CN117917427A - Polypeptides for modulating oral flora balance - Google Patents
Polypeptides for modulating oral flora balance Download PDFInfo
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- CN117917427A CN117917427A CN202310002737.6A CN202310002737A CN117917427A CN 117917427 A CN117917427 A CN 117917427A CN 202310002737 A CN202310002737 A CN 202310002737A CN 117917427 A CN117917427 A CN 117917427A
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides polypeptides for modulating the balance of oral flora. The sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptide can selectively inhibit harmful bacteria in oral cavity, ensure normal physiological activity of oral probiotics, further effectively maintain dynamic balance between oral microorganisms and between microorganisms and host, be beneficial to maintaining normal organism metabolism, immunity, nutrition and oral health, and effectively prevent dental caries, periodontal disease, halitosis, recurrent oral ulcer, etc.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly, it relates to polypeptides for modulating the balance of oral flora.
Background
Billions of microorganisms are living in the oral cavity, and various microorganisms are co-inhabited, competing and antagonized at different positions, so that a self-stable state of dynamic balance is maintained in population quantity and functions, and a complex oral cavity microecological micro-world of human beings is constructed. Among them, as the oral flora of the second largest flora of the human body, bacterial species are up to 700 more, most of them are beneficial, can resist diseases, help digestion and regulate body functions, and have an important role in oral health, called oral Probiotics (Probiotics).
Oral probiotics, a class of active microorganisms beneficial to the host, exert their probiotic efficacy mainly through processes of secretion of antibacterial substances, competitive colonization with oral harmful bacteria, regulation of host immune response, regulation of biofilm pH, etc., even specific probiotic strains such as lactobacillus acidophilus (Lactobacillus acidophillus) and lactobacillus rhamnosus (Lactobacillus rhamnosus) have been demonstrated to have antibacterial and antifungal effects, lactobacillus salivarius (Lactobacillus salivarizs) is believed to inhibit the occurrence of dental caries and antagonize periodontal disease-causing bacteria (Yan Ziheng, hu Xiaofang, wu Xin. Research on the effects of probiotics on oral common diseases [ J ]. Stomatology, 2018,38 (02): 185-188.doi: 10.13591/j.cnki.kqyx.2018.02.021.).
Under normal physiological conditions, dynamic balance exists between oral microorganisms and between microorganisms and hosts, and the oral microorganisms participate in metabolism, immunity, nutrition and maintenance of oral health. However, at present, many oral antibacterial products kill oral harmful bacteria and also kill oral probiotics too much, so that the balance of an oral microbial system is broken, the oral probiotics are reduced and cannot proliferate rapidly, the oral harmful bacteria are increased and toxins produced by the oral harmful bacteria damage oral health, and then related oral diseases such as decayed teeth, periodontal disease, halitosis, recurrent oral ulcer and the like are caused, and the oral diseases further aggravate the imbalance of oral micro-ecology, so that oral problems enter into vicious circle, therefore, the search of an oral antibacterial product capable of effectively regulating the balance of oral flora is necessary for oral health.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the polypeptide for regulating and controlling the balance of oral flora, which can selectively inhibit harmful bacteria in the oral cavity, ensure the normal physiological activity of probiotics in the oral cavity and further effectively maintain the balance of micro-ecology in the oral cavity.
A first object of the present invention is to provide a polypeptide having a homology of 90% or more with the polypeptide shown in SEQ ID NO. 1.
It is a second object of the present invention to provide polypeptides for modulating the balance of oral flora.
The third object of the present invention is to provide a polypeptide obtained by subjecting the N-terminus of the above polypeptide to acetylation modification.
A fourth object of the present invention is to provide the use of the above-mentioned polypeptides in the preparation of bacteriostatic products.
It is a fifth object of the present invention to provide the use of the above polypeptide for the preparation of a product for regulating the balance of oral flora.
A sixth object of the present invention is to provide the use of the above polypeptide in the preparation of an oral product.
A seventh object of the present invention is to provide an oral appliance.
The above object of the present invention is achieved by the following technical scheme:
The polypeptide shown in SEQ ID NO. 1 comprises 36 amino acids, has a molecular weight of 4050.85, a molecular formula of C 189H310N50O48 and a total average hydrophilicity of-0.411, and the polypeptide can be found for the first time to ensure normal physiological activity of oral probiotics while selectively inhibiting oral harmful bacteria, thereby effectively maintaining balance of oral micro-ecology. Therefore, the invention provides a polypeptide with more than 90% of homology with the polypeptide shown in SEQ ID NO. 1, a polypeptide with a sequence shown in SEQ ID NO. 1 for effectively regulating the balance of oral cavity flora, a polypeptide obtained by carrying out acetylation modification on the N end of the polypeptide, and application of the polypeptide in preparing antibacterial products.
Preferably, the antibacterial is for inhibiting oral harmful bacteria.
Further preferably, the oral pest comprises one or more of staphylococcus aureus, fusobacterium nucleatum, escherichia coli, actinobacillus concomitans, fostana, praecox intermedia, porphyromonas gingivalis, treponema denticola, streptococcus mutans, candida albicans, or actinomyces viscosus.
More preferably, the oral pest is one or more of staphylococcus aureus, escherichia coli or porphyromonas gingivalis.
The invention also provides application of the polypeptide in preparing a product for regulating the balance of oral flora.
Preferably, the modulating oral flora balance comprises inhibiting oral harmful bacteria and maintaining oral beneficial bacteria activity.
Further preferably, the oral pest comprises one or more of staphylococcus aureus, fusobacterium nucleatum, escherichia coli, actinobacillus concomitans, fostana, praecox intermedia, porphyromonas gingivalis, treponema denticola, streptococcus mutans, candida albicans, or actinomyces viscosus.
Further preferably, the oral probiotics comprise one or more of bifidobacterium longum, enterococcus faecalis, streptococcus salivarius, lactobacillus salivarius, lactococcus lactis, lactobacillus acidophilus, bifidobacterium adolescentis, streptococcus sanguinis, lactobacillus rhamnosus.
The invention also provides application of the polypeptide in preparing an oral product and an oral product containing the polypeptide.
Preferably, the oral product is one or more of oral care liquid, mouthwash, toothpaste or mouthwash effervescent tablets.
The invention has the following beneficial effects:
The polypeptide can selectively inhibit harmful bacteria in the oral cavity, ensure normal physiological activities of the probiotics in the oral cavity, further effectively maintain dynamic balance among oral microorganisms and between microorganisms and hosts, be beneficial to maintaining normal organism metabolism, immunity, nutrition and oral health of organisms, and effectively prevent oral diseases such as decayed teeth, periodontal disease, halitosis, recurrent oral ulcer and the like.
Drawings
FIG. 1 is an HPLC plot of the acetylated polypeptide of example 1.
FIG. 2 is a LCMS spectrum of the acetylated polypeptide of example 1.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Human oral epithelial cells HOEC: from Shenzhen Haodihua Biotechnology Inc.
Bifidobacterium longum: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 15707.
Enterococcus faecalis: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 29212.
Streptococcus salivarius: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 13419.
Lactobacillus salivarius: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 11741.
Lactococcus lactis: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 11007.
Lactobacillus acidophilus: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 4355.
Bifidobacterium adolescentis: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 15704.
Streptococcus sanguis: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 10556.
Lactobacillus rhamnosus: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 11981.
Staphylococcus aureus: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 29213.
Fusobacterium nucleatum: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 10953.
Coli: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 15597.
Actinobacillus: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 700685.
Fusarium (Fusarium): american type culture Collection (AMERICAN TYPE culture collection), number ATCC 43037.
Praecox intermedia: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 25611.
Porphyromonas gingivalis: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 33277.
Dense tooth scale screw: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 35405.
Streptococcus mutans: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 25175.
Candida albicans: american type culture Collection (AMERICAN TYPE culture collection), number ATCC 10231.
Actinomycetes viscosus: american type culture Collection (AMERICAN TYPE culture collection), accession number ATCC 27044.
Example 1 Polypeptides for modulating oral flora balance
Selecting 1-16 amino acids at the N-terminal of cecropin A (the sequence is shown as SEQ ID NO: 2: KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK), replacing 2 amino acids at the N-terminal of cecropin A with lysine, and marking as a sequence fragment 1;
Selecting 22-41 amino acids at the C-terminal of cecropin D (the sequence is shown as SEQ ID NO: 3: MKISKIFVFVFAIVFATASVSAAPGNFFKDLVSIVLDVSGS), replacing 38-40 amino acids at the C-terminal of cecropin D with 3 prolines, replacing 41 amino acids at the C-terminal of cecropin D with asparagine, and marking as a sequence fragment 2;
Connecting the sequence segment 1 and the sequence segment 2 to obtain a target polypeptide sequence, wherein the N end of the sequence segment 1 is used as the N end of the target polypeptide sequence, and the C end of the sequence segment 2 is used as the C end of the target polypeptide sequence: KKKLFKKIEKVGQNIRAAPGNFFKDLVSIVLDPPPN (shown as SEQ ID NO: 1).
The target polypeptide with the sequence shown as SEQ ID NO. 1 is synthesized by a protein chemical synthesis method (dehydration condensation) by the Minkangde New drug development limited company of Shanghai medicine, and in order to improve the stability of the target polypeptide in the following test experiment, the first lysine at the N end of the target polypeptide is subjected to acetylation modification to obtain the acetylated polypeptide. And then HPLC analysis and LCMS analysis are carried out on the obtained product, wherein the obtained HPLC spectrum and LCMS spectrum are respectively shown in fig. 1 and 2, and the basic physicochemical properties of the molecular weight, molecular formula and the like are shown in table 1.
TABLE 1
| Molecular weight | 4050.85 |
| Molecular formula | C189H310N50O48 |
| Amino acid number | 36 |
| Theoretical isoelectric point | 10.12 |
| Total number of positively charged residues (Arg+Lys) | 8 |
| Total number of negatively charged residues (Asp+Glu) | 3 |
| Total average hydrophilicity | 0.411 (Hydrophilic protein, soluble in water) |
| Instability index | 23.51<40.00, Classified as stable protein |
| Fat coefficient | 94.72 (Good stability) |
As can be seen from the combination of FIGS. 1-2 and Table 1, the acetylated polypeptides have a molecular weight of 4050.85, a molecular formula of C 189H310N50O48 and a purity of 98.52%.
Test example 1 cytotoxicity of acetylated polypeptides
The test example detects cytotoxicity of the acetylated polypeptides by an MTT colorimetric method, and is specifically as follows:
(1) Amplification culture: human oral epithelial cells HOEC were digested with 1mL of 0.25wt% trypsin for 2min, centrifuged at 800rpm for 5min, resuspended in MEM complete medium and then flask-expanded at 37℃under 5% volume fraction CO 2 and saturated humidity for 24h.
(2) And (3) paving: the cells in the flask were found to grow to 90% of the area of the bottom, and the cells were collected, then inoculated into 96-well plates at 10 4 cells/well, and further cultured for 24 hours.
(3) And (3) intervention: after removal of the medium, 30mL of MEM complete medium containing cecropin A at a final concentration of 0.5wt% was added, respectively, as test group No. 1; 30mL of MEM complete medium containing cecropin D at a final concentration of 0.5wt% was added, respectively, as test group No. 2; 30mL of MEM complete medium containing 0.5wt% final concentration of acetylated polypeptide was added, respectively, as experiment group No. 3; a blank (30 mL of MEM complete medium containing 10wt% fetal bovine serum without the test sample), a negative (30 mL of 0.9wt% sodium chloride solution) and a positive (30 mL of 5% volume fraction dimethyl sulfoxide solution) were simultaneously established, and the culture was continued under the same culture conditions of (1) for 24 hours.
(4) And (3) measuring: mu.L of 5mg/mL MTT solution was added to each well, the culture was continued for 4 hours, the liquid in the well was discarded, 150. Mu.L of DMSO was added, and after shaking for 10 minutes, absorbance was measured at 570nm and 630nm using an ELISA reader.
The relative proliferation rate (RGR) of each group of cells was calculated according to the formula "rgr=experimental group absorbance/blank group absorbance×100%". The results are shown in Table 2.
TABLE 2
| Sample of | Relative proliferation (%) |
| Experiment group 1 | 81.44±3.95 |
| Experiment group 2 | 79.64±5.30 |
| Experiment group 3 | 99.59±4.89 |
| Negative group | 99.52±3.71 |
| Positive group | 12.31±0.15** |
| Blank group | 100 |
* P <0.01 compared to the blank group.
It can be seen that the proliferation of HOEC is inhibited to some extent in the experimental group 1 and the experimental group 2, and that the proliferation of HOEC is hardly affected in the experimental group 3, which indicates that the toxicity of the polypeptide of the invention is low.
Test example 2 haemolysis of acetylated polypeptides
The test for evaluating the hemolysis of the acetylated polypeptide obtained in example 1 was carried out with reference to the guidelines for research on drug irritation, allergy and hemolysis.
Firstly, 20mL of rabbit heart blood is taken and placed in an Erlenmeyer flask (the Erlenmeyer flask is filled with glass beads), the Erlenmeyer flask is shaken for 10min, fibrinogen is removed, the defibrinated blood is formed, 10 times of 0.9wt% sodium chloride solution is added, the mixture is shaken uniformly, centrifugation is carried out for 10min at a rotating speed of 1500r/min, the supernatant is removed, the precipitated red blood cells are washed for 2 times by using 0.9wt% sodium chloride solution, and the obtained red blood cells are diluted into a 2% red blood cell suspension by using 0.9wt% sodium chloride solution according to volume.
2.5ML of purified water was used as a positive control group, 2.5mL of a 0.9wt% sodium chloride solution was used as a negative control group, 2.5mL of an original cecropin A was used as a sample concentration of 0.5wt% sodium chloride solution (sodium chloride concentration of 0.9 wt%) was used as a test group No. 1, 2.5mL of an original cecropin D was used as a sample concentration of 0.5wt% sodium chloride solution (sodium chloride concentration of 0.9 wt%) was used as a test group No. 2, and 2.5mL of the acetylated polypeptide obtained in example 1 was used as a sample concentration of 0.5wt% sodium chloride solution (sodium chloride concentration of 0.9 wt%) was used as a test group No. 3. After 2.5mL of rabbit erythrocyte suspension is added respectively, the mixture is shaken and mixed uniformly, the mixture is placed in a water bath with the temperature of 37+/-0.5 ℃ for 3 hours, the mixture is taken out after the water bath is finished, and the mixture is centrifuged for 10 minutes at the rotating speed of 3000r/min, and the supernatant is sucked into a cuvette. The experiment was repeated three times and the results averaged. The absorbance of the positive control group (a positive), the absorbance of the negative control group (a negative) and the absorbance of the experimental group (a real) were measured at 542nm using a colorimetric method with the negative control group as a blank, and the hemolysis rate was calculated as = (a real-a negative)/(a positive-a negative) ×100%, and the results are shown in table 3.
TABLE 3 Table 3
| Sample of | Experiment group 1 | Experiment group 2 | Experiment group 3 |
| Hemolysis rate (%) | 3.01 | 3.05 | 1.04 |
It can be seen that the test group No. 3 has lower hemolysis, and the hemolysis rate is as low as 1.04%, which is significantly lower than that of the test group No. 1 and the test group No. 2, indicating that the hemolysis of the polypeptide of the present invention is lower.
Test example 3 significant reduction in probiotic inhibition
Referring to the antibacterial effect test method (suspension quantification method) of 7.3 antibacterial daily chemical products under the item QBT 2738-2012-evaluation method of antibacterial effect of daily chemical products, the test example tests the inhibition effect of acetylated polypeptide, cecropin A and cecropin D (the action concentration is 0.5 wt%) on several common oral probiotics for 2min, and establishes a positive group (cetylpyridinium chloride with the final concentration of 0.1 wt%) and a negative group (sodium chloride solution with the final concentration of 0.09 wt%). The results are shown in Table 4.
TABLE 4 Table 4
#: P <0.05 compared to cecropin A; # #. P <0.01 compared to cecropin A; * : p <0.05 compared to cecropin D; * *: p <0.01 compared to cecropin D.
It can be seen that compared to cecropin a and D, the inhibitory effect of the acetylated polypeptides of the present invention on oral probiotics is significantly reduced.
Test example 4 significantly enhanced inhibition of harmful bacteria
Referring to the antibacterial effect test method (suspension quantification method) of 7.3 antibacterial daily chemical products under the item QBT 2738-2012-evaluation method of antibacterial effect of daily chemical products, the test example tests the inhibition effect of acetylated polypeptide, cecropin A and cecropin D (the action concentration is 0.5 wt%) on several common oral harmful bacteria for 2min, and establishes a positive group (cetylpyridinium chloride with the final concentration of 0.1 wt%) and a negative group (sodium chloride solution with the final concentration of 0.09 wt%). The results are shown in Table 5.
TABLE 5
#: P <0.05 compared to cecropin A; # #. P <0.01 compared to cecropin A; * : p <0.05 compared to cecropin D; * *: p <0.01 compared to cecropin D.
Compared with cecropin A and cecropin D, the inhibiting effect of the acetylated polypeptide on oral harmful bacteria is obviously improved.
In conclusion, the polypeptide can selectively inhibit harmful bacteria in the oral cavity, ensure normal physiological activities of probiotics in the oral cavity, further effectively maintain dynamic balance among oral microorganisms and between microorganisms and hosts, be beneficial to maintaining normal organism metabolism, immunity, nutrition and oral health of organisms, and can effectively prevent oral diseases such as decayed teeth, periodontal disease, halitosis, recurrent oral ulcer and the like.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A polypeptide with more than 90 percent of homology with the polypeptide shown in SEQ ID NO. 1.
2. The polypeptide for regulating the balance of oral flora is characterized by having a sequence shown in SEQ ID NO. 1.
3. A polypeptide obtained by acetylating the N-terminus of the polypeptide of claim 2.
4. Use of a polypeptide according to any one of claims 1 to 3 for the preparation of a bacteriostatic product.
5. The use according to claim 4, wherein the antibacterial is against oral harmful bacteria.
6. The use according to claim 5, wherein the oral pest comprises one or more of staphylococcus aureus, fusobacterium nucleatum, escherichia coli, actinobacillus concomitans, fosetyl, praecox intermedia, porphyromonas gingivalis, treponema denticola, streptococcus mutans, candida albicans, or actinomyces viscosus.
7. Use of a polypeptide according to any one of claims 1 to 3 for the preparation of a product for regulating the balance of oral flora.
8. Use of a polypeptide according to any one of claims 1 to 3 in the preparation of an oral product.
9. The use according to claim 8, wherein the oral product is one or more of an oral care solution, a mouthwash, a toothpaste or a mouthwash effervescent tablet.
10. An oral product comprising the polypeptide of any one of claims 1 to 3.
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