CN115895967B - Lactobacillus rhamnosus for resisting human papillomavirus HPV and application thereof - Google Patents
Lactobacillus rhamnosus for resisting human papillomavirus HPV and application thereof Download PDFInfo
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- 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
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Abstract
The application provides lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.25853 and provides application of the lactobacillus rhamnosus in inhibiting human papillomavirus HPV. The lactobacillus rhamnosus CGMCC No.25853 can remarkably inhibit the growth and reproduction of human papillomavirus HPV; the preparation method has excellent application prospects in products such as probiotic powder, probiotic stick, probiotic cream and/or probiotic dressing gel.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus for resisting human papillomavirus HPV and application thereof.
Background
Lactobacillus rhamnosus (LactobacillusrhamnosusGG, LGG) is lactobacillus and a strain of Li Tanggan, is a gram positive bacterium which is plasmid-free, anaerobic, acid-resistant and spore-free, is commonly existing in intestinal tracts of human beings and animals, and has the biological advantages of strong tolerance, antibiotics resistance and the like.
Experiments and documents in related technologies show that lactobacillus rhamnosus can inhibit the expression of inflammatory factor IL-8 by downregulating the expression level of phosphorylated proteins in MAPK, JAK/STAT and NF-kB pathways, thereby playing roles in regulating intestinal flora and preventing and treating gastrointestinal tract infection and diarrhea.
In summary, although the application of lactobacillus rhamnosus in relieving enteritis is disclosed at the present stage, the application and report of lactobacillus rhamnosus in resisting HPV are not yet known, so that the screening of lactobacillus rhamnosus with HPV resisting effect is urgently needed.
Disclosure of Invention
In order to fill the blank of the lactobacillus rhamnosus in terms of HPV resistance, the application specifically screens and provides the lactobacillus rhamnosus with the HPV resistance effect of human papillomavirus and application thereof.
In a first aspect, the present application provides a human papillomavirus HPV resistant lactobacillus rhamnosus LK-08 (lactobacillus rhamnosus) deposited under the accession number: CGMCC No.25853, the preservation unit is: CGMCC.
The screened lactobacillus rhamnosus LK-08 (Lactobacillus rhamnosus) not only effectively fills the blank application of the strain in HPV resistance, but also has anti-inflammatory and antibacterial effects by inhibiting the expression of human papilloma virus HPVE6 and E7 proteins, and has extremely high application value.
In a second aspect, the present application provides the use of lactobacillus rhamnosus for the preparation of a product for alleviating bacterial and fungal gynaecological inflammation and/or inhibiting the proliferation and expression of HPV viruses.
Preferably, the human papilloma HPV virus inhibited by lactobacillus rhamnosus comprises HPV-16, HPV-18, HPV-30, HPV-31 and HPV-33.
Preferably, the product is selected from the group consisting of probiotic powder, probiotic bar, probiotic cream and/or probiotic dressing gel.
The probiotic powder, the probiotic stick agent, the probiotic cream and/or the probiotic dressing gel containing the lactobacillus rhamnosus can effectively utilize the epitheliophilic characteristics of HPV viruses, and exert the effects of relieving bacterial and fungal gynecological inflammation and/or inhibiting proliferation and expression of the HPV viruses by being retained on an affected part and entering target tissues.
In a third aspect, the present application provides a probiotic dressing gel, which adopts the following technical scheme:
a probiotic dressing gel comprises Lactobacillus rhamnosus LK-08 (Lactobacillus rhamnosus), and has viable count of no less than 5×10 5 CFU/g。
Preferably, the composition comprises the following components in percentage by weight: lactobacillus rhamnosus 0.1-10%, protein lysate 0-10%, interferon 0-1.0%, and dressing gel as the rest.
Preferably, the protein lysate is prepared by inactivating and lysing one or more of tea polysaccharide, HPVE6 protein and HPVE7 protein.
Preferably, the interferon is one or more of IFN- α, IFN- β, IFN- γ, IFN-T and IFN- ω.
Preferably, the polymer gel is formed by copolymerizing polyethylene glycol and polyglycolide and lactide according to a weight ratio of 1 (1.2-1.8).
By adopting the technical scheme, the probiotic dressing gel compounded by the raw materials has the advantages of convenient administration, long-acting slow release and stable performance, can be effectively retained on an affected part to exert the effects, is not easy to damage the organism and is easy to wash away;
the reason for this may be analyzed as follows: lactobacillus rhamnosus, protein lysate and interferon loaded in polymer gel formed by copolymerizing polyethylene glycol and polyglycol lactide are not easily interfered by adverse factors of affected parts, and can act on the affected parts for a long time through slow release and compound action, so that the relief of bacterial and fungal gynecological inflammation and/or the inhibition of HPV virus are realized.
In a fourth aspect, the present application provides a process for preparing a probiotic dressing gel:
a preparation method of the probiotic dressing gel comprises the following specific steps:
s1, vacuum dewatering polyethylene glycol at 100-120 ℃ for 1-2h, introducing argon gas, cooling to 80 ℃, and then adding a polyglycolide monomer and a catalyst stannous octoate;
s2, after the reaction temperature is increased to 140-156 ℃, and the reaction is carried out for 4-8 hours under the atmosphere of normal pressure argon, unreacted monomers and low boiling point products are removed to obtain polymer gel, lactobacillus rhamnosus, protein lysate and interferon are added, and then washing and freeze drying are carried out, so as to obtain the probiotic dressing gel.
By adopting the technical scheme, the preparation process is obviously simplified, and meanwhile, no additional additive and auxiliary agent are added, so that the gel performance of the probiotic dressing is relatively stable, the gel has excellent effects of relieving inflammation and resisting HPV, and the gel has the advantages of less possibility of interference of the amount of the live bacteria and longer efficacy due to the specificity of a gel system, so that the gel has higher economic benefit.
In summary, the present application has the following beneficial effects:
1. lactobacillus rhamnosus LK-08 (Lactobacillus rhamnosus) with a preservation number of CGMCC No.25853 screened in the application has extremely high application value in preparing products for relieving bacterial and fungal gynecological inflammation and/or inhibiting proliferation expression of HPV virus;
2. the probiotic dressing gel compounded by the raw materials has the advantages of convenient administration, long-acting slow release, stable performance and the like, can exert the effects of resisting inflammation and HPV by being effectively retained on an affected part, and is not easy to cause damage to a body;
3. the preparation process of the probiotic dressing gel not only remarkably simplifies the process, but also avoids the influence of heating and additives, and the obtained probiotic dressing gel has relatively stable performance, long-acting excellent anti-inflammatory and anti-HPV effects, and is suitable for industrialized high-benefit production.
Detailed Description
The present application is described in further detail below with reference to examples.
Preparation example
Preparation example 1
Lactobacillus rhamnosus LK-08 (Lactobacillus rhamnosus) against human papillomavirus HPV, deposited under accession number: CGMCC No.25853, and the preservation date is No. 3 of 9 months in 2022; the preservation units are as follows: CGMCC; the preservation unit addresses are: sampling, activating, and performing anaerobic amplification culture on MRS solid medium at 30deg.C for 48 hr.
Preparation examples 2 to 8
Protein lysate, which has different inactivated cleaved protein raw materials, is specifically shown in the following table, and is prepared by the following cleavage steps:
A. firstly, placing the protein raw material after inactivation for 5min at 120 ℃ in 0.01mol/LPBS buffer solution, and regulating the total protein content in the inactivation solution to 2000 mug/mL;
B. adding a cracking agent TritonX-100 accounting for 0.8 percent of the total weight of the inactivating liquid and sodium deoxycholate accounting for 0.6 percent of the total weight of the inactivating liquid into the inactivating liquid, and cracking for 120 minutes at 26 ℃ to obtain a cracking liquid;
C. firstly removing a cracking agent TritonX-100 and deoxycholate sodium in the cracking liquid by using a 300KD membrane ultrafiltration dialysis, and then centrifuging at 12000rpm for 2 hours to remove foreign proteins, collecting the supernatant and filtering and sterilizing to obtain the protein lysate.
Table: preparation examples 2 to 8 inactivated cleaved protein raw material
Performance test the probiotic dressing gels prepared in each example and comparative example were selected for testing and tested for in vitro effect against human papilloma HPV virus, the specific test steps and test conditions were as follows;
infecting human cervical cancer HeLa cells provided by the institute of biochemistry and cell biology of Shanghai life sciences of China academy of sciences with HPV-16 pseudovirus particles, and culturing by a conventional method;
then taking HeLa cells with good growth phase and growth condition, inoculating the HeLa cells into a 24-hole plate, after the cells are attached to the wall for 24 hours, feeding probiotic dressing gel (1 g), and respectively testing HPV content (copies/cell) after the cells are interfered for 12 hours, 24 hours, 48 hours and 72 hours.
Examples
Examples 1 to 6
The probiotic dressing gel is prepared from the following raw materials in parts by weight as shown in the table (g) by the following preparation process:
s1, vacuum dewatering polyethylene glycol at 120 ℃ for 1h, introducing argon gas, cooling to 80 ℃, and then adding a polyglycolide monomer and stannous octoate accounting for 0.5% of the total weight of the polyethylene glycol;
wherein the weight ratio of polyethylene glycol to polyglycolide is 1:1.2;
s2, after the reaction temperature is increased to 148 ℃, and the reaction is carried out for 6 hours under the atmosphere of normal pressure argon, firstly removing unreacted monomers and low boiling point products to obtain polymer gel, then adding lactobacillus rhamnosus, protein lysate and interferon, and then washing and freeze-drying to obtain the probiotic dressing gel;
wherein lactobacillus rhamnosus is prepared from preparation 1, and the viable count is 1×10 8 CFU/g; protein lysate was prepared from preparation 2; the interferon consisted of IFN- α, IFN- γ and IFN-T and was 1:1:0.8.
Table: examples 1-6 Probiotics dressing gel Components and weight (g)
Comparative example 1
A dressing gel differs from example 1 in that it does not contain Lactobacillus rhamnosus from preparation 1 and is replaced by an equivalent amount of polymer gel.
Comparative example 2
A probiotic gel differs from example 1 in that the lactobacillus rhamnosus contained is lactobacillus rhamnosus (lactobacillus rhamnosus) with deposit number: CGMCC No.24049, the preservation unit is: CGMCC.
The probiotic dressing gels of examples 1-6 and comparative examples 1-2 above were extracted and tested for HPV content (copies/cell) after 12h, 24h, 48h, 72h of intervention according to the test procedure and criteria described above, and the average (mean±sd, n=8) was reported in the table below.
Table: examples 1 to 6, comparative example 1 HPV content (copies/cell)
As can be seen from the above table, the probiotic dressing gels prepared in examples 1-6 all have excellent anti-HPV virus effects, and HPV contents after 12h, 24h, 48h and 72h of intervention are 8.62+ -0.22-9.40+ -0.21 respectively; 7.25+/-0.15-8.22+/-0.25; 3.76+/-0.20-7.18+/-0.52; 2.71+ -0.11-6.76+ -0.48;
compared with the comparative example 1 (blank control group) without lactobacillus rhamnosus and the comparative example 2 without special screening of lactobacillus rhamnosus, the anti-HPV virus performance of the anti-HPV virus composition is obviously improved, and particularly, the inhibition effect under the intervention of the equal time periods of 48 hours and 72 hours is most obvious;
also to be specified are: the weak inhibition effect of comparative example 2 under the intervention of 48h and 72h and the like is probably due to the antibacterial, anti-inflammatory efficacy and immunoregulatory capacity of lactobacillus rhamnosus, and has a certain inhibition effect on proliferation and expression of HPV virus.
In conclusion, the screened lactobacillus rhamnosus LK-08 (Lactobacillus rhamnosus) effectively fills the blank application of the strain in the aspect of HPV resistance, and can exert stable and efficient HPV resistance effect by inhibiting the expression of human papillomavirus HPVE6 and E7 proteins;
when the gel loaded with lactobacillus rhamnosus, protein lysate and interferon polymer is used as dressing gel, the gel benefits from the shape and the characteristics of the gel, has the advantages of convenience in administration, long-acting slow release, stable performance and the like, and can exert the anti-HPV effect by long-acting retention on an affected part in clinical application.
In addition, it can be seen from examples 1 to 4 that the greater the amount of lactobacillus rhamnosus LK-08, the higher the anti-HPV effect thereof, but above 5%, the better the effect thereof is, the same or equivalent, even at the initial stage, so that the amount of lactobacillus rhamnosus LK-08 is preferably 0.1 to 10%, and the protein lysate and interferon can further enhance the anti-HPV performance of the dressing gel, see examples 5 to 6.
Examples 7 to 13
A probiotic dressing gel differs from example 5 in the use of protein lysates used, the specific correspondence being shown in the following table.
Table: table comparing the usage of protein lysates in examples 7-13
The probiotic dressing gels of examples 7-13 above were extracted and tested for HPV content (copies/cell) after 12h, 24h, 48h, 72h of intervention according to the test procedure and criteria described above, and the average (mean±sd, n=8) was recorded in the table below.
Table: examples 5, 7-13 HPV content (copies/cell)
As can be seen from the above table, the probiotic dressing gels prepared in examples 5, 7-13 all have excellent anti-HPV virus effects, and the HPV content after 12h, 24h, 48h, 72h of intervention is 7.51±0.16-9.11±0.16, respectively; 6.42+/-0.13-8.10+/-0.11; 5.24+/-0.24-6.72+/-0.31; 4.88+ -0.42-6.28+ -0.22;
the lactobacillus rhamnosus, protein lysate and interferon polymer gel loaded are used as dressing gel, and the protein lysate has the advantages of convenient administration, long-acting slow release, stable performance and the like, and has the effect of strengthening the anti-HPV effect besides benefiting from the form and the characteristics of the protein lysate;
the properties of the protein lysate are different according to the selection of the cracking raw materials, and the multicomponent protein raw materials have a certain compounding effect, see examples 10-13, wherein tea polysaccharide, HPVE6 protein and/or HPVE7 protein are used as the optimal compounding, and the reasons for the analysis are probably as follows:
are well known in the art: HPV is a non-envelope double-chain small molecule circular DNA virus, and consists of a protein capsid and genetic material DNA, wherein the protein capsid consists of L1 and L2, wherein the C end of L1 protein and the N end of L2 protein are rich in positive charge amino acids (R and K), namely nuclear localization signals;
the infection process is also completed mainly by the combination of the nuclear localization signal and the corresponding receptor in the HeLa cell, so that when the protein lysate used in the application carries a large amount of glycoprotein with negative charges and a cleavage sequence similar to the nuclear localization signal, the protein lysate can respectively and cooperatively inhibit the proliferation and expression of HPV virus by combining with the virus coat protein carrying positive charges and combining with the corresponding receptor in the HeLa cell in advance.
Example 14
A probiotic dressing gel differs from example 5 in that the interferon used consists of IFN- α, IFN- β and IFN- γ and is 1:2:1.
Example 15
A probiotic dressing gel differs from example 5 in that the interferon used consists of IFN- α, IFN- β, IFN- γ and IFN-T in a 1:2:1:0.8 ratio.
The probiotic dressing gels of examples 14-15 above were extracted and tested for HPV content (copies/cell) after 12h, 24h, 48h, 72h of intervention according to the test procedure and criteria described above, and the average (mean±sd, n=8) was recorded in the table below.
Table: examples 5, 14-15 HPV content (copies/cell)
As can be seen from the above table, the probiotic dressing gels prepared in examples 5, 7-13 all have excellent anti-HPV virus effects, and the HPV content after 12h, 24h, 48h, 72h of intervention is 9.10±0.15 to 9.15±0.15, respectively; 8.02+/-0.11-8.18+/-0.12; 6.70+/-0.30-6.75+/-0.28; 6.25+/-0.20-6.30+/-0.24;
the lactobacillus rhamnosus, protein lysate and interferon polymer gel loaded are used as dressing gel, and the interferon has the advantages of convenient administration, long-acting slow release, stable performance and the like, and has the effect of strengthening the anti-HPV effect when being used as dressing gel;
the properties of the interferon are slightly different according to the selection and the compounding of the interferon, and a certain compounding effect is also achieved among various interferons, see examples 5 and 14-15, but the more the types of the non-interferons are, the better the analysis is, the possible reasons are as follows:
the interferon is combined with an interferon receptor on the surface of a target cell to induce a plurality of antiviral proteins in the target cell, prevent the synthesis of the viral proteins and inhibit the replication and transcription of viral nucleic acid, so that the aim of resisting viruses is fulfilled, and the target cell is damaged when the interferon is excessively classified;
and the purpose of enhancing the autoimmunity is achieved by activating the phagocytic activity of macrophages, but the test is an in vitro test, and thus is not shown in the data of examples 5, 14-15, but the addition of the interferon has extremely high practical value in the actual clinical application process.
Example 16
A probiotic dressing gel is different from example 1 in that the polymer gel used is copolymerized from polyethylene glycol and polyglycolide in a weight ratio of 1:1.5.
Example 17
A probiotic dressing gel is different from example 1 in that the polymer gel used is copolymerized from polyethylene glycol and polyglycolide in a weight ratio of 1:1.8.
Example 18
A probiotic dressing gel is different from example 1 in that the polymer gel used is copolymerized from polyethylene glycol and polyglycolide in a weight ratio of 1:2.0.
Example 19
The probiotic dressing gel differs from example 1 in that the polymer gel used has different preparation step parameters, as shown in detail below: s1, vacuum dewatering polyethylene glycol at 100 ℃ for 2 hours, introducing argon gas, cooling to 80 ℃, and then adding a polyglycolide monomer and stannous octoate accounting for 0.5% of the total weight of the polyethylene glycol;
s2, after the reaction temperature is increased to 140 ℃, and the reaction is carried out for 8 hours under the atmosphere of normal pressure argon, unreacted monomers and low boiling point products are removed, and the polymer gel is obtained.
Example 20
The probiotic dressing gel differs from example 1 in that the polymer gel used has different preparation step parameters, as shown in detail below: s1, vacuum dewatering polyethylene glycol at 120 ℃ for 1h, introducing argon gas, cooling to 80 ℃, and then adding a polyglycolide monomer and stannous octoate accounting for 0.5% of the total weight of the polyethylene glycol;
s2, after the reaction temperature is increased to 156 ℃, and the reaction is carried out for 4 hours under the atmosphere of normal pressure argon, unreacted monomers and low boiling point products are removed, and the polymer gel is obtained.
The probiotic dressing gels of examples 16-20 above were extracted and tested for HPV content (copies/cell) after 12h, 24h, 48h, 72h of intervention according to the test procedure and criteria described above, and the average (mean±sd, n=8) was recorded in the table below.
Table: examples 1, 16-20 HPV content (copies/cell)
As can be seen from the above table, the probiotic dressing gels prepared in examples 1, 16-20 all have excellent anti-HPV virus effects, and the HPV content after 12h, 24h, 48h, 72h of intervention is 9.38±0.22 to 9.48±0.22, respectively; 8.20+/-0.22-8.31+/-0.28; 7.18+/-0.40-7.31+/-0.40; 6.70+/-0.35-6.85+/-0.42;
it can be seen that the polymer gel loaded with lactobacillus rhamnosus, protein lysate and interferon, thanks to its morphology and characteristics, is more favourable for long-lasting anti-HPV, with different degree of improvement at 48h, 72h, the reasons for this analysis may be as follows, with reference to examples 1, 16-20: the polymer gel not only ensures that the number of internal effective living bacteria is not easily interfered by external adverse factors, but also can inhibit HPV virus through slow release and compounding of each effective component for a long time and stability, thus being extremely suitable for clinical application, being easy to damage organisms and being washed out by clean water.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (9)
1. Lactobacillus rhamnosus (Lactobacillus rhamnosus) LK-08 against human papillomavirus HPV, deposited under the accession number: CGMCC No.25853, and the preservation unit is China general microbiological culture Collection center.
2. Use of lactobacillus rhamnosus against human papillomavirus HPV according to claim 1 for the preparation of a product for inhibiting the proliferative expression of HPV virus selected from the group consisting of probiotic powder, probiotic stick, probiotic cream or probiotic dressing gel.
3. The use according to claim 2, wherein the lactobacillus rhamnosus-inhibited human papilloma HPV viruses comprise HPV-16, HPV-18, HPV-30, HPV-31, HPV-33.
4. A probiotic dressing gel comprising lactobacillus rhamnosus LK-08 as described in claim 1, having a viable count of not less than 5×10 5 CFU/g。
5. The probiotic dressing gel according to claim 4, characterized by consisting of the following components in weight percentage: lactobacillus rhamnosus 0.1-10%, protein lysate 0-10%, interferon 0-1.0%, and polymer gel as the rest.
6. The probiotic dressing gel of claim 5, wherein the protein lysate is prepared by inactivated lysis of one or more of tea polysaccharide, HPV E6 protein, HPV E7 protein.
7. The probiotic dressing gel of claim 5, wherein the interferon is one or more of IFN- α, IFN- β, IFN- γ and IFN-T.
8. The probiotic dressing gel according to claim 5, wherein the polymer gel is formed by copolymerizing polyethylene glycol and polyglycolide in a weight ratio of 1 (1.2-1.8).
9. A method for preparing a probiotic dressing gel according to any one of claims 4 to 8, characterized by the specific steps of:
s1, vacuum dewatering polyethylene glycol at 100-120 ℃ for 1-2h, introducing argon gas, cooling to 80 ℃, and then adding a polyglycolide monomer and a catalyst stannous octoate;
s2, after the reaction temperature is increased to 140-156 ℃, and the reaction is carried out for 4-8 hours under the atmosphere of normal pressure argon, unreacted monomers and low boiling point products are removed to obtain polymer gel, lactobacillus rhamnosus, protein lysate and interferon are added, and then washing and freeze drying are carried out, so as to obtain the probiotic dressing gel.
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| CN112315899A (en) * | 2020-11-04 | 2021-02-05 | 湖南天根乐微君科技有限公司 | Biological gel dressing for preventing/treating HPV (human papilloma Virus) and gynecological inflammation as well as preparation method and application thereof |
| CN112760247A (en) * | 2020-11-10 | 2021-05-07 | 深圳华大生命科学研究院 | Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss |
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