Active biofilm of sturgeon functional polypeptide and preparation thereof

Abstract

The present application relates to the technical field of biology, in particular to a functional polypeptide of sturgeon and an active biomembrane prepared from the functional polypeptide.The functional polypeptide has good anti-inflammatory and bone cell proliferation and differentiation promoting activities, and can be used for the preparation of products with the above functions, has good application value, and is conducive to the high-value utilization of sturgeon cartilage and the improvement of the utilization rate of sturgeon raw materials.The active biomembrane containing the functional polypeptide has good bacteriostatic, anti-inflammatory, wound healing promoting, fresh-keeping, pesticide adsorbing and other functions, has various biological activities, has good mechanical properties, and has excellent membrane performance.In addition, the preparation process of the biomembrane is simple, the types of raw materials used are less, the cost is low, and the preparation efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a functional polypeptide of sturgeon and an active biomembrane prepared therefrom. BACKGROUND

[0002] Polypeptide is a kind of active substance with special physiological function composed of various amino acids. Functional polypeptide is a kind of polypeptide with specific biological activity, which has the characteristics of easy absorption, easy processing, low allergenicity, low osmotic pressure, etc., and has important application value in the fields of food, medicine, cosmetics, etc. Developing functional polypeptide from animal processing by-products as raw materials has important significance for improving the utilization rate of animal raw materials and promoting human health.

[0003] Active biomembrane made of polysaccharide has transparency and certain mechanical properties. Polysaccharide film combined with other active components can endow the film with other properties, such as antibacterial, antioxidant, fresh-keeping, etc. At present, the polysaccharide raw materials used in active biomembrane are mainly chitosan and cellulose.

[0004] Cartilage polysaccharide is a natural acidic mucopolysaccharide, usually combined with protein and existing in the form of glycosaminoglycan, the basic unit of which is a disaccharide composed of D-glucuronic acid and N-acetyl-D-galactosamine connected by β-1, 3 glycosidic bond, and the disaccharides are further connected by β-1, 4 glycosidic bond to form a biological macromolecule. It has gel properties, antibacterial and biocompatible properties, and also has biological activities such as anti-atherosclerosis, treatment of arthritis and anti-tumor. At present, there are few active biomembranes developed from cartilage polysaccharide as raw material. SUMMARY

[0005] The first object of the present application is to provide a functional polypeptide and its application.

[0006] The second object of the present application is to provide an active biomembrane containing the above-mentioned functional polypeptide, its preparation method and application.

[0007] Specifically, the present application provides the following technical solutions:

[0008] In the first aspect, the present application provides a functional polypeptide, which is a polypeptide as described in 1) or 2) below:

[0009] 1) a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1;

[0010] 2) a polypeptide obtained by adding a protein tag to the N-terminus and / or C-terminus of a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1.

[0011] The sequence as shown in SEQ ID NO. 1 is specifically as follows:

[0012] SEQ ID NO. 1: STESLQANVQR.

[0013] The protein tag described above can be any protein tag, including but not limited to His, Flag, GST, c-Myc, eGFP / eCFP / eYFP / mCherry eGFP, HA, SUMO tag, etc. The protein tags carried by the N-terminus and C-terminus of the polypeptide can be the same or different.

[0014] The functional polypeptide described above has been experimentally verified to have high anti-inflammatory, anti-infection, and bone cell proliferation and differentiation promoting activities.

[0015] The functional polypeptide described above can be a pure natural small molecule active peptide isolated from sturgeon cartilage.

[0016] The functional polypeptide described above can be obtained by chemical synthesis, host cell expression and purification, or extraction from sturgeon cartilage raw materials.

[0017] In a second aspect, the present application provides a nucleic acid molecule encoding the functional polypeptide.

[0018] According to the amino acid sequence and codon rules of the functional polypeptide described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the functional polypeptide. Based on the degeneracy of codons, the above-mentioned nucleotide sequence can not be unique, but all nucleic acid molecules capable of encoding the functional polypeptide are within the scope of protection of the present application.

[0019] In a third aspect, the present application provides a biological material comprising the nucleic acid molecule, which is an expression cassette, a vector or a host cell.

[0020] The expression cassette is a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule to transcription or translation regulatory elements.

[0021] The vector includes but is not limited to a plasmid vector, a viral vector, and a transposon.

[0022] The host cell includes microbial cells (e.g., E. coli, yeast, etc.) or animal cells or cell lines.

[0023] In a fourth aspect, the present application provides the use of the functional polypeptide described above or the nucleic acid molecule or the biological material in the preparation of food, medicine or packaging material.

[0024] In some embodiments of the present application, the medicine has anti-inflammatory, anti-infection and / or bone cell growth promoting functions.

[0025] The present application also provides use of the functional polypeptide or the nucleic acid molecule or the biomaterial in the preparation of a product having the functions of anti-inflammation, anti-infection, promoting bone cell proliferation, promoting wound healing and / or preservation.

[0026] The product is preferably a medicine.

[0027] In a fifth aspect, the present application provides a product comprising the functional polypeptide as described above.

[0028] In a sixth aspect, the present application provides an active biomembrane comprising the functional polypeptide as described above.

[0029] Specifically, the biomembrane further comprises chondroitin sulfate and chitosan in a mass ratio of (1-4) : 1.

[0030] Preferably, in the raw materials for preparing the biomembrane, the mass ratio of chondroitin sulfate, chitosan and the functional polypeptide is (1-4) : 1 : (1-4).

[0031] Preferably, the chondroitin sulfate is sturgeon chondroitin sulfate (extracted from sturgeon cartilage) and has a molecular weight of 25-30 kDa.

[0032] The chitosan has a molecular weight of 1800-2200 Da.

[0033] In a sixth aspect, the present application provides a method for preparing the active biomembrane as described above, which comprises mixing chondroitin sulfate, chitosan and the functional polypeptide with water and then drying to form a film.

[0034] Preferably, the volume of water (mL) is in a ratio of 10 mL : (0.3-0.9) g to the total mass (g) of chondroitin sulfate, chitosan and the functional polypeptide.

[0035] Preferably, the drying to form a film is performed at 20-45℃.

[0036] Further preferably, the drying to form a film is performed at 20-30℃ or 35-45℃.

[0037] Preferably, after mixing chondroitin sulfate, chitosan and the functional polypeptide with water, the mixture is stirred for 3-5 h, the bubbles in the mixture are removed, and then the mixture is dried to form a film.

[0038] In a seventh aspect, the present application provides use of the active biomembrane as described above in the preparation of a product for promoting wound healing, anti-inflammation, anti-infection, preservation and / or adsorption of pesticides.

[0039] The preservation can be preservation of food.

[0040] The functional polypeptide provided by the application has good anti-inflammatory and bone cell proliferation and differentiation promoting activities, and can be used for the preparation of drugs with the above functions, has the advantages of simple preparation process, high yield, good application value, and is conducive to realizing high-value utilization of sturgeon cartilage and improving utilization of sturgeon raw materials.

[0041] The active biological membrane containing the functional polypeptide has good bacteriostatic, anti-inflammatory, wound healing promoting and other functions, and has various biological activities. The in-vitro test proves that the active biological membrane has bactericidal activity, and at the same time, the mouse local infection model proves that the biological membrane has a significant therapeutic effect on wound infection caused by Staphylococcus aureus, can promote skin cell proliferation, and promote wound healing. The biological membrane also has good mechanical properties and excellent membrane performance. In addition, the biological membrane also has good preservation and pesticide adsorption functions. The preparation process of the biological membrane is simple, the types of raw materials used are less, the cost is low, and the preparation efficiency is high. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0043] Figure 1 The mass spectrum Basepeak diagram of the sturgeon cartilage polypeptide in Example 1 of the application.

[0044] Figure 2 The biological membrane finished product in Example 4 of the application, wherein (A) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 4:1:4; (B) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 2:1:2; (C) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 1:1:2; (D) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 1:1:1; (E) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 1:0:0; (F) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 0:1:0; (G) the mass ratio of chondroitin sulfate: chitosan: functional polypeptide is 0:0:1.

[0045] Figure 3 The mouse wound treatment schematic diagram in Example 6 of the application.

[0046] Figure 4 The MRSA bacterial quantity detection result of the mouse skin wound tissue in Example 6 of the application. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below in combination with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the protection scope of the present application.

[0048] The chondroitin sulfate (chondroitin sulfate) used in the following examples is sturgeon chondroitin sulfate, and the molecular weight is 26.57 kDa. The specific preparation method is as follows: after the freeze-dried cartilage is ground, chondroitin sulfate extraction enzyme is added, and enzyme hydrolysis is carried out for 4 h (20 mg of chondroitin sulfate special extraction enzyme per gram of sturgeon cartilage; the enzyme hydrolysis conditions are: pH 6-8, 58-60°C, enzyme hydrolysis for 4-5 h), the enzyme is inactivated, and chondroitin sulfate AC enzyme is continuously used for enzyme hydrolysis, and the amount of chondroitin sulfate AC enzyme is 0.05-0.15 IU / mg of substrate, and the enzyme hydrolysis is carried out at pH 5-7, 34-38°C for 80-90 min. The enzyme hydrolysis product is precipitated with anhydrous ethanol for 10-14 h, and the precipitate is collected to obtain the required sturgeon chondroitin sulfate. The molecular weight of chitosan used in the following examples is about 2000 Da.

[0049] Example 1: Obtaining of functional polypeptide

[0050] 1. Preparation of sturgeon cartilage active peptide

[0051] The sturgeon cartilage is washed clean, freeze-dried and ground, then water is added at a ratio of 1:25, the pH is adjusted to 7, alkaline protease is added at a dosage of 150 IU / g of sturgeon cartilage, and enzyme hydrolysis is carried out for 4 h. After the enzyme is inactivated, chondroitin lyase is added at a dosage of 200 IU / g of sturgeon cartilage, and enzyme hydrolysis is carried out for 3 h. After 100°C enzyme inactivation treatment, the supernatant is obtained by centrifugation at 10,000 r / min for 15 min. After adding 3 times the volume of anhydrous ethanol, alcohol precipitation is carried out at 4°C overnight. After centrifugation at 10,000 r / min for 15 min, the supernatant is separated, concentrated, freeze-dried, and the sturgeon cartilage rich in amino acid active peptide mixture is obtained, with a yield of 64.38%.

[0052] 2. Gel chromatography separation

[0053] 5 mL of sturgeon cartilage rich in amino acid active peptide mixture is added to a Sephadex G-15 gel chromatography column, and the gel column is continuously eluted with deionized water (flow rate is 1.0 mL / min). Each component is collected according to the elution curve peak observed at 280 nm, and the collected components are freeze-dried and evaluated for functional activity (the detection method of functional activity is the same as that in Examples 2-3).

[0054] The anti-inflammatory activity and the bone cell proliferation activity of the above collected components 1-4 are shown in Table 1.

[0055] Table 1

[0056] Component Proportion Anti-inflammatory activity (NO production) Bone cell proliferation rate 1 37.97％ 11.22 μmol / L 12.33％ 2 17.02％ 5.05 μmol / L 19.32％ 3 18.74％ 3.38 μmol / L 48.05％ 4 26.27％ 36.66 μmol / L 32.74％

[0057] 3. Reverse phase high performance liquid chromatography (RP-HPLC) separation

[0058] The components obtained from gel chromatography were further purified using RP-HPLC on a Kromasil 100-5-C18 semi-preparative column. The chromatographic conditions are shown below:

[0059] Mobile phase A: ultra-pure water (containing 0.1% trifluoroacetic acid);

[0060] Mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid);

[0061] Injection volume: 50 μL;

[0062] Sample concentration: 5 mg / mL;

[0063] Detection wavelength: 280 nm;

[0064] Elution program: 0-5 min, mobile phase B 0%; 5-10 min, mobile phase B from 0% to 20%; 10-45 min, mobile phase B from 20% to 45%; 45-65 min, mobile phase B from 45% back to 20%.

[0065] Fractions of each eluted peak were collected using an automatic fraction collector and then lyophilized. The proportion and activity of each component (activity detection method same as Examples 2 and 3) are shown in Table 2.

[0066] Table 2

[0067] Component Proportion Anti-inflammatory activity (NO production) Bone cell proliferation rate 1 6.24％ 7.74 μmol / L 10.99％ 2 8.30％ 7.77 μmol / L 50.82％ 3 9.18％ 20.04 μmol / L 18.84％ 4 10.42％ 4.02 μmol / L 15.32％ 5 11.33％ 10.11 μmol / L 57.39％ 6 9.85％ 3.19 μmol / L 55.38％ 7 10.52％ 30.00 μmol / L 48.86％ 8 7.00％ 32.95 μmol / L 55.95％ 9 3.76％ 3.80 μmol / L 39.44％ 10 12.57％ 5.93 μmol / L 15.97％ 11 10.83％ 33.33 μmol / L 20.38％

[0068] 4. LC-MS / MS analysis

[0069] The samples of each component obtained by the above-mentioned reversed-phase high-performance liquid chromatography separation were subjected to chromatographic separation using a nanoliter flow rate Easy nLC1200 chromatographic system (Thermo Scientific). The buffer solution was 0.1% formic acid aqueous solution for A liquid and a mixed solution of acetonitrile and water containing 0.1% formic acid (80% acetonitrile) for B liquid. The chromatographic column was equilibrated with 100% A liquid. After the sample was injected into the Trap Column (100 μm*20 mm, 5 μm, C18, Dr. Maisch GmbH), it was subjected to gradient separation through the chromatographic analysis column (75 μm*150 mm, 3 μm, C18, Dr. Maisch GmbH) at a flow rate of 300 nl / min. The liquid separation gradient was as follows: 0 min-2 min, B liquid linear gradient from 2% to 5%; 2 min-44 min, B liquid linear gradient from 5% to 28%; 44 min-51 min, B liquid linear gradient from 28% to 40%; 51 min-53 min, B liquid linear gradient from 40% to 100%; 53 min-60 min, B liquid maintained at 100%.

[0070] After the peptide segment separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60 min, and the detection mode was positive ion, the parent ion scan range was 350-1800 m / z, the first mass spectrometry resolution was 60,000@m / z 200, the AGC target was 3e6, and the first maximum IT was 50 ms. The peptide segment second mass spectrometry analysis was collected according to the following method: after each full scan, the second mass spectrometry spectrum (MS2 scan) of 20 highest intensity parent ions was triggered to be collected, the second mass spectrometry resolution was 15,000@m / z 200, the AGC target was 1e5, the second maximum IT was 50 ms, the MS2 Activation Type was HCD, the Isolation window was 1.6 m / z, and the Normalized collision energy was 28.

[0071] The mass spectrometry Basepeak of the polypeptide is shown in Table 1. Figure 1

[0072] After the above separation, purification and identification, a plurality of polypeptides with different sequences, molecular weights and properties were obtained. Functional analysis was performed on these polypeptides to screen functional polypeptides with anti-inflammatory and cell proliferation and differentiation promoting functions. The sequences of the polypeptides screened and having higher anti-inflammatory and cell proliferation and differentiation promoting activities are shown in Table 3 (SEQ ID NO. 1). As an example, some polypeptides with poor activity and their sequences (SEQ ID NO. 2-4) separated are also listed in Table 3.​

[0073] Table 3

[0074] Sequence No. Peptide segment sequence SEQ ID NO. 1 STESLQANVQR SEQ ID NO. 2 IGGIGTVPVGR SEQ ID NO. 3 RYPIVTPR SEQ ID NO. 4 GKTGPVGPQ

[0075] Detection of anti-inflammatory activity of functional polypeptide of Example 2

[0076] The polypeptide with sequence as shown in SEQ ID NO. 1 was detected for anti-inflammatory activity, and the specific method and results are as follows:

[0077] After using C57 mice, the mice were orally administered with different polypeptide samples of the same concentration for 2 weeks, and the whole blood of the mice was collected by the method of orbital blood collection. The expression levels of inflammatory factor NO, anti-inflammatory factor IL-10 and pro-inflammatory factor IL-1β in the serum of the mice were determined by ELISA kit, and the specific experimental steps were operated according to the kit instructions.

[0078] (1) Preparation of serum sample: Take the whole blood and place it in a 1.5 mL centrifuge tube, stand at room temperature for 2 h, centrifuge at 1000 g for 15 min to collect the serum and transfer it to a new centrifuge tube, and store it at -80℃ for standby.

[0079] (2) ELISA: Use diluent to configure different concentration gradient standards. The experiment sets standard wells, sample wells and blank control wells, 50 μL of different concentration standards are added to each standard well to establish the standard curve; the polypeptide sample to be tested is diluted 5 times with diluent, and the diluent is used as a blank control, then 50 μL of sample / test solution is slowly added to the bottom of the 96-well enzyme-labeled plate, 100 μL of enzyme-labeled reagent is added to each well, after incubation at 37℃ for 60 min, the liquid in the plate is aspirated and discarded, and after spinning, 200 μL of washing solution is added to each well, stand for 30 s, then spin and discard the washing solution, repeat the above washing steps 5 times, after drying, add 50 μL of color developing agent A and 50 μL of color developing agent B to each well, mix well on a shaker at low speed, and after reaction at 37℃ for 15 min, add an equal volume of stop solution to terminate the reaction, use the blank hole to zero the enzyme-labeled instrument, then measure the absorbance value of the sample at 450 nm wavelength and record it.

[0080] The results are shown in Table 4.

[0081] Table 4

[0082]

[0083] Detection of activity of functional polypeptide for promoting cell proliferation and differentiation of Example 3

[0084] The polypeptide with sequence as shown in SEQ ID NO. 1 was detected for activity of promoting cell proliferation and differentiation, and the specific method and results are as follows:

[0085] 1. Cell culture:

[0086] MC3T3-E1 osteoblasts were subcultured and grown to a confluence rate of 80-90% before the next passage. The cells were digested with 0.25% trypsin (without EDTA) and seeded at a density of 2 x 10 4 cell / mL into 100 x 100 mm culture dishes or six-well plates and cultured for 24-72 h before subculture and storage.

[0087] 2. Cell proliferation experiment:

[0088] The osteoblasts were subcultured and counted when they were in good condition. The cells were seeded at a density of 4 x 10 4 cell / mL into 96-well cell culture plates and cultured in complete medium for 24 h. The original medium was then discarded and the cells were starved in serum-free medium. After 12 h of incubation at 37°C and 5% CO2, different polypeptide samples at the same concentration were added to the cells, and a blank control group was set up. Each treatment group had five replicates. After 36 h of incubation, 10 μL of CCK-8 was added to each well, and the reaction was carried out at 37°C in the dark. The A450 value was determined using a microplate reader, and the results were calculated using the following formula:

[0089]

[0090] 3. Determination of alkaline phosphatase (ALP) activity for promoting cell differentiation:

[0091] The preosteoblast cell line MC3T3-E1 was seeded at a density of 2 x 10 4 cell / mL into 24-well cell culture plates and cultured normally for 2 days until the confluence rate reached 90%. The medium was then replaced with differentiation medium every other day, and the cells were cultured for 4 days. The medium was then replaced with serum-free medium overnight, and different polypeptide samples at the same concentration were added to the cells. The control group was not treated. The original medium was removed, and the cells were washed three times with pre-cooled PBS at 4°C. Cell lysis solution was added to the cells on ice, and the cells were scraped off with a cell scraper. The cells were dispersed by repeated pipetting with a pipette gun, and the cells were treated with a cell sonicator for 1 min. The cells were centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was collected and the protein concentration was determined. The activity of ALP in each sample was determined using a Roche alkaline phosphatase diagnostic kit, and the results were corrected for protein concentration. The ALP activity was calculated relative to the blank control group, and the fold change of each group relative to the blank control group was calculated.

[0092] The results are shown in Table 5.

[0093] Table 5

[0094] Polypeptide sequence No. Bone cell proliferation rate ALP activity SEQ ID NO. 1 59.95％ 50.23％ SEQ ID NO. 2 26.07％ 20.52％ SEQ ID NO. 3 40.11％ 32.06％ SEQ ID NO. 4 29.95％ 25.37％

[0095] Preparation of active biofilm

[0096] Chondroitin sulfate, chitosan and functional polypeptide (amino acid sequence as shown in SEQ ID NO. 1) were mixed in a ratio of 4:1:4, 2:1:2, 1:1:2, 1:1:1, 1:0:0, 0:1:0 and 0:0:1 by mass ratio in 10 mL of deionized water (in each of the above mass ratio groups, the mass-volume ratio (g:mL) of chondroitin sulfate to water was 4%, 2%, 1%, 1%, 1%, 0%, 0%, respectively), stirred for 4 h, and then ultrasonically degassed to remove air bubbles. Then, the mixture was poured into a petri dish, adjusted to a thickness of 0.5 cm, and dried into a film at room temperature or in an oven at 40°C.

[0097] After drying, the biofilm was carefully removed and stored in a desiccator for future use.

[0098] The active biofilm prepared as above was characterized as shown in Table 1. Figure 2

[0099] Example 5: Detection and characterization of active biofilm

[0100] The tensile force and puncture force were measured using a texture analyzer, and the water absorption of the active biofilm was also detected. The specific method and results are as follows:

[0101] 1. Tensile force and puncture force detection

[0102] Experimental method: The mechanical properties of the active biofilm were tested using a texture analyzer. The prepared biofilm sample was cut into a 0.9 x 6 cm strip, and the two ends were clamped with a clamp. The test parameters were as follows: the test type was tension mode, the test target was distance, the target value was 100 mm, the waiting time was 0 s, the trigger point load was 70 mN, the test speed was 0.5 mm / s, and the clamp was TA-DGA. The tensile strength and elongation at break were calculated according to the following formula:

[0103] Tensile strength (Mpa) = F / (h x d);

[0104] Tensile elongation at break (%) = (L-L0) / L0 x 100;

[0105] Where F is the maximum tension at break of the film sample, h (mm) represents the width of the film sample, d (mm) represents the thickness of the film sample; L represents the length of the film sample after stretching, and L0 represents the length of the film before stretching.

[0106] ​The edibility of the packaged food depends on the type of polymer matrix used in the packaging film, the type of additive and the interaction between them. The mechanical properties of the packaging film can be characterized by tensile strength and elongation at break. The experimental results show (Table 6) that the addition of functional polypeptide and chondroitin sulfate significantly improves the tensile strength and elongation at break of the active biofilm compared to other films. It can be seen that chondroitin sulfate as a polymer can produce a strong interaction between polypeptide and chitosan, so it can be used as a crosslinking agent to make the molecules arrange densely and promote the increase of the tensile strength of the film.

[0107] Table 6 Mechanical properties of the film

[0108]

[0109] 2. Water absorption test

[0110] Experimental method: accurately cut the same size of active biofilm, and weigh the weight of the active biofilm m1. The weighed active biofilm is immersed in ultrapure water at room temperature, and after 4h, the active film is carefully taken out, the surface water is absorbed by filter paper, and then the weight of the film at this time is accurately weighed m2. The calculation of water absorption rate is carried out according to the following formula:

[0111] W% = (m2-m1) / m1x100%;

[0112] Experimental results: the experimental results are shown in Table 7. The prepared biofilm has good water absorption capacity, can effectively reduce water evaporation, and has good application prospect.

[0113] Table 7 Experimental results of water absorption rate

[0114]

[0115] 3. Film thickness detection

[0116] Experimental method: the film thickness of 6 random positions on the film sample is measured by a manual digital micrometer with a precision of 0.001mm, and the average value is taken. Each sample is repeated 8 times.

[0117] Experimental results: the thickness detection results of the biofilm are shown in Table 8. Moreover, through the test analysis of preparing biofilm by using different amounts and proportions of chondroitin sulfate, chitosan and polypeptide, the thickness of the film increases with the increase of the solid content of the film.

[0118] Table 8 Thickness of film sample

[0119]

[0120]

[0121] 4. Water vapor transmission rate detection

[0122] Experimental method: seal the intact film sample in a weighing cup (diameter 4 cm, depth 4 cm) containing 3 g of CaCl2, then place the cup in a desiccator containing saturated potassium iodide (20℃, 70% relative humidity), weigh the cup every 4 h, for 48 h. Calculate the water vapor permeability according to the following formula:

[0123] Water vapor permeability = (M x x) / (t x A x Δp);

[0124] Where M is the weight difference of the weighing cup (g), x is the thickness of the film (m), A is the permeation area of the film (m2), Δp is the vapor pressure difference of the permeation area (2339 Pa at 20℃), t is the permeation time (s).

[0125] The experimental results are shown in Table 9.

[0126] Table 9 Water vapor permeability test results

[0127]

[0128] Barrier performance is directly related to the food preservation ability of the biofilm, and is usually determined by the water vapor permeability, which mainly determines the ability of the biofilm to prevent water loss during food preservation, and is mainly related to the permeability of the active biofilm. The permeability of the film is related to the degree of cross-linking between the components of the film, and a high degree of cross-linking can increase the path of water vapor transmission and reduce the water vapor permeability. The above experimental results show that the water vapor permeability of the active biofilm of the present application is significantly lower than that of the traditional chitosan film, and the reduction in water vapor permeability of the biofilm can improve its food preservation ability, especially for postharvest fruit preservation applications, the enhancement of barrier performance can reduce the respiration rate and water evaporation of the fruit, and more effectively delay the aging of postharvest fruit.

[0129] Example 6 Analysis of the biological activity of the active biofilm

[0130] 1. Evaluation of wound healing and anti-infection effect promotion

[0131] In order to evaluate the effect of the active biofilm prepared in the above examples on the treatment of wound infection, a mouse wound infection model was constructed to evaluate the treatment effect of the active biofilm on bacterial inflammation, and the specific method and results are as follows:

[0132] BALB / c mice aged 6-8 weeks were placed in cages with standard temperature (24℃) and humidity (60-70%) for 3 days, and randomly divided into 3 groups (6 mice per group): normal group, PBS blank group, and active biofilm group. The mouse back hair was treated with depilatory cream and a razor, and then a 10 mm long wound was made on the back of the mouse with scissors (such asFigure 3 The skin wound was covered with 20 μL of bacteria (methicillin-resistant Staphylococcus aureus, MRSA, 10 7 CFU / mL). Different groups of mice were treated with PBS buffer solution and active biofilm, respectively (wherein the normal group was subjected to skin wound making, the PBS blank group was subjected to skin wound making and then the skin wound was smeared with sterile PBS, and the bioactive film group was subjected to skin wound making and then the skin wound was smeared with a film with a size corresponding to that of the skin wound and completely covered on the skin wound), and the mice were placed under simulated sunlight for 10 min. After 4 days of treatment, the mice were sacrificed and the skin wound tissues were obtained.

[0133] After the skin wound tissues of the mice were obtained, they were placed in 5 mL of sterile normal saline for culture. After the tissues were homogenized by crushing, the obtained solution was cultured at 37°C overnight, and the number of bacteria was determined by plate counting method.

[0134] The results are shown in Figure 4 The number of MRSA bacteria in the skin wound tissues of the mice was determined by standard plate counting method, and the number of bacteria on the skin surface in the PBS treatment group was about 5.26 log 10 CFU / mL, and after treatment with the biofilm, the cell density of the living bacteria was only 0.51 log 10 CFU / mL, which was significantly lower than that in the PBS treatment group and close to that in the normal group, indicating that the number of bacteria on the skin surface was significantly reduced after treatment with the biofilm, and the reduction of MRSA was beneficial to the healing of the skin tissue.

[0135] 2. Effect on the proliferation activity of skin tissue cells

[0136] The effect of the active biofilm on the proliferation of skin tissue cells was detected, and the experimental method was as follows:

[0137] 1) The skin tissue was placed in 1xPBS solution pre-cooled at 4°C for washing, and non-target tissue components were removed.

[0138] 2) The skin was cut into particles with a size of 0.5 cm 3 , and washed to remove debris.

[0139] 3) A small opening was made in the skin epidermis layer with a thickness of 200 μm, which helped the penetration of the enzyme solution.

[0140] 4) The treated skin tissue was placed in a centrifuge tube containing 50 mL of RPMI+2 U / ml Dispase II.

[0141] 5) The tube opening was sealed with a sealing film, and the centrifuge tube containing the enzyme solution and the skin tissue was placed in a water bath at 37°C and 60 rpm / min for digestion.

[0142] 6) 37°C water bath for 1 hour, during which the centrifuge tube was turned over every 10 minutes to ensure the skin was fully exposed to the dissociation solution.

[0143] 7) After 1 hour, the first round of enzyme solution was removed by rinsing with 1x PBS.

[0144] 8) The epidermis was carefully peeled from the dermis, and the peeling was performed in 1x PBS in an ice bath.

[0145] 9) The peeled epidermis and dermis were placed in a cell culture dish containing 20 ml of RF10 (RPMI + 10% FBS + 1% Penicillin-Streptomycin + 1% L-Glutamine) and 1.5 mg / ml collagenase; and they were placed in a 37°C, 5% CO2 incubator for overnight or more than 12 hours.

[0146] 10) The supernatant was collected using a pipette and filtered using a filter with a pore size of 100 μm, and the cells in the dermis and epidermis were collected.

[0147] 11) After counting the cells, the cells were added to a 96-well plate at a density of 5x10 4

[0148] Results are shown in Table 10.

[0149] Table 10 Skin cell proliferation rate

[0150]

[0151] The above experimental results show that, compared with normal and PBS-treated mice, the proliferation rate of epidermal cells and dermal cells in the tissues of the mice treated with the biofilm has a significant growth trend, indicating that the prepared biofilm has the ability to promote the proliferation and differentiation of skin tissue cells in mice, and is beneficial to wound healing.

[0152] 3. Bio-compatibility test of biofilm

[0153] ​The above results show that the prepared biofilm has good photodynamic bactericidal performance and the ability to promote wound healing, but the biological safety of the photodynamic bactericidal system should also be considered. In order to evaluate the biocompatibility of the biofilm, a cytotoxicity test was performed using NIH 3T3 mouse embryonic fibroblasts.

[0154] Experimental method: Mouse embryonic fibroblasts (NIH 3T3) were used in the experiment, and the cells were cultured in 3T3 cell culture medium at 37°C, 5% CO2. According to 2x10 4 cells / mL density into a 24-well plate, and the adherent cells were cultured overnight in a carbon dioxide incubator. The original cell culture medium was discarded, and an equal volume of biofilm solution (200 μg / mL) of the same concentration was added to each well. The control group and the blank group were set up, and the culture was continued for 24 h. Then 10 μL of CCK-8 was added to each well, and the reaction was carried out at 37°C in the dark. The A450 value was measured by a microplate reader, and the cell survival rate was calculated according to the following formula: cell survival rate = (A treatment - A blank) / (A control - A blank) * 100%.

[0155] The results are shown in Table 11.

[0156] Table 11 Cell survival rate

[0157]

[0158] The results of the cytotoxicity test show that after 3T3 cells are treated with a certain concentration of biofilm for 24 h, they still exhibit a high survival rate and a high cell viability value, and the active biofilm exhibits good biocompatibility.

[0159] 4. Fresh-keeping effect evaluation

[0160] Fresh and intact plums were selected as the test object and were placed in beakers with open, ordinary PE preservative film, chitosan film, chondroitin sulfate, active peptide and active biofilm, respectively. The beakers were stored at room temperature of 25°C for 0, 2, 4 and 6 days. The L*, a* and b* values of the film samples were measured by diffuse reflection spectrophotometer in L*, a* and b* modes. Before measurement, the white standard plate was calibrated. The fruit hardness was measured by a fruit hardness tester (GY-3, Zhejiang Top Instrument Co., Ltd.). During the measurement process, two points were taken symmetrically on each fruit at an equal distance, and the peel of the test site was gently scraped off with a sharp blade. The hardness tester probe was pressed into the outer peel until the probe ring reached the outer peel. The fruit hardness of each group was randomly sampled and expressed in kg cm -2 .

[0161] The results are shown in Table 12, and the results show that in all treatments, the hardness of the fruit decreases with the increase of storage time. However, the biological membrane treatment can more effectively maintain the hardness of the fruit during storage than other control treatments. With the increase of storage time, the L* value of the fruit decreases, and the a* value increases. Compared with the control group, the a* value after the biological membrane treatment is lower than that of other treatments, indicating that the red flesh of the fruit during storage is significantly delayed, and the biological membrane prepared by the application has a fresh-keeping effect.

[0162] Table 12 Fruit property detection

[0163]

[0164]

[0165] 5. Adsorption of pesticides

[0166] Experimental method: The adsorption capacity of the biological membrane for pesticides is detected by static adsorption method. The specific steps are as follows: 50 mg of common adsorbents: activated carbon particles, activated alumina, diatomite, macroporous adsorption resin, cellulose powder, chitosan, and biological membrane are weighed and added to 1 L of pesticide solution (100 mg / L), and stirred and adsorbed by a magnetic stirrer. After 24 h of adsorption, centrifugation (10000 rpm, 4℃, 10 min) is performed to take the supernatant, and after filtration through a 0.22 μm filter membrane, a kit is used to test the content of each component in the residual pesticide.

[0167] As shown in Table 13, the biological membrane has a certain adsorption capacity for each pesticide, and compared with the simple use of chitosan, the adsorption capacity is significantly enhanced. The active biological membrane prepared by the application has a good application prospect as a pesticide adsorbent.

[0168] Table 13 Adsorption capacity of various adsorbents for pesticide residues

[0169]

[0170] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a functional polypeptide in the preparation of an anti-inflammatory medicament; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO.

1.

2. Use of a nucleic acid molecule encoding a functional polypeptide or a biological material comprising the nucleic acid molecule in the preparation of an anti-inflammatory medicament; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO. 1; the biological material is an expression cassette, a vector or a host cell.

3. Use of a functional polypeptide in the preparation of an agent for promoting the proliferation of bone cells in vitro; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO.

1.

4. Use of a nucleic acid molecule encoding a functional polypeptide or a biological material comprising the nucleic acid molecule in the preparation of an agent for promoting the proliferation of bone cells in vitro; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO. 1; the biological material is an expression cassette, a vector or a host cell.

5. Use of a functional polypeptide in the preparation of an agent for promoting the differentiation of bone cells in vitro; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO.

1.

6. Use of a nucleic acid molecule encoding a functional polypeptide or a biological material comprising the nucleic acid molecule in the preparation of an agent for promoting the differentiation of bone cells in vitro; the amino acid sequence of the functional polypeptide is shown as SEQ ID NO. 1; the biological material is an expression cassette, a vector or a host cell.

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