CN116444611B - Milk active peptide TDPLFKG and preparation method and application thereof - Google Patents
Milk active peptide TDPLFKG and preparation method and application thereof Download PDFInfo
- Publication number
- CN116444611B CN116444611B CN202211530344.4A CN202211530344A CN116444611B CN 116444611 B CN116444611 B CN 116444611B CN 202211530344 A CN202211530344 A CN 202211530344A CN 116444611 B CN116444611 B CN 116444611B
- Authority
- CN
- China
- Prior art keywords
- small peptide
- peptide
- composition
- tdplfkg
- milk
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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Classifications
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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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention provides a milk-derived active peptide TDPLFKG, a preparation method and application thereof. The invention firstly provides a small peptide or a derivative thereof, wherein the amino acid sequence of the small peptide is shown as SEQ ID NO. 1; SEQ ID NO.1: TDPLFKG. The small peptide is milk-source active peptide, and has good antioxidation and immunity regulation effects.
Description
Technical Field
The invention relates to an active peptide, a preparation method and application thereof, in particular to a milk-derived active small peptide TDPLFKG, a derivative thereof, a preparation method and application thereof.
Background
Bioactive peptide refers to a peptide having a specific physiological activity to a living body, wherein milk protein is one of important sources of bioactive peptide. In recent years, milk-derived active peptides have become a well-known word. On one hand, the milk-derived active peptide has the characteristics of small fragments and easy absorption; on the other hand, it has many potential biological functions, so that it attracts more and more attention, and is one of the hot spots of scientific research. Many milk-derived active peptides have also been well demonstrated for their beneficial effects, including antibacterial, anticancer, antihypertensive, cholesterol-lowering, antidiabetic, antiinflammatory, antidepressant properties. More than 4000 different milk-derived active peptides have been reported in the currently most authoritative milk-derived active peptide database BIOPEP-UMW. However, novel milk-derived active peptides having antioxidant or immunoregulatory functions different from existing polypeptides remain the direction of current need for further research.
Disclosure of Invention
It is an object of the present invention to provide milk-derived active small peptides and derivatives thereof.
It is another object of the present invention to provide a process for the preparation of said small peptides and derivatives thereof.
It is a further object of the present invention to provide the use of said small peptides and derivatives thereof.
In one aspect, the invention provides a small peptide or a derivative thereof, wherein the amino acid sequence of the small peptide is shown as SEQ ID NO. 1;
SEQ ID NO.1：TDPLFKG。
The small peptide is active peptide, and has good antioxidation and immunity regulation effects.
According to a specific embodiment of the invention, the derivative is a derivative peptide obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification and/or glycosylation modification on the basis of the side chain group, amino-terminus or carboxy-terminus of the amino acid sequence shown in SEQ ID NO. 1. Preferably, the derivative peptide has substantially the same biological activity (e.g. antioxidant and/or immunity modulating efficacy) as the small peptide of the amino acid sequence shown in SEQ ID NO. 1.
In another aspect, the present invention provides a method for preparing the small peptide or derivative thereof, comprising:
preparing the small peptide or the derivative thereof by a microbial fermentation method; or alternatively
Preparing the small peptide or its derivative by genetic engineering (which can be obtained directly from cells by separation and purification); or alternatively
The small peptide or derivative thereof is synthesized by chemical means.
According to a specific embodiment of the present invention, there is provided a method for preparing the small peptide or the derivative thereof, comprising:
the small peptide or the derivative thereof is obtained by taking emulsion containing milk protein as a fermentation substrate, inoculating lactobacillus paracasei for fermentation and separating from a fermentation product.
Lactobacillus paracasei (Lactobacillus paracasei) is a gram positive bacterium, is usually subjected to abnormal fermentation, has the characteristics of facultative anaerobism, no movement and no spore, is in the form of bacillus or longbacterium, and is singly or in pairs, and has the width of 2.0-4.0 mu m and the length of 0.8-1.0 mu m. In the invention, lactobacillus paracasei can be utilized to ferment and hydrolyze milk proteins to produce the small peptide, and the small peptide derivative can be obtained by modification optionally.
According to a specific embodiment of the present invention, in the method for producing a small peptide or a derivative thereof according to the present invention, the fermentation substrate comprises: 3 to 10 percent of cow milk concentrated protein and 1 to 10 percent of lactose.
According to a specific embodiment of the present invention, in the method of the present invention for producing a small peptide or a derivative thereof, fermentation conditions are: the inoculation amount is 1-3% at 30-45 ℃, and the bacterial count in the seed liquid is more than 1.5X10 11 CFU/mL.
According to a specific embodiment of the present invention, in the method for producing a small peptide or a derivative thereof of the present invention, the lactobacillus paracasei includes lactobacillus paracasei K56. Lactobacillus paracasei K56 is a public strain of CN107916236a, and has been shown to have antibacterial, anti-obesity, antioxidant, blood pressure regulating, intestinal flora regulating, immune system regulating, etc. effects.
The lactobacillus paracasei K56 strain is adopted for natural fermentation to produce the milk-source active peptide TDPLFKG, so that the concentration of the produced target small peptide can be increased, and the production cost can be reduced. The method comprises the following steps: the method comprises the steps of taking cow milk concentrated protein as a raw material, fermenting by using lactobacillus paracasei, concentrating by adopting a membrane technology, and separating and purifying to obtain target small peptide. In the present invention, the small peptide finished product in the form of powder can be obtained by a vacuum freeze-drying technique or a low-temperature spray-drying technique. The amino acid sequence of the peptide can be checked by UPLC-MS.
In some embodiments of the invention, the method for producing milk-derived active peptide TDPLFKG by natural fermentation using lactobacillus paracasei K56 strain comprises:
dissolving 3-10% of milk concentrated protein and 1-10% of lactose in an aqueous solution with the pH value of 7.0-8.0 and the temperature of 25-90 ℃, uniformly mixing, and cooling to room temperature to obtain a milk concentrated protein solution;
Inoculating fermentation strain, fermenting to obtain fermentation liquor;
Centrifuging the fermentation liquor, collecting supernatant, and performing ultrafiltration filtration by adopting a membrane with a molecular weight cutoff of less than 3kDa to obtain filtered fermentation liquor;
Further separating and purifying the filtered fermentation liquor to obtain the target small peptide.
In another aspect, the invention also provides the use of said small peptides or derivatives thereof for the preparation of a composition having antioxidant and/or immunomodulating efficacy.
In another aspect, the invention also provides a composition comprising a small peptide and/or derivative thereof according to the invention, optionally together with an adjuvant.
According to a specific embodiment of the invention, the composition is a food composition, a pharmaceutical composition or a cosmetic composition.
The milk-derived active peptide TDPLFKG has the beneficial effects that: on the one hand, the anti-oxidation capability is higher; on the other hand, the preparation has better immunoregulatory activity and can improve the quality of life. The small peptide and the derivatives thereof have very important significance in developing foods, health-care products and medicines with the functions of antioxidation and immunoregulation.
Drawings
Fig. 1 is a first order mass spectrum of a fragment with a mass to charge ratio of 454.732 (m/z= 454.732).
FIG. 2 shows the secondary mass spectrum of a fragment with a mass to charge ratio 454.732 and the fragmentation of polypeptides az, by.
FIG. 3 is a pie chart of in vitro digestion product analysis of milk-derived active peptide TDPLFKG.
Detailed Description
Before the embodiments of the invention are further described, it is to be understood that the invention is not limited in its scope to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art, see in particular Sambrook et al MOLECULAR CLONING：ALABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989and Third edition,2001;Ausubel et al ,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; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (P.B. Becker, ed.) Humana Press, totowa,1999, et al.
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1, obtaining of active peptide TDPLFKG
1. Preparation of milk concentrate protein solution
10% Milk protein concentrate, 10% lactose, 0.5% NACL (all by mass fraction) are dissolved in an aqueous solution having a pH of 7.0-8.0. Sterilizing at 95deg.C for 10min, and cooling to 37deg.C in ice water bath;
2. Preparation of fermentation broths
Adding lactobacillus paracasei K56 lyophilized powder to make the addition amount reach 1% of milk concentrated protein concentration (i.e. 1.5X10 11 CFU/L), mixing, placing into an incubator at 37deg.C, standing and fermenting for 4 hr to obtain fermentation broth.
3. Extraction of polypeptides
Heating the fermentation broth in 95 ℃ water bath for 10min for inactivation, cooling by using ice water bath, and centrifuging at low temperature under the following conditions: 8000rcf, 4 ℃, centrifuging for 10min, discarding bottom sediment, and taking supernatant. Taking supernatant, transferring into an inner tube of an ultrafiltration tube for ultrafiltration, and selecting an ultrafiltration membrane with a molecular weight cutoff of 3kDa, wherein the ultrafiltration centrifugation condition is 4800rcf, 4 ℃ and 30min. Collecting the bottom ultrafiltrate, and preserving the obtained polypeptide solution at low temperature under the condition of 4 ℃.
4. Preparation of polypeptide powder
And (3) carrying out spray drying on the polypeptide solution obtained in the step (5) to obtain the cow milk concentrated protein peptide powder, wherein the spray drying conditions are as follows: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the flow rate is 25mL/min.
5. Screening of active peptides TDPLFKG
1) UPLC analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the analysis method, the chromatographic analysis and the mass spectrometry are carried out on the cow milk concentrated protein peptide by utilizing an ultra-high performance liquid phase, electrospray, a quaternary rod and a time-of-flight mass spectrum, so that the amino acid sequence of all polypeptides in the fermentation broth is obtained.
The same amino acid sequence in the parallel control group is selected, and then the amino acid sequence with lower biological activity probability score in PEPTIDE RANKER is removed from the disclosed amino acid sequence, so as to obtain the sequence of the active peptide TDPLFKG.
Example 2 Synthesis of active peptide TDPLFKG
1. Synthesis of bioactive peptides
1. RINK g (substitution degree 0.3 mmol/g) of the resin was weighed into a 150ml reactor and immersed in 50ml of Dichloromethane (DCM).
After 2.2 hours, the resin was washed with 3 times the resin volume of nitrogen-Dimethylformamide (DMF), then drained, and the resin was drained and ready for use.
3. An amount of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and shaken on a decolorizing shaker for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the resin was washed four times with 3 volumes of DMF and then drained.
4. A small amount of resin is taken and detected by ninhydrin (ninhydrin nine-well) method (two drops of each of detection A and detection B are reacted for 1min at 100 ℃), and the resin is colored, which indicates that the deprotection is successful.
5. The method comprises the steps of weighing an appropriate amount of amino acid Asn and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Asn and the 1-hydroxy-benzotriazole (HOBT), adding 3ml of N, N-Diisopropylcarbodiimide (DIC), shaking uniformly for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ to react.
After 6.2 hours, a certain amount of acetic anhydride head (acetic anhydride: DIEA: dcm=1:1:2, v: v) was used for half an hour, then washed four times with 3 times the resin volume of DMF, and dried for use.
7. An amount of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and shaken on a decolorizing shaker for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the mixture was washed four times with DMF and then dried.
8. A small amount of resin is taken and detected by ninhydrin (ninhydrin nine-well) method (two drops of each of detection A and detection B are reacted for 1min at 100 ℃), and the resin is colored, which indicates that the deprotection is successful.
9. Weighing the right amount of the second amino acid and the right amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the second amino acid and the right amount of HOBT, adding 2.5ml of DIC, shaking uniformly for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ for reaction.
After 10.1 hours, a small amount of resin is taken for detection, ninhydrin method is used for detection (two drops of detection A and detection B are respectively reacted for 1min at 100 ℃), and if the resin is colorless, the reaction is complete; if the resin is colored, this indicates incomplete condensation and the reaction is continued.
11. After completion of the reaction, the resin was washed four times with DMF and then drained, a quantity of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and placed on a decolorizing shaker and shaken for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the sample was washed four times with DMF and then drained to examine whether the protection had been removed.
12. Amino acids Gly, lys, phe, leu, pro, asp and Thr were added sequentially according to steps 9-11.
13. After the last amino acid has been taken up, the deprotection is achieved, the resin is washed four times with DMF and then pumped off with methanol. Then with 95 cutting fluid (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropyl silane: the bioactive peptide was cleaved from the resin (10 ml cleavage solution per gram of resin) and spun down four times with glacial ethyl ether (cleavage solution: ethyl ether=1:9, v: v).
Thus, bioactive peptide TDPLFKG was synthesized artificially.
2. Confirmation of bioactive peptides
1) UPLC analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the above analysis methods, the bioactive peptide TDPLFKG is subjected to chromatographic analysis and mass spectrometry using ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide TDPLFKG is shown in figure 1, the secondary mass spectrum of the extracted peak and az and by breaking conditions are shown in figure 2, and the bioactive peptide of the peak can be obtained with a mass-to-charge ratio of 454.732 and a retention time of 27.92min.
3) Results
As can be seen from FIG. 2, according to the breaking condition of az and by, the fragment sequence with mass to charge ratio 454.732 is Thr-Asp-Pro-Leu-Phe-Lys-Gly (TDPLFKG) and is recorded as SEQ ID NO.1 by analysis and calculation of Mascot software. The fragment corresponds to the residue sequence of the Serum amyloid A protein protein at 69-74 th positions, and the sequence is shown in SEQ ID NO.2.
SEQ ID NO.2：QRWGTFLKEAGQGAKDMWRAYQDMKEANYR GADKYFHARGNYDAARRGPGGAWAAKVISNARETIQGITD PLFKGMTRDQ VREDSKADQFANEWGRSGKDPNHFRPAGLPDK.
Example 3 antioxidant Activity assay of milk-derived active peptides
1. ABTS method for measuring in vitro antioxidant capacity of milk active peptide TDPLFKG
1. Experimental reagent and instrument
Cow milk concentrated protein (MPC) powder, a constant natural commercial company (Shanghai); lactobacillus paracasei (K56), biosciences inc; neutral protease (enzyme activity 1.1X106 u/g), xia Cheng (Shanghai) Biotechnology Co., ltd; lactose, national pharmaceutical group chemical company, inc; sodium chloride, shanghai Lingfeng chemical reagent Co., ltd; gastropeptone Peptone, BBI life sciences ltd; total antioxidant capacity test kit (ABTS method), shanghai Biyun Tian Biotech company.
Electronic balance, sartorius germany; constant temperature water bath box, shanghai-a constant technology company; 3kda ultrafiltration tube, millipore company; DR-200Bc enzyme labeling instrument, dekkera instruments, inc.; JB-VS-1300U ultra-clean bench, shanghai Yingming clean equipment limited company; LRH-250F biochemical incubator, shanghai-Heng science instruments Co., ltd; GI36T autoclave, xiamen micro instruments inc; froma 700 fridge, sameiser's technology (china) limited; 96-well cell culture plate, millipore company, usa; RO15 pure water system, likang biomedical science and technology control Co., ltd; GL-22M high-speed refrigerated centrifuge, shanghai Lu Xiangyi centrifuge instruments Inc.
2. Experimental method
According to the specification of the total antioxidant capacity detection kit, mixing the ABTS solution and the ABTS oxidant solution in a ratio of 1:1, and storing for 12-16 hours in a dark place for use. The prepared ABTS mother solution is stored at room temperature in dark place and is stable within 2-3 days. Before use, the ABTS working mother solution is diluted by 38-42 times by PBS, so that after the absorbance of the ABTS working solution minus the corresponding PBS blank control, A734 is 0.7+/-0.05, and the ABTS working solution is stored in a dark place and is prepared for use.
200. Mu.L of ABTS working solution was added to each well of a 96-well plate, 10. Mu.L of tocopherol (Trolox) diluted with PBS was added to the standard curve assay wells in a concentration gradient, 10. Mu.L of PBS was added to the blank wells, and the mixture was gently mixed. After incubation for 4min at room temperature, absorbance was measured at 734 nm.
200 Mu L of ABTS working solution is added into each detection hole of a 96-well plate, 10 mu L of samples with different concentrations are added into sample detection holes according to the requirement of the required concentration, 10 mu L of PBS is added into blank control holes, and the mixture is gently mixed. After incubation for 4min at room temperature, absorbance was measured at 734 nm. And calculating the total antioxidant capacity of the sample according to a standard curve. The total antioxidant capacity is expressed in terms of the concentration of the Trolox standard solution, formula:
total antioxidant capacity (mmol/g) =c Trolox/CS
Wherein: c Tr olox-Trolox standard solution concentration (mmol/L) with the same absorbance as the sample
C S -concentration of synthetic polypeptide sample (mg/mL)
3. Experimental results and analysis
TABLE 1 Total antioxidant capacity of milk-derived active peptides TDPLFKG
| Experimental grouping | Total antioxidant capacity (mmol/g) |
| Positive control group phytic acid 1mg/ml | 0.0031±0.0011 |
| TDPLFKG 1mg/ml | 0.0049±0.0006** |
| TDPLFKG 0.5mg/ml | 0.0038±0.0004* |
| TDPLFKG 0.1mg/ml | 0.0022±0.0005 |
Note that: * Compared with the negative control group, there was a very significant difference (P < 0.01); * Compared with the negative control group, the negative control group has significant difference (P < 0.05).
The experimental principle of the ABTS method is that the ABTS can be oxidized into green ABTS+ under the action of a proper oxidant, the generation of the ABTS+ can be inhibited when an antioxidant exists, the total antioxidant capacity of the polypeptide can be calculated by measuring the absorbance of the ABTS+ at 734nm, and the smaller the absorbance value is, the stronger the antioxidant capacity of the substance is. These oxidants are mainly reactive oxygen species including hydroxyl radicals, hydrogen peroxide and superoxide radicals, and the ABTS method is the measure of the total capacity to inhibit these reactive oxygen species. Trolox, a water-soluble vitamin E, has a strong antioxidant power and is used as a reference substance for the total antioxidant capacity of other antioxidants.
The experimental results are shown in table 1, and the in vitro total antioxidant activity of the polypeptide TDPLFKG is measured by the ABTS method, so that the experimental group added with the milk-derived active peptide TDPLFKG has a certain degree of reduction of the light absorption value compared with the positive control group, and has better capability of reducing oxidized substances. As can be seen from Table 1, it was found that the total antioxidant capacity of milk-derived active peptide TDPLFKG increased with increasing polypeptide concentration, and that the total antioxidant level of milk-derived active peptide TDPLFKG was optimal at a concentration of 1 mg/mL. Thus, the milk-derived active polypeptide TDPLFKG of the invention is considered to have significant antioxidant capacity.
EXAMPLE 4 immunomodulatory Activity assay of milk-derived active peptides
1. Determination of proliferation rate of in vitro macrophages by milk-derived active peptide TDPLFKG
1. Experimental reagent and instrument
PBS, nanj Kaiki Biotech Co., ltd; DMEM incompletely high sugar culture broth; nanj Kaiki Biotech Co., ltd; fetal bovine serum, GIBCO limited; macrophage RAW264.7, cell site of Shanghai national academy of sciences; lipopolysaccharide (LPS, E.Coli O111: B4), sigma Co., ltd; 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide, amresco; bovine serum albumin (bovine serum albumin, BSA), genebase.
Centrifuge 5414D mini high-speed Centrifuge, eppendorf limited; GL-22M high-speed refrigerated centrifuge, shanghai Lu Xiangyi centrifuge instruments Inc.; electronic balances Sartorius, germany; constant temperature water bath box, shanghai-a constant technology company; DR-200Bc enzyme labeling instrument, dekkera instruments, inc.; JB-VS-1300U ultra-clean bench, shanghai YingMing clean equipments Limited company.
2. Experimental method
DMEM incomplete medium (containing penicillin 80U/mL; streptomycin 0.08 g/L) containing 10% fetal bovine serum was prepared, RAW264.7 cells were prepared to a concentration of 2X 10 5/m L using this medium, and the cell suspension was inoculated in 25cm 2 disposable flasks or 96-well plates. Culturing in a saturated vapor carbon dioxide incubator with the temperature of 37 ℃ and the concentration of CO 2 being 5%. After 24h the medium was changed. Passaging was performed when the flask was observed with an inverted microscope to be substantially full of cells in the flask bottom. At the time of passage, the old culture medium was carefully aspirated, 1.5ml of trypsin digest was added, the mixture was placed in an incubator at 37℃for accurate digestion for 2min, and 2ml of medium was added to terminate the reaction. Repeatedly and gently blowing the cells to make the cells growing on the bottom wall fall off and scatter. The cells at the bottom of the tube were collected by centrifugation at 800g for 2min, and the cells were resuspended to a concentration of 2X 10 5 by adding the medium, and cultured.
Early stage of experiment: RAW264.7 cells were cultured in DMEM medium containing milk-derived active peptide TDPLFKG at a concentration of 0g/L, 0.1g/L, 0.5g/L, and 1.0g/L for 24 hours, LPS solution of 100. Mu.g/L was added to each flask at the 24 th hour, and after further culturing for 2 hours, the cell culture solution was carefully discarded, the bottom of the wells was washed with PBS, 5% MTT 20. Mu.L was added for 48 hours, and then 100. Mu.L of a solution was added to terminate the culture, absorbance (OD 570) was measured with a microplate reader at a wavelength of 570nm after the overnight dissolution, and the BSA group was a negative control, and the growth index (GI: growth Indices) was calculated according to the following formula.
Gi= (small peptide OD value-blank OD value)/(blank control OD value-blank OD value).
3. Experimental results and analysis
TABLE 2 influence of milk-derived active peptides TDPLFKG on macrophage proliferation after LPS stimulation
| Experimental grouping | Experimental results (OD 570) |
| TDPLFKG 0mg/ml | 0.586±0.012 |
| TDPLFKG 1mg/ml | 0.544±0.009* |
| TDPLFKG 0.5mg/ml | 0.556±0.004* |
| TDPLFKG 0.1mg/ml | 0.567±0.012 |
Note that: * Compared with the negative control group, there was a very significant difference (P < 0.01); * Compared with the negative control group, the negative control group has significant difference (P < 0.05).
When the organism is stimulated by external LPS, immune cells including macrophages can proliferate, enhance the phagocytic function of the macrophages, promote the secretion of cytokines, thereby improving the capability of the organism for resisting the infection of external pathogens, reducing the morbidity of the organism, but possibly generating inflammation to endanger the homeostasis of the organism. The experimental results are shown in table 1, and the in vitro immunomodulatory activity of polypeptide TDPLFKG is measured by measuring the proliferation rate of macrophages after stimulation by LPS, so that the proliferation rate of the macrophages in the experimental group added with the milk-derived active peptide TDPLFKG is reduced to a certain extent compared with that in the blank group, which indicates that the proliferation level of the cells is reduced under the condition of macrophage inflammation, and the damage of the inflammation to the organism is reduced. As can be seen from table 1, the immunomodulatory capacity of the milk-derived active peptide TDPLFKG was significantly lower than that of the blank group at a concentration of 1.0mg/mL, and was optimal. Thus, the milk-derived active polypeptide TDPLFKG of the invention can be considered to have significant immunomodulatory capacity.
Example 5 stability of milk-derived active peptide and core fragment verification experiment
1. In vitro simulated gastrointestinal digestion experiment of milk-derived active peptide TDPLFKG
1. In vitro digestion of lyophilized powder of milk-derived active peptide TDPLFKG
1.5Mg of the sample powder was dissolved in 1.5mL of ddH 2 O, pepsin (enzyme: substrate=1:50, w/w) was added to the sample (concentration: 1 mg/mL), and then the pH was adjusted to 2.0, and the mixture was subjected to a water bath at 37℃for 90 minutes. Subsequently, the pH of the hydrolysate was adjusted to 7.5, pancreatin (enzyme: substrate=1:25, w/w) was added, and the mixture was water-bath at 37℃for 150min. Finally, the reaction was stopped by inactivating the enzyme with a hot water bath at 95℃for 5 min. In the experiment, a control group was set up and the same pH and temperature treatments were performed at the same time, except that pepsin and pancreatin were not added. The control and sample groups were lyophilized in vacuo to a powder and stored at-80 ℃ for subsequent analysis.
UPLC-MS analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the analysis method, chromatographic analysis and mass spectrometry are carried out on the in-vitro digestion products of the milk active peptide TDPLFKG by utilizing an ultra-high performance liquid phase, electrospray, a quaternary rod and a time-of-flight mass spectrum. Comparing the polypeptide sequences of the polypeptides in the control group with those in the experimental group to detect the stability of the milk-derived active peptide.
3. Experimental results and analysis
FIG. 3 is a pie chart of in vitro digestion product analysis of milk-derived active peptides TDPLFKG, illustrating how much by content.
As shown in FIG. 3, the ratio of the original sequence of the milk-derived active peptide TDPLFKG in the experimental group and the control group is over 99%, which indicates that pepsin and pancreatin are not hydrolyzed, and the in vivo acidic environment is not hydrolyzed. Experiments prove that the milk-derived active peptide TDPLFKG has stability in the gastrointestinal tract digestion process.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (8)
1. A small peptide, the amino acid sequence of which is shown as SEQ ID NO. 1;
SEQ ID NO.1：TDPLFKG。
2. A derivative of a small peptide, wherein the derivative is a derivative peptide which is obtained by methylation and/or acetylation modification on the basis of the amino terminal or the carboxyl terminal of the amino acid sequence shown in SEQ ID NO.1 and has the same biological activity as the small peptide with the amino acid sequence shown in SEQ ID NO.1, and the biological activity is antioxidation and/or immunity regulation.
3. A method of preparing the small peptide of claim 1, the method comprising:
preparing the small peptide by a genetic engineering method; or alternatively
The small peptides are synthesized by chemical means.
4. A method of preparing the small peptide of claim 1, the method comprising:
inoculating Lactobacillus paracasei deposited with the German collection of microorganisms under the accession number DSM27447 to ferment using an emulsion containing milk protein as a fermentation substrate, and separating the small peptide of claim 1 from the fermentation product;
wherein the fermentation substrate comprises: 3% -10% of cow milk concentrated protein and 1% -10% of lactose.
5. The method of claim 4, wherein the fermentation conditions are: the inoculation amount is 1% -3% at 30-45 ℃, and the bacterial count in the seed liquid is more than 1.5X10 11 CFU/mL.
6. Use of the small peptide of claim 1 for the preparation of a composition having antioxidant and/or immunomodulating efficacy, said composition being a food composition.
7. Use of the small peptide of claim 1 for the preparation of a composition having antioxidant efficacy, said composition being a cosmetic composition.
8. A composition comprising the small peptide of claim 1, optionally together with an adjuvant;
the composition is a food composition or a cosmetic composition.
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