arg-Tyr-Leu-Gly-Tyr-Leu, research have shown that two small peptide not only has opioid activity, it may have immune to adjust
Section activity, can strengthen lymphopoiesis, improve natural killer cells (NK) ability, promote to bite the movement of neutrocyte.
Few brightness et al. obtains biologically active polypeptide QEPVL and its catabolite QEPV using Lactobacillus helveticus fermentation skimmed milk, passes through
Antioxidation in vitro experiment, ion vitro immunization function promote experiment, demonstrate polypeptide QEPV with preferable antioxidation biology activity
With the activity for promoting cellular immunity, free radical that on the one hand can be in removing machine body reduces injury of the free radical to human body；It is another
Aspect, biologically active polypeptide QEPVL and QEPV can also strengthen immunity of organisms, enhancing lymphocyte, the propagation of macrophage
Ability, makes the increase of the macrophage nitric oxide amount of inducing, and promotees Factor of Macrophage.
Although being implied with many biologically active peptide sequences in cow's milk protein, these bio-active peptide sequences only have
Competence exertion is discharged by certain means to act on.The main means for obtaining lactoprotein active peptide have two kinds：Hair
Ferment, is obtained using microorganism (lactic acid bacteria) fermentation milk protein；Enzymolysis, including the digestive enzyme from animal and from plant and
The protease of microorganism.
Result of study shows milk-derived biologically active peptide as small molecule material, with its molecular weight it is low, it is active it is strong, use
Few unique advantage is measured, is more and more attracted people's attention.So they are used not only as functional food ingredient
There is adjusting immune response, antitumor etc. in the manufacture of various functions food, and as immunomodulator；Can be clear
Except free radical has effects that to slow down aging.In addition, most milk-derived biologically active peptides can resist the digestion of intestines and stomach, small
Enteral is absorbed by organisms in the form of complete, without being broken down into single amino acids, has absorption easy to digest, edible safety
It is high.Therefore the body that can improve of milk-derived biologically active peptide resists the stimulation of extraneous undesirable element, reduces body incidence
Deng functional characteristic attention, it is expected to become the non-medication thing with removing free radical, reducing cancer probability and anti-aging
Matter raw material, has boundless application prospect in functional food and field of medicaments, from now on will become international food circle and
The most popular research topic of biopharmaceutical production industry, to improve level of human health, not falling ill and fall ill less in other words, delay to decline
Always, the extension service life plays an important role.
The content of the invention
It is an object of the invention to provide：
A kind of separated biologically active polypeptide NTVPA, it includes
(a) by Asn-Thr-Val-Pro-Ala (SEQ ID NO：1) polypeptide of the amino acid composition shown in；
(b) or in SEQ ID NO：One or several amino acid are substituted, lack or added in amino acid sequence shown in 1
And with isoreactivity as derived from (a) polypeptide；
A kind of separated biologically active polypeptide VVTIL, it includes
(c) by Val-Val-Thr-Ile-Leu (SEQ ID NO：2) polypeptide of the amino acid composition shown in；
(d) or in SEQ ID NO：One or several amino acid are substituted, lack or added in amino acid sequence shown in 2
And with isoreactivity as derived from (c) polypeptide；
A kind of separated biologically active polypeptide PKKNQ, it includes
(e) by Pro-Lys-Lys-Asn-Gln (SEQ ID NO：3) polypeptide of the amino acid composition shown in；
(f) or in SEQ ID NO：One or several amino acid are substituted, lack or added in amino acid sequence shown in 3
And with isoreactivity as derived from (e) polypeptide.
Preferably, the source of the biologically active polypeptide is milk-derived.
The amino acid sequence of κ-casein is existing technology, specific amino acid sequence such as SEQ ID NO:Shown in 4：
Met Met Lys SerPhePheLeu Val ValThr Ile LeuAlaLeuThrLeuGlyAlaGlnGlu
GlnAsnGlnGluGlu Pro Ile ArgCysGlu Lys Asp GluArgPhePheSer Asp Lys Ile Ala Lys
Tyr Ile Pro Ile Gln Tyr Val LeuSerArg Tyr Pro Ser Tyr GlyLeuAsn Tyr Tyr
GlnGln Lys Pro Val AlaLeu Ile AsnAsnGlnPheLeu Pro Tyr Pro Tyr TyrAla Lys Pro
AlaAla Val ArgSer Pro AlaGln Ile LeuGlnTrpGln Val LeuSerAsnThr Val Pro Ala
Lys SerCysGlnAlaGln Pro ThrThr Met AlaArg His Pro His Pro His Leu SerPhe Met
Ala Ile Pro Pro Lys LysAsnGln Asp Lys ThrGlu Ile Pro Thr Ile Asn Thr Ile
AlaSerGlyGlu Pro ThrSerThrSerThr Pro ThrThrGluSerThr Val Leu Gly Asp Ser Pro
Glu Val Ile GluSer Pro ProGlu Ile AsnThr Val Gln Val ThrSer ThrAla Val。
The biologically active polypeptide NTVPA of the present invention is milk-derived, is derive specifically from κ caseins, and be κ caseins (SEQ
ID NO:4) amino acid residue of the 102nd~106.
The biologically active polypeptide VVTIL of the present invention is milk-derived, is derive specifically from κ caseins (SEQ ID NO:The 8th 4)~
The amino acid residue of 12.
The biologically active polypeptide PKKNQ of the present invention is milk-derived, is derive specifically from κ caseins (SEQ ID NO:4) the 131st
The amino acid residue of~135.
Preferably, described biologically active polypeptide NTVPA, VVTIL, PKKNQ are respectively provided with antioxidation activity in vitro and enhancing machine
The function of body immunity.
Biologically active polypeptide NTVPA, VVTIL, PKKNQ of the present invention can pass through the method for genetic engineering and chemistry side
Method is artificial synthesized, can also from dairy products by isolating and purifying, enzyme degraded method obtain.
The invention also discloses the nucleotide fragments of coding aforementioned biological active peptides NTVPA, VVTIL, PKKNQ.
The amino acid sequence and nucleotides sequence of κ-casein are classified as existing technology, encode κ-casein the 102nd~106
The biologically active polypeptide NTVPA of the nucleotide fragments energy encoding mature of amino acid residue, encodes the 8th~12 bit amino of κ-casein
The biologically active polypeptide VVTIL of the nucleotide fragments energy encoding mature of sour residue, encodes the 131st~135 bit amino of κ-casein
The biologically active polypeptide PKKNQ of the nucleotide fragments energy encoding mature of sour residue.
Second aspect of the present invention discloses the preparation method of aforementioned biological active peptides, and step is as follows：
1) ferment：Lactobacillus helveticus (Lactobacillus helveticus) is added to progress anaerobism hair in skimmed milk
Ferment, obtains Lactobacillus helveticus acidified milk；
2) the thick of polypeptide carries：Low-temperature centrifugation separation is carried out to the Lactobacillus helveticus acidified milk of step 1), takes supernatant；
3) purifying of polypeptide：
A. hyperfiltration treatment is carried out to the supernatant of step 2), collects filtrate；
B. Solid Phase Extraction post separation：The filtrate of collection uses Waters Sep-pak C18 Solid Phase Extraction column extractings, collects
Biologically active polypeptide mixture；
4) digestion of polypeptide and stability：Using two step enzymatic isolation method enzymolysis steps 3) biologically active polypeptide mixture, obtain
Biologically active polypeptide mixture after must being digested；Enzyme is pepsin used by the first step digests, and second step enzymolysis is adopted
Enzyme is pancreatin.
Skimmed milk of the present invention is cow's milk by ungrease treatment, and fat content is less than 0.1% in usual skimmed milk.
Preferably, in step 1), Lactobacillus helveticus for Lactobacillus helveticus (Lactobacillus helveticus,
CICC6024)。
Preferably, in step 1), the condition of the anaerobic fermentation is：36~38 DEG C of fermentation temperature, 6~8h of fermented and cultured；
More preferably 37 DEG C of fermentation temperature, fermented and cultured 7h.
Preferably, in step 2), the condition of the low-temperature centrifugation is：4 DEG C, 8000~10000rpm, centrifugation 15~
30min。
Preferably, it is the super filter tube that molecular cut off is respectively 3kDa in step 3) a, used by the hyperfiltration treatment.
More preferably, in step 3) a, in the process of ultrafiltration treatment, rotating speed 4800r/min, time 30min, centrifugation
Temperature is 4 DEG C.
Preferably, in step 3) b, Solid Phase Extraction post separation, specific method is：Activation and balance Waters Sep-pak
C18 solid-phase extraction columns；The filtrate collected in step 3) a is diluted rear loading, using elution, is collected obtained
Eluent, i.e., comprising biologically active polypeptide mixture.
Preferably, in step 3) b, in solid-phase extraction column partition method, used eluent is methanol and ddH2The mixing of O
Liquid, the methanol and ddH2Methanol and ddH in the mixed liquor of O2The volume ratio of O is 80:20, the methanol and ddH2The mixed liquor of O
In contain 0.1% (v/v) formic acid.
Preferably, in step 3) b, in solid-phase extraction column partition method, consolidated using methanol activation Waters Sep-pak C18
Phase extraction column；It is preferred that 2ml methanol.
Preferably, in step 3) b, in solid-phase extraction column partition method, using ddH2O balances Waters Sep-pak C18 consolidate
Phase extraction column；It is preferred that 1mlddH2O。
Preferably, in step 3) b, in solid-phase extraction column partition method, the filtrate collected in step 3) a is diluted 50 times
Loading afterwards, using 400ul elutions.
Preferably, in step 3) b solid-phase extraction columns partition method, first using 2mL methanol activation Waters Sep-pak C18
Solid-phase extraction column, using 1mLddH2O balances Waters Sep-pak C18 solid-phase extraction columns；Loading sample is 2mL；By step
3) filtrate collected in a dilutes 50 times, using 400 μ L elutions.
Preferably, it is molten to concretely comprise the following steps the mixture containing polypeptide for obtaining step 3) for step 4) the two steps enzymatic isolation method
In sterile deionized water, it is 2.0 ± 0.1 to adjust pH value, adds pepsin, reaction solution is obtained, in 37 ± 0.5 DEG C of perseverance
Insulation reaction 90min in tepidarium, obtains first step enzymolysis liquid；The pH value of first step enzymolysis liquid is adjusted to 7.5 ± 0.1, and
Pancreatin is added, the insulation reaction 150min in 37 ± 0.5 DEG C of water bath with thermostatic control, obtains second step enzymolysis liquid；Second step is digested
Liquid inactivates enzyme using Boiling bath method, time 5min, obtains enzymolysis product；Powdery enzymolysis product is obtained using freeze-drying.
It is furthermore preferred that the Boiling bath method is 95 DEG C of immersion methods.
Preferably, the additive amount of the step 4) pepsin is pepsin 10~30mg/g substrates；The pancreatin
Additive amount is pancreatin 30~50mg/g substrates.
More preferably, the additive amount of the step 4) pepsin is pepsin 20mg/g substrates；The addition of the pancreatin
Measure as pancreatin 40mg/g substrates.
Preferably, the eluting peak for the polypeptide that molecular weight is 501.26Da is extracted, is biologically active polypeptide NTVPA；Extraction
Molecular weight is the eluting peak of the polypeptide of 544.38Da, is biologically active polypeptide VVTIL；It is the more of 614.37Da to extract molecular weight
The eluting peak of peptide, is biologically active polypeptide PKKNQ.
In the present invention, it is known that the molecular weight of NTVPA, the eluting peak that extraction molecular size is 501.26Da, is this hair
Bright biologically active polypeptide NTVPA.Specifically, NTVPA molecular sizes of the present invention are its retention time of the eluting peak of 501.26Da
For 1.93min.
In the present invention, it is known that the molecular weight of VVTIL, the eluting peak that extraction molecular size is 544.38Da, is this hair
Bright biologically active polypeptide VVTIL.Specifically, NTVPA molecular sizes of the present invention are its retention time of the eluting peak of 544.38Da
For 7.30min.
In the present invention, it is known that the molecular weight of PKKNQ, the eluting peak that extraction molecular size is 614.37Da, is this hair
Bright biologically active polypeptide PKKNQ.Specifically, PKKNQ molecular sizes of the present invention are its retention time of the eluting peak of 614.37Da
For 10.36min.
Third aspect present invention discloses aforementioned biological active peptides NTVPA, VVTIL, PKKNQ or derivatives thereof and is preparing
Application in anti-oxidant and/or enhancing immunity of organisms food, health products and medicine.
The present invention biologically active polypeptide NTVPA, VVTIL, PKKNQ, or derivatives thereof can be used for the dairy products such as Yoghourt
And various food, reduction free radical are to the cosmetics of skin damage；And due to the present invention biologically active polypeptide NTVPA,
VVTIL, PKKNQ can directly be absorbed by intestines and stomach not to be degraded, therefore can be used for preparing the health products for improving immunity,
Or it is used to prepare with anti-oxidant and/or enhancing immunity of organisms medicine.
Fourth aspect present invention discloses a kind of anti-oxidation medicine, comprising aforementioned biological active peptides NTVPA, VVTIL,
The derivative of PKKNQ or aforementioned biological active peptides.
Fifth aspect present invention discloses a kind of enhancing immunity of organisms medicine, comprising aforementioned biological active peptides NTVPA,
The derivative of VVTIL, PKKNQ or aforementioned biological active peptides.
Sixth aspect present invention discloses a kind of health product of potential anti-aging/anti-aging, includes aforementioned biological
The derivative of active peptides NTVPA, VVTIL, PKKNQ or aforementioned biological active peptides.
The derivative of the polypeptide, refers on the amino acid side groups of polypeptide, aminoterminal or c-terminus carry out hydroxyl
Change, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide of the present invention has the beneficial effect that：Milk-derived biologically active polypeptide NTVPA, VVTIL of the present invention,
PKKNQ has good antioxidation activity and promotes immunity of organisms activity；On the one hand free radical that can be in removing machine body, subtracts
Few injury of the free radical to human body；On the other hand, biologically active polypeptide of the invention can also strengthen immunity of organisms, and enhancing is huge
The phagocytic function of phagocyte, improves the ability that body resists extraneous pathogenic infection, reduces body incidence, and simulate digestion
Test result indicates that three biologically active polypeptides found are stablized under the conditions of common animal body is digested, will not be further
Degraded, can directly be absorbed by animal body and play its bioactive functions for having, to exploitation with anti-oxidation function,
Strengthen immune function, the food to slow down aging, health products and medicine tool is of great significance and application value.
Brief description of the drawings
Fig. 1：The elution collection of illustrative plates of Lactobacillus helveticus acidified milk digestion product
Fig. 2：Mass chromatography extraction figure (m/z=501.26)
Fig. 3：Mass-to-charge ratio is the first mass spectrometric figure of 501.26 fragment
Fig. 4：Mass chromatography extraction figure (m/z=544.38)
Fig. 5：Mass-to-charge ratio is the first mass spectrometric figure of 544.38 fragment
Fig. 6：Mass chromatography extraction figure (m/z=614.37)
Fig. 7：Mass-to-charge ratio is the first mass spectrometric figure of 614.37 fragment
Fig. 8：Mass-to-charge ratio is the second order ms figure of 501.26 fragment
Fig. 9：The az for the sequence that mass-to-charge ratio obtains for 501.26 predictions, by crack conditions (NTVPA)
Figure 10：Mass-to-charge ratio is the second order ms figure of 544.38 fragment
Figure 11：The az for the sequence that mass-to-charge ratio obtains for 544.38 predictions, by crack conditions (VVTIL)
Figure 12：Mass-to-charge ratio is the second order ms figure of 614.37 fragment
Figure 13：The az for the sequence that mass-to-charge ratio obtains for 614.37 predictions, by crack conditions (PKKNQ)
Figure 14：Control group mass chromatography extraction figure (m/z=544.38)
Figure 15：Control group mass-to-charge ratio is the first mass spectrometric figure of 544.38 fragment
Figure 16：Control group mass-to-charge ratio is the second order ms figure of 544.38 fragment
Figure 17：The az for the sequence that control group mass-to-charge ratio obtains for 544.38 predictions, by crack conditions (VVTIL)
Figure 18：Trolox standard curves
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to the prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of embodiment 1 active peptide NTVPA, VVTIL, PKKNQ
First, the preparation of Lactobacillus helveticus acidified milk
Using the skimmed milk (degreasing of 12wt% of skimmed milk power (New Zealand NZMP boards skimmed milk power) and water configuration 12wt%
Breast is prepared as 12g skimmed milk powers being added in 88g water, similarly hereinafter).Then, the actication of culture of purchase is connect with 2% amount
Kind is cultivated into the sterilized non-fat breast of 12% (W/V), 37 DEG C, incubation time 7h of temperature, that is, completes the activation of Lactobacillus helveticus, even
It is continuous to activate 2 times, acidified milk is made, it is spare as Lactobacillus helveticus acidified milk leavening.
Lactobacillus helveticus leavening prepared by 10mL is taken to be inoculated into the sterilized 12wt% skimmed milks of 500mL (inoculation
Rate is 2v/v%), 37 DEG C fermentation 7 it is small when after, after stirring out curdled milk, preserved under the conditions of 4 DEG C, obtain Lactobacillus helveticus acidified milk.
2nd, the obtained and confirmation of biologically active polypeptide mixture
1. experimental method
1) sample treatment
Lactobacillus helveticus acidified milk and the control fermentation breast respectively prepared by previous step, and the skimmed milk dress of 12wt%
Enter and low-temperature centrifugation is carried out in centrifuge tube, centrifugal condition 9000rpm/min, 4 DEG C, 20min.Precipitation is abandoned after centrifugation, takes supernatant.
Supernatant is poured into super filter tube respectively, the molecular cut off of filter membrane is 3kDa, and in ultra-filtration process, rotating speed is
4800r/min, time 30min, centrifuging temperature are 4 DEG C.The filtrate of Lactobacillus helveticus acidified milk is collected, is protected in -4 DEG C of freezings
Deposit.
Solid Phase Extraction after 2mL ultrafiltrates are diluted 50 times, with 1mL C18 pillars, 2mL methanol activation pillar, 1mLddH2O
Pillar is balanced, loading sample dilutes the filtrate after 50 times for 2mL, and last 400 μ L elutions, wherein eluent are 80:
The formic acid of 20v/v methanol/waters and 0.1%v/v.
Dry powder is lyophilized into after obtained elution liquid nitrogen is blown, it is rear to simulate pipe intestinal digesting experiment：First, using sterilizing go from
Sub- water dissolves dry powder, adds pepsin (being purchased from Sigma companies) in the solution, and ratio is that every gram of sample adds pepsin
20mg, adjusts the pH value of reaction solution to 2.0,90min is kept the temperature in 37 DEG C of waters bath with thermostatic control；Then by the pH value of reaction solution adjust to
7.5, pancreatin (Corolase PP, purchased from German AB companies) is added, ratio is that every gram of sample adds pancreatin 40mg, in 37 DEG C of perseverances
150min is kept the temperature in tepidarium；Finally being placed in heating 5min in 95 DEG C of water-baths inactivates enzyme.Control group is set at the same time, except being added without
Outside pepsin and trypsase, identical pH and Temperature Treatment are in same time.By control group and sample sets vacuum refrigeration
After being dried to powder, be stored in -80 DEG C it is spare.
2) liquid matter is analyzed
Liquid phase chromatogram condition：
Instrument：Waters ACQUITY UPLC Ultra Performance Liquid Chromatography instruments
Chromatographic column specification：CSH C18 chromatographic columns
Flow velocity：0.4mL/min
Temperature：45℃
Ultraviolet detection wavelength：220nm
Sample size：5μL
Mobile phase A liquid：ddH2O
Mobile phase B liquid：Acetonitrile solution
Gradient condition：0min-2.5min keeps 99%A liquid, 1%B liquid；2.5min-5minB liquid is changed into 5%, A liquid from 1%
It is changed into 95% from 99%；5min-10minB liquid is changed into 10%, A liquid from 5% and is changed into 90% from 95%；10min-17min%B liquid
It is changed into 25%, A liquid from 10% and is changed into 75% from 90%；17min-22min, B liquid are changed into 40%, A liquid from 25% and are changed into from 75%
60%；22min-27min, B liquid are changed into 80%, A liquid from 40% and are changed into 20% from 60%；27min-29min, B liquid become from 80%
It is 0% for 100%, A liquid, keeps 2min；31min-31.5min, B liquid are changed into 5%, A liquid from 100% and are changed into 95% from 0%；
31.5min-32min, B liquid are changed into 1%, A liquid from 5% and are changed into 99% from 95%；32min-34min, keeps 99%A liquid, 1%B
Liquid.
Mass Spectrometry Conditions：
Ionic means：ES+
Mass range (m/z)：50-2000
Capillary voltage (Capillary) (kV)：3.0
Sampling spiroid (V)：35.0
Ion source temperature (DEG C))：105
Remove solvent temperature (DEG C)：350
Taper hole throughput (L/Hr)：50.0
Go solvent stream (L/hr)：600.0
Collision energy (eV)：6.0
Impinging air flows (ml/min)：0.6
Sweep time (sec)：0.26
Interior sweep time (sec)：0.02
According to above-mentioned experiment condition, using Masslynx software analysis, obtain in Lactobacillus helveticus acidified milk digestion product
Peptide material retention time and molecular weight, as shown in table 1, with the mass chromatography extraction figure of Masslynx softwares extraction polypeptide, one
Level mass spectrogram, is shown in Fig. 2-7.
Polypeptide nucleocytoplasmic ratio in 1 Lactobacillus helveticus acidified milk digestion product of table
3rd, the amino acid sequence of biologically active polypeptide determines method
1. experimental method
(1) foundation of cow's milk source κ-casamino acid sequence library
κ-casamino acid sequence searches for obtain by BIOPEP, and the casamino acid sequence that search obtains is built up
One database.
2) peptide masses is right
The quality analyzed UPLC-MS, scans in the amino acid sequence database of milk protein, obtains phase
The polypeptide sequence of pass.The step realizes that specific requirement is as follows by JAVA programs and MySQL database：Input is by UPLC-MS
Obtained quality, polypeptide sequence of the output quality error in ± 0.01, the quality of the sequence, the specific albumen of the sequence come
Source.
3) verification of polypeptide sequence
Obtained amino acid sequence is verified by the Biolynx in Masslynx softwares, prediction is obtained into polypeptide sequence
The actual second order ms figure that theoretic second order ms figure and MS/MS are obtained contrasts, and software passes through main in second order ms figure
Whether peak provides score value to success, to confirm polypeptide that prediction obtains.Second order ms are to the sequence scheming and predict
Az, by crack conditions figure are as shown in Fig. 8-13.
2. experimental result
Obtain biologically active polypeptide NTVPA, VVTIL, PKKNQ by verification, be milk-derived, be derive specifically from cow's milk κ-
Casein, wherein NTVPA are the amino acid residue of κ-casein the 102nd~106, and VVTIL is κ-casein the 8th~12
Amino acid residue, PKKNQ are the amino acid residue of κ-casein the 131st~135, are denoted as SEQ ID NO respectively：1-3.
In addition, understand polypeptide NTVPA, PKKNQ not in the identical reservation of control group by the experimental result of control group
Between locate appearance, i.e., NTVPA, PKKNQ both of which are to simulate digestion degraded through intestines and stomach to obtain, and are not present in Lactobacillus helveticus hair
In kefir milk.And polypeptide VVTIL appearances at the identical retention time of control group, as shown in Figure 14~17, and peak area changes
Become little, it is to decompose the biologically active polypeptide that κ-casein obtains in cow's milk, Er Qie by Lactobacillus helveticus to illustrate polypeptide VVTIL
Stablize relatively under the action of digestive ferment, be not easy to be degraded after intestines and stomach simulate digestion trial.
The antioxidation activity experiment of 2 biologically active peptide of embodiment
Using removing free radical method (DPPH methods) and total antioxidant capacity method (ABTS methods), the life obtained to embodiment 1
The antioxidation activity of thing active peptides is tested.
1st, the antioxidation activity in vitro of [DPPH] method measure biologically active peptide
1) experiment reagent and instrument
Reagent：1,1- diphenyl -2- trinitrophenyl-hydrazines (1,1-Diphenyl-2-picrylhydrazyl [DPPH]),
Japanese Wako companies production；Methanol, Shanghai traditional Chinese medicines company provide；The milk-derived biologically active polypeptide obtained in synthetic example 1.
Key instrument：Pro200 microplate reader, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products.
2) experimental method
(1) 0.1mmol/L [DPPH] methanol solution
19.72mg [DPPH] is weighed with assay balance to be dissolved in 500mL methanol solutions, the 0.1mmol/L prepared
[DPPH] methanol solution, tinfoil are kept in dark place, i.e., with i.e. use.
(2) antioxidation activity of [DPPH] method measure biologically active peptide
500 μ L concentration are added in 1.5mL EP pipes to be separately added into for 0.1mmol/L [DPPH] methanol solution, by table 2
The sample to be tested and deionized water of 500 μ L various concentrations are as blank control.
After detected sample is loaded, it is uniformly mixed, takes 200 microlitres into 96 orifice plates after being stored at room temperature 30min, use enzyme
Mark instrument detects light absorption value at 517nm.Free radical scavenging activity is calculated according to the following formula, and experimental result is shown in Table 1.
Formula：[DPPH] free radical scavenging activity=(A0-As)/A0× 100%
Wherein A0Represent the light absorption value of blank control group, AsRepresent the light absorption value of sample sets.
2 DPPH methods of table measure the total antioxidant capacity result of biologically active polypeptide
The external total antioxidant activity of biologically active polypeptide NTVPA, VVTIL, PKKNQ is surveyed by [DPPH] method
It is fixed, it is found that polypeptide NTVPA has Scavenging ability, its specific IC50(mg/mL)Value is shown in Table 2.According to the standard of oxidation resistance
Can assert the biologically active polypeptide of invention has oxidation resistance.
2nd, the antioxidation activity in vitro of ABTS methods measure biologically active peptide
1) experiment reagent and instrument
Reagent：Antioxidative activities kit (ABTS methods), green skies company production；Methanol, Shanghai traditional Chinese medicines company provide；Close
The milk-derived biologically active polypeptide obtained into embodiment 1.
Key instrument：Pro200 microplate reader, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture.
2) experimental method
(1) configuration of ABTS working solutions
40 microlitres of ABTS solution and 40 microlitres of oxidizing agent solutions are taken to be configured to ABTS working stocks, lucifuge is stored after preparation
Used after when 12-16 is small, the ABTS working stocks prepared at room temperature store by lucifuge, stablizes in 2-3 days.Before use, ABTS
Working stocks are diluted to ABTS working solutions with PBS, about 40 times, it is desirable to which it is empty that the absorbance of ABTS working solutions subtracts corresponding PBS
After white control, A734 is 0.7 ± 0.05.
(2) making of Trolox standard curves
With sample preparation solution dilution standard product, 10mM Trolox standard solution is diluted to 0.005,0.01,0.03,
0.05th, 0.1,0.25,0.5 and 1.0mM.200 microlitres of ABTS working solutions, standard curve are added in each detection hole of 96 orifice plates
The Trolox standard solution of 10 microlitres of various concentration is added in detection hole, is gently mixed.After incubation at room temperature 4 minutes, microplate reader is used
Light absorption value is measured at 734nm.
Trolox concentration and light absorption value are in good proportional relation, and concentration is higher, and light absorption value is lower.Trolox marks of the present invention
Directrix curve the result is shown in Figure 14, the linear relationship of standard curve is good, related coefficient 0.9981, the precision of Trolox standard curves
Degree and accuracy meet testing requirements, are calculated available for follow-up.
(3) antioxidation activity of ABTS methods measure biologically active peptide
200 microlitres of ABTS working solutions are added in each detection hole of 96 orifice plates, 10 microlitres of PBS are added in blank control wells
Solution, 10 microlitres of various samples are added in sample detection hole, are gently mixed.After incubation at room temperature 4 minutes, with microplate reader in 734nm
Place's measure light absorption value.Measure calculates the total antioxidant capacity of sample according to standard curve.Total antioxidant capacity representation with
The concentration of Trolox standard solution represents.Total antioxidant capacity is calculated according to the following formula, and experimental result is shown in Table 3：
3 ABTS methods of table measure the total antioxidant capacity result of biologically active polypeptide
By total antioxidant capacity method (ABTS methods) to the external total antioxidation of biologically active polypeptide NTVPA, VVTIL, PKKNQ
Activity is determined, it is found that biologically active polypeptide NTVPA has stronger oxidation resistance：In the case of concentration is 5mg/mL
Trolox concentration corresponding to polypeptide NTVPA is 1.0554mmol/L.Therefore, the biologically active polypeptide NTVPA of invention can be assert
With significant oxidation resistance, and VVTIL, PKKNQ have certain oxidation resistance, but opposite NTVPA is weaker.
Illustrate have from biologically active polypeptide NTVPA, VVTIL, PKKNQ of κ-casein according to above experimental result
Oxidation resistance, can remove the free radical in animal body, have potential anti-aging/anti-senescence function.
The above, be only presently preferred embodiments of the present invention, above-described embodiment be only illustrated the principle of the present invention and
Its effect, and not to the present invention in any form with substantial limitation, it is noted that for the common skill of the art
Art personnel, on the premise of the method for the present invention is not departed from, can also make some improvement and supplement, these are improved and supplement
It should be regarded as protection scope of the present invention.All those skilled in the art, are not departing from the feelings of the spirit and scope of the present invention
It is this when the equivalent variations for a little variation, modification and evolution made using disclosed above technology contents under condition
The equivalent embodiment of invention；Meanwhile all any equivalent variations made according to substantial technological of the invention to above-described embodiment
Variation, modification and evolution, in the range of still falling within technical scheme.
Claims (6)
1. a kind of separated biologically active polypeptide NTVPA, its amino acid sequence such as SEQ ID NO：Shown in 1.
2. encode the nucleotide fragments of biologically active polypeptide NTVPA described in claim 1.
3. biologically active polypeptide NTVPA described in claim 1 is preparing anti-oxidant and/or enhancing immunity of organisms food, is protecting
Application in strong product and medicine.
4. a kind of anti-oxidation medicine, includes biologically active polypeptide NTVPA as claimed in claim 1.
5. one kind enhancing immunity of organisms medicine, includes biologically active polypeptide NTVPA as claimed in claim 1.
6. a kind of health-oriented products of potential anti-aging/anti-aging, include biologically active polypeptide as claimed in claim 1
NTVPA。
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| CN105254740B (en) * | 2015-10-16 | 2019-01-08 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide NQFYQKF and its preparation and application |
| CN105254743B (en) * | 2015-10-16 | 2019-04-26 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide SAEP and its preparation and application |
| CN105254751B (en) * | 2015-10-16 | 2019-02-05 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide WNIPMGLIV and its preparation and application |
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| CN107141346B (en) * | 2017-06-22 | 2020-04-14 | 浙江辉肽生命健康科技有限公司 | Bioactive polypeptide ATLEDSPEVI, and preparation method and application thereof |
| CN107236031B (en) * | 2017-07-06 | 2020-04-24 | 浙江辉肽生命健康科技有限公司 | Bioactive polypeptide PMIGVNQELAY, and preparation method and application thereof |
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| CN107840880A (en) * | 2017-11-14 | 2018-03-27 | 上海交通大学 | A kind of biologically active polypeptide GLNYYQQKPVA and its preparation method and application |
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| CN107840878A (en) * | 2017-11-14 | 2018-03-27 | 上海交通大学 | A kind of biologically active polypeptide NNQFLPYPYYAKPA and its preparation method and application |
| CN107827972A (en) * | 2017-12-11 | 2018-03-23 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide SPEVIESPPEIN and its preparation method and application |
| CN110999979B (en) * | 2019-12-20 | 2021-06-29 | 云南农业大学 | Moringa seed protease capable of promoting sheep milk coagulation and polypeptide prepared by same and application thereof |
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| CN112707949A (en) * | 2021-01-19 | 2021-04-27 | 浙江辉肽生命健康科技有限公司 | 33 bioactive peptides derived from lymphocytes and having blood sugar reducing effect, and application thereof |
| CN112661810A (en) * | 2021-01-19 | 2021-04-16 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide derived from lymphocyte and having ACE (angiotensin converting enzyme) inhibitory activity and application thereof |
| CN116444611B (en) * | 2022-11-30 | 2024-06-04 | 内蒙古伊利实业集团股份有限公司 | Milk active peptide TDPLFKG and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101917866A (en) * | 2007-04-16 | 2010-12-15 | 索莱有限责任公司 | Protein hydrolysate compositions with organoleptic attribute and physical characteristic of improvement |
| ES2354784A1 (en) * | 2009-08-06 | 2011-03-18 | Grupo Leche Pascual S.A.U. | Enzymatic procedure for the production of bioactive peptides. (Machine-translation by Google Translate, not legally binding) |
| CN103012552A (en) * | 2012-12-12 | 2013-04-03 | 上海交通大学 | Bioactive polypeptide QEPV, and preparation and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009121176A1 (en) * | 2008-03-31 | 2009-10-08 | The University Of British Columbia | Insulin-induced gene (insig) peptide compositions and methods for cytoprotection |
| CN102964427B (en) * | 2012-12-12 | 2014-01-15 | 上海交通大学 | Bioactive polypeptide QEPVL, and preparation and application thereof |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| ES2354784A1 (en) * | 2009-08-06 | 2011-03-18 | Grupo Leche Pascual S.A.U. | Enzymatic procedure for the production of bioactive peptides. (Machine-translation by Google Translate, not legally binding) |
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