Lactobacillus paracasei GM-080 and antibacterial application of peptide of lactobacillus paracasei GM-080

Abstract

The invention provides an application of lactobacillus paracasei GM-080 and a peptide of the lactobacillus paracasei GM-080 in preparation of an antibacterial composition. The composition is a pharmaceutical composition, a nutritional supplement or a health-care food. The invention further provides an application of the lactobacillus paracasei GM-080 and the peptide of the lactobacillus paracasei GM-080 in preparation of the antibacterial composition. Wherein the storage code of the lactobacillus paracasei GM-080 is BCRC (Bull Cyclic Redundancy Check) 910220 or CCTCC (China Center For Type Culture Collection) M 204012. In addition, the invention also provides a peptide of the lactobacillus paracasei GM-080 and a composition of the peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of Lactobacillus paracasei (also known as Lacticaseibacillus paracasei) against Streptococcus pyogenes, especially inhibiting Streptococcus pyogenes by secreting antibacterial peptides. Background Art

[0002] Upper Respiratory Tract Infections (URTIs) are the most common diseases for which antibiotics are used worldwide. Upper respiratory tract infections include rhinitis, nasal congestion (including suppuration) (about 44.4%); acute pharyngitis (about 10.8%); acute otitis media (about 5.1%); otitis media with effusion (about 1.2%); acute sinusitis (about 8.2%). Acute pharyngitis is prone to occur during the alternation of autumn and winter and spring and summer seasons. Viral infection is the most common cause of acute pharyngitis. However, most viral infection symptoms are relatively mild and will heal on their own.

[0003] Pharyngitis caused by bacterial infection is prone to cause serious complications. In the population aged 5 - 15 years old, the main pathogenic bacteria are Streptococcus pyogenes, also known as Group A Streptococcus (GAS), and it is easy to invade the population aged 5 - 15 years old, and may cause complications such as rheumatic heart disease, myocarditis, polyarthritis, glomerulonephritis, septicemia, etc., which may all endanger life. The main pathogenic bacteria of acute otitis media and acute sinusitis are mainly Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.

[0004] Clinically, antibiotics are mostly used to relieve upper respiratory tract infections caused by pathogenic bacteria. However, with the side effects of antibiotics and the continuous emergence of antibiotic - resistant strains in clinical practice, and since antibiotics cannot be used for daily health care of the body, scientists have begun to look for methods to enhance mucosal immunity and inhibit pathogenic bacteria without using antibiotics. Among them, probiotics are regarded as a safe, effective and side - effect - free positive means and can be used to prevent upper respiratory tract infections.

[0005] The mechanism of probiotics in preventing respiratory tract infections, in addition to directly inhibiting the growth of common respiratory tract pathogenic bacteria, can also inhibit the damage of pathogenic bacteria toxins, inhibit the adhesion and invasion of pathogenic bacteria to host cells, and enhance mucosal immunity; past animal and clinical studies have also pointed out that some probiotic strains can reduce the symptoms and frequency of upper respiratory tract infections caused by viruses.

[0006] Therefore, the use of probiotics for preventing the occurrence of upper respiratory diseases should be a very good and reasonable option. In the past, the most famous strain for inhibiting Streptococcus pyogenes was Streptococcus salivarius K12 discovered in 2001. There are also many other strains, such as S. salivarius M18, PS4, JIM8777, CCHSS3, 57.I, etc. Their common antibacterial mechanism is that Streptococcus salivarius can secrete bacteriocins such as megaplasmid-encoded class I lantibiotics, salivaricin A2, and salivaricin B, thereby inhibiting the growth of streptococci.

[0007] Therefore, finding probiotics with antibacterial effects and their active ingredients is the subject to be solved by the present invention. Summary of the Invention

[0008] In view of this, after years of research, the inventor of this case has finally successfully isolated a probiotic with the ability to inhibit Streptococcus pyogenes. This probiotic is Lactobacillus paracasei, and this probiotic and its peptide have the effect of inhibiting Streptococcus pyogenes, providing a novel and safe prevention strategy for upper respiratory diseases.

[0009] To achieve the above object, the present invention provides a peptide with antibacterial effects, and the peptide sequence is SEQ ID NO:1.

[0010] In an embodiment of the present invention, the peptide is secreted by Lactobacillus paracasei GM-080, and the deposit number of Lactobacillus paracasei GM-080 is BCRC 910220 or CCTCC M 204012.

[0011] The present invention further provides a composition with antibacterial effects, which contains the peptide as described above.

[0012] In an embodiment of the present invention, the composition may further contain a pharmaceutically acceptable carrier.

[0013] In an embodiment of the present invention, the composition is a solution, suspension, emulsion, powder, lozenge, pill, syrup, troche, tablet, chewing gum, paste or capsule.

[0014] In an embodiment of the present invention, the composition may further contain an edible material.

[0015] The present invention further provides a use of Lactobacillus paracasei GM-080 or its peptide for preparing an antibacterial composition. The Lactobacillus paracasei has a deposit number of BCRC 910220 or CCTCC M 204012. The antibacterial activity is to inhibit the growth of microorganisms, and the peptide sequence is SEQ ID NO:1.

[0016] In one embodiment of the present invention, the microorganism to be inhibited is Streptococcus.

[0017] In one embodiment of the present invention, the microorganism to be inhibited is Streptococcus pyogenes.

[0018] In one embodiment of the present invention, the composition is a pharmaceutical composition, a nutritional supplement or a health food.

[0019] In one embodiment of the present invention, the peptide is secreted by Lactobacillus paracasei GM-080.

[0020] The present invention provides Lactobacillus paracasei GM-080, which can inhibit the growth of Streptococcus pyogenes and its ability to adhere to throat cells. Lactobacillus paracasei GM-080 can colonize in the respiratory tract and effectively reduce the viable count of Streptococcus pyogenes locally, especially by inhibiting Streptococcus pyogenes with the peptide AMP-01026 (SEQ ID NO:1) secreted by Lactobacillus paracasei GM-080. The present invention further provides a composition comprising Lactobacillus paracasei GM-080 or peptide AMP-01026, which can inhibit the growth of Streptococcus pyogenes and its ability to adhere to throat cells, as an active ingredient. Since the composition provided by the present invention uses probiotics or peptides as active ingredients, it has the advantage of low side effects. Brief Description of the Drawings

[0021] Figure 1 Analysis of the ability to inhibit the growth of Streptococcus pyogenes and inhibit adhesion to throat cells;

[0022] Figure 2 Analysis of the ability of GM-080 to colonize in the throat of mice in the form of viable bacteria;

[0023] Figure 3 Experimental procedure for evaluating the protective ability of animals infected with Streptococcus pyogenes;

[0024] Figure 4 Analysis of the ability of GM-080 to protect mice from weight loss caused by Streptococcus pyogenes and the number of bacteria in the throat;

[0025] Figure 5 Analysis of the DNA and amino acid sequences of the antibacterial peptides AMP-01026 and AMP-01240 of GM-080 and their differential Q-PCR expression levels compared with other Lactobacillus paracasei;

[0026] Figure 6 Results of chromatographic separation and mass spectrometry analysis of AMP-01026;

[0027] Figure 7 Results of chromatographic separation and mass spectrometry analysis of AMP-01240;

[0028] Figure 8 Analysis of the ability of AMP-01026 and AMP-01240 to inhibit Streptococcus pyogenes evaluated by co-culture method;

[0029] Figure 9 Evaluation of the ability of AMP-01026 and AMP-01240 to inhibit Streptococcus pyogenes by the disk diffusion method. Detailed implementation mode

[0030] All technical and scientific terms described in this specification, unless otherwise defined, shall have the meanings commonly understood by those of ordinary skill in the relevant art.

[0031] The singular terms "a", "an", and "the" as used in this specification and the claims, unless otherwise stated, may refer to more than one object.

[0032] The terms "or", "and", and "with" as used in this specification, unless otherwise stated, shall refer to "or / and". In addition, the terms "comprising" and "including" are open-ended conjunctions without limitation. The foregoing paragraphs are for systematic reference only and should not be construed as limiting the subject matter of the invention.

[0033] The compositions provided by the present invention can be prepared into a dosage form suitable for the compositions of the present invention by using techniques well-known to those with ordinary knowledge in the technical field to which the present invention pertains, combining the active ingredients or compositions provided in this case with at least one pharmaceutically acceptable vehicle. The dosage form includes, but is not limited to, solutions, emulsions, suspensions, powders, lozenges, troches, tablets, chewing gums, capsules, and other similar or suitable dosage forms for the present invention.

[0034] The term "pharmaceutically acceptable" means that a substance or composition must be compatible with other components of its pharmaceutical formulation and not exacerbate the symptoms of the patient.

[0035] The term "pharmaceutically acceptable vehicle" includes one or more types of components selected from the following: solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and other similar or suitable vehicles for the present invention.

[0036] In the aforementioned composition, one or more solubilizing aids, buffering agents, coloring agents, flavoring agents, etc., which are commonly used in the field of pharmaceutical preparations, may also be appropriately added as needed.

[0037] The term "pharmaceutical composition" refers to a solid or liquid composition that is in a form, concentration, and degree of purity suitable for administration to a patient and, after administration, can induce the desired physiological changes; the pharmaceutical composition is sterile and / or non-pyrogenic.

[0038] Example 1. Analysis of the ability of GM-080 to inhibit Streptococcus pyogenes NZ131

[0039] (1) Inhibition zone experiment:

[0040] An inhibition zone test was performed using the clinical strain NZ131 of Streptococcus pyogenes. The pathogen was activated and cultured for two days in TSA + 5% defibrinated sheep blood; multiple strains of lactic acid bacteria (as shown in Table 1) were cultured in small amounts using MRS medium. The lactic acid bacteria solution on the day after activation (adjusted to 1×10 7 CFU / ml) was used for secondary activation and cultured overnight for later use; Streptococcus salivarius K12 and M18 were cultured and activated in BHI medium. After adjusting the pathogen NZ131 to an appropriate cell count (5×10 8 CFU / ml), 100 μl was evenly spread on the plate. After it was slightly dried, sterile 11-mm glass tubes were used to punch holes. 100 μl of the whole bacterial solution (the concentration of lactic acid bacteria adjusted to 1×10 9 CFU / ml) was added to each well. After incubation at 37°C for 48 hours, the size of the inhibition zone was measured with a vernier caliper. The size of the inhibition zone was the value obtained by subtracting the inner circle from the outer circle. The larger the value, the stronger the inhibitory ability.

[0041] Eighteen probiotic strains were screened using the inhibition zone experiment of Streptococcus pyogenes, and it was found that Lactobacillus paracasei GM-080 had the best inhibitory effect (Table 1).

[0042] Table 1 Comparison of different strains in inhibiting Streptococcus pyogenes NZ131

[0043]

[0044] (2) Evaluation of the efficacy of lactic acid bacteria in inhibiting the adhesion of Streptococcus pyogenes NZ131 to cells by plate culture:

[0045] FaDu cells were seeded at 4x10 5Seed 6-well culture plates with cells / well. After overnight culture, replace the medium with serum-free medium. Adjust the Lactobacillus and pathogenic bacteria to 4x10 8 cfu / ml. Mix the pathogenic bacteria and Lactobacillus at a ratio of 1:1. Take 100 μl of the mixed bacteria and add it to the 6-well culture plate. Incubate at 37 °C for 2 hours. After the reaction, aspirate the medium, wash three times with PBS, add 0.5 mL of Trypsin / PBS to detach the cells, and serially dilute with PBS by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Then perform 5% blood / TSA plating. After incubation at 37 °C for 48 hours, count the bacteria. Set the number of bacteria in the group infected only with Streptococcus pyogenes as 100%, and calculate the relative number of bacteria in other groups.

[0046] Since 2001, Streptococcus salivarius K12 and M18, which were discovered, have been well-known strains that can inhibit Streptococcus pyogenes in past studies. Therefore, further compare the ability of Lactobacillus paracasei GM-080, Streptococcus salivarius K12, and M18 to inhibit Streptococcus pyogenes Figure 1 For the analysis of the ability to inhibit the growth of Streptococcus pyogenes and inhibit adhesion to throat cells, from Figure 1 The results show that the ability of GM-080 to inhibit Streptococcus pyogenes is slightly better than that of Streptococcus salivarius.

[0047] Example 2. Testing the colonization of probiotic gel in the throat

[0048] Prepare a 1.26% agar aqueous solution. After boiling and dissolving, let it cool to room temperature and then to 37 °C. Add the live GM-080 bacterial powder to make the final bacterial count 5×10 per gram of gel 10CFU, and stir evenly. Wait for the agar gum to solidify into a solid state before use. Place the probiotic agar gum on the stainless steel mesh cover of the mouse cage to replace the water bottle for supplying water, and continuously give the mice agar gum containing lactic acid bacteria for 2 days. Disinfect the faces and mouths of the mice with alcohol cotton swabs, then use a wet sterile cotton swab to reach into the mice's mouths until the pharynx, and rotate the cotton swab 3 times to collect throat bacteria. Immediately smear the cotton swab after collecting the sample directly on the MRS (deMan, Rogosa, and Sharpe) agar plate, place it in an anaerobic incubator at 37°C for 48 hours, observe the growth of lactic acid bacteria colonies, and pick up the lactic acid bacteria colonies for random amplified polymorphic DNA (RAPD) analysis to evaluate the ability of the strain to colonize the larynx. The DNA template concentration used in RAPD PCR: 10 ng; the RAPD primer used is LacP2 primer: ACGCGCCCT (SEQ ID NO: 7). If the RAPD bands run out by the PCR product are exactly the same as the standard product, it is the same strain.

[0049] Evaluate the colonization ability of GM-080 in the larynx of mice by testing the oral administration of GM-080 gel. Figure 2 For the analysis that GM-080 can colonize the throat of mice in the form of live bacteria, from Figure 2 The results can confirm the presence of live GM-080 bacteria in the throat from the live bacteria in the mouse throat samples by RAPD molecular methods, indicating that this strain can colonize the respiratory tract and can achieve better effects in inhibiting respiratory bacteria.

[0050] Example 3. Analysis of the protective ability and efficacy of GM-080 against Streptococcus pyogenes infection

[0051] Experimental groups (4 mice in each group). First, feed the mice with 0.3 mg / mL antibiotic enrofloxacin gel for two days, and then feed the experimental groups with probiotic GM-080 (5×10 10 CFU / g) gel for 7 days; the control groups are first fed with 0.3 mg / mL antibiotic enrofloxacin gel for two days and then directly infected with the pathogenic bacteria. On the 8th day, Streptococcus pyogenes 2x10 9 cfu / 0.04 mL / one nasal cavity (infect both nasal cavities) is given by intranasal instillation, and observe the changes in the body weights of the mice before and within 2 days after infection and take out the pharyngeal tissues to evaluate the number of Streptococcus pyogenes in them. The experimental procedure is as Figure 3 shown.

[0052] Further establish a mouse model of Streptococcus pyogenes infection. First, use antibiotic gel to clear the original flora in the throat and then infect with Streptococcus pyogenes. Figure 4GM-080 can protect against weight loss in mice caused by Streptococcus pyogenes and analyze the number of bacteria in the throat. As Figure 4 The results showed that Streptococcus pyogenes infection in the throat of mice caused weight loss in mice; however, Figure 4 Results A and B showed that oral administration of GM-080 gel could slow down the weight loss caused by Streptococcus pyogenes; and Figure 4 The culture of pharyngeal specimens in C found that the administration of GM-080 gel could effectively reduce the number of viable Streptococcus pyogenes bacteria locally.

[0053] Example 4. Prediction of Antimicrobial Peptides from the Whole Genome Sequencing of GM-080

[0054] The whole genome sequencing of strain GM-080 was completed using the second-generation + third-generation sequencing. The assembled gene sequence was used for subsequent prediction analysis at the Antimicrobial peptide (AMPs) (AMP: https: / / awi.cuhk.edu.cn / dbAMP) website to identify potential antimicrobial peptides. The presence of these peptides in the Lactobacillus genome was verified by fluorescence quantitative PCR. The DNA template concentration used in fluorescence quantitative PCR (Q-PCR) was 10 ng; the primers for Q-PCR of AMP-01026 were: Forward primer: GGCCGTGTAGGATTCGGATAA (SEQ ID NO: 3); Reverse primer: GTGGTAAACGAAAGTGCCCC (SEQ ID NO: 4); the product size was 119 bp. The primers for Q-PCR of AMP-01240 were: Forward primer: AGATGCGAGCGAAAACTCGT (SEQ ID NO: 5); Reverse primer: ATTGGAGGTGACAGCATCCG (SEQ ID NO: 6); the product size was 116 bp.

[0055] Table 2 Q-PCR Primer Sequences of AMP-01026 and AMP-01240

[0056]

[0057]

[0058] Figure 5 For the DNA and amino acid sequences of GM-080 antimicrobial peptides AMP-01026 and AMP-01240, and the analysis of the difference in Q-PCR expression levels with other Lactobacillus paracasei, Figure 5B It was found that compared with other Lactobacillus paracasei, GM-080 did express these two antimicrobial peptides in large quantities, especially the expression level of AMP-01026 was much higher than that of other Lactobacillus paracasei.

[0059] Example 5: Confirmation of the antibacterial effect of antimicrobial peptides

[0060] Mingxin Company was commissioned to synthesize two antimicrobial peptides, including AMP-01026: MELLNEKELAHVIGGKRKCPKTPFDNTPGAWFAHLILGC (SEQ ID NO: 1), and the purity and analysis report are as Figure 6 shown, and AMP-01240: MKQFDEQKMVNMSDEELLEFIGGDSIRDVSPTFNKIRRWFDGLFK (SEQ ID NO: 2), and the purity and analysis report are as Figure 7 shown. The powder of the antimicrobial peptide was diluted with DMSO to different concentrations, and then co-cultured with pathogenic streptococcus at different time points. At the same time, the OD600nm or plate coating method was used to confirm whether it was the GM-080 antimicrobial peptide that could inhibit Streptococcus pyogenes NZ131.

[0061] Table 3 Sequences and molecular weights of peptides AMP-01026 and AMP-01240

[0062] Peptide Name Sequence Number Amino Acid Sequence Molecular Weight AMP-01026 SEQ ID NO:1 MELLNEKELAHVIGGKRKCPKTPFDNTPGAWFAHLILGC 4336.13 AMP-01240 SEQ ID NO:2 MKQFDEQKMVNMSDEELLEFIGGDSIRDVSPTFNKIRRWFDGLFK 5399.18

[0063] Figure 8 For the analysis of the ability of AMP-01026 and AMP-01240 to inhibit Streptococcus pyogenes by co-culture method, from Figure 8 A It was found that when co-cultured with different concentrations (10 - 200 mg / mL) of AMP-01026 (SEQ ID NO: 1) for 2 - 3 hours, it had a good inhibitory effect on Streptococcus pyogenes.

[0064] Figure 9 For the evaluation of the ability of AMP-01026 and AMP-01240 to inhibit Streptococcus pyogenes by plate coating method, the viable count of the pathogen was further confirmed by plate coating method. From Figure 9 A It was found that at different concentrations (1 - 200 mg / mL), AMP-01026 could significantly inhibit the growth of Streptococcus pyogenes starting from 2 or 5 hours, indicating that the antimicrobial peptide AMP-01026 produced in large quantities by GM-080 has the ability to inhibit the growth of Streptococcus pyogenes.

[0065] From Figure 8 B, Figure 9As a result, another antimicrobial peptide, AMP-01240 (SEQ ID NO:2), could not inhibit the growth of Streptococcus pyogenes at different concentrations (1 - 200 mg / mL).

[0066] The results showed that the GM-080 antimicrobial peptide has selectivity for different pathogenic bacteria, and whether it has antibacterial ability needs to be confirmed by experiments.

[0067] Summarizing the above results, we can know that:

[0068] 1. In the antibacterial circle experiment of Streptococcus pyogenes, compared with 18 other probiotic strains, Lactobacillus paracasei GM-080 had the best effect in inhibiting Streptococcus pyogenes.

[0069] 2. The Lactobacillus paracasei GM-080 strain can colonize the respiratory tract and achieve better results in inhibiting respiratory bacteria.

[0070] 3. Administering GM-080 gel can effectively reduce the viable count of Streptococcus pyogenes locally.

[0071] 4. In addition, the peptide containing the SEQ ID NO:1 sequence provided by the present invention also has the effect of resisting Streptococcus pyogenes.

[0072] Therefore, the Lactobacillus paracasei GM-080 and its antibacterial peptide provided by the present invention are very suitable for developing and preparing compositions for resisting Streptococcus pyogenes, which helps the development of products and industries for preventing upper respiratory diseases.

[0073] Regarding the content disclosed in the preferred embodiments of this specification, those with ordinary knowledge in the technical field to which the present invention pertains can clearly know that the foregoing embodiments are only illustrative; those with ordinary knowledge in the technical field to which the present invention pertains can implement through various transformations and substitutions without differing from the technical features of the present invention. According to the embodiments of the specification, the present invention can have various transformations without hindering implementation. The scope of the present invention is defined by the claims provided in this specification, and this scope covers the foregoing methods and structures and equivalent inventions thereof.

[0074] The above-mentioned multiple effects fully meet the legal patent requirements of novelty and progressiveness. Therefore, an application is filed according to law. We earnestly request your bureau to approve this invention patent application to encourage inventions.

[0075]

Deposit of Biological Material

[0076] Lactobacillus paracasei (also known as Lacticaseibacillus paracasei) GM-080 is deposited at the Food Industry Research and Development Institute in Taiwan, China, with the deposit number BCRC 910220, and at the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC M 204012.

Claims

1. A peptide with antibacterial effect, characterized in that: The peptide sequence is SEQ ID NO:

1.

2. The peptide according to claim 1, characterized in that The peptide is secreted by Lactobacillus paracasei GM-080, and the deposit number of Lactobacillus paracasei GM-080 is BCRC 910220 or CCTCC M204012.

3. A composition with antibacterial effect, characterized in that: The composition comprises the peptide according to claim 1 or 2.

4. The composition according to claim 3, characterized in that The composition may further comprise a pharmaceutically acceptable carrier.

5. The composition according to claim 3, characterized in that The composition is a solution, suspension, emulsion, powder, lozenge, pill, syrup, lozenge, tablet, chewing gum, syrup or capsule.

6. The composition according to claim 3, characterized in that The composition may further comprise an edible material.

7. A use of Lactobacillus paracasei GM-080 or its peptide for preparing an antibacterial composition, characterized in that: The Lactobacillus paracasei deposit number is BCRC 910220 or CCTCC M 204012, the antibacterial agent is to inhibit the growth of microorganisms, and the peptide sequence is SEQ ID NO:

1.

8. The use according to claim 7, characterized in that The microorganism is of the genus Streptococcus.

9. The use according to claim 7, characterized in that: The microorganism is Streptococcus pyogenes.

10. The use according to claim 7, characterized in that The composition is a pharmaceutical composition, a nutritional supplement or a health food.

11. The use according to claim 7, characterized in that: The peptide is secreted by Lactobacillus paracasei GM-080.

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