KR20230045675A - Novel Lactobacillus plantarum Probio-88, extract amd composition thereof - Google Patents

Abstract

The present invention provides a Lactobacillus plantarum probio-88 strain, and extracts and compositions containing the same. The strains, extracts and compositions according to the present invention inhibit SARS-CoV-2 infection and replication, reduce inflammation and intracellular accumulation of reactive oxygen species (ROS), reduce extracellular signal-regulated kinase (ERK) activity and improve antiviral activity, and has an effect of being useful as a coronavirus treatment agent or auxiliary treatment agent.

Description

Novel Lactobacillus plantarum Probio 88, extract and composition thereof {Novel Lactobacillus plantarum Probio-88, extract amd composition thereof}

The present invention relates to a novel Lactobacillus plantarum Probio 88, an extract and a composition thereof, and more particularly, inhibits replication of SARS-CoV-2 virus and production of reactive oxygen species (ROS), and is used as a treatment for coronavirus. It relates to novel Lactobacillus plantarum Probio 88, extracts and compositions thereof that can be used.

Coronaviruses (CoVs) have caused two large-scale pandemics in the past 20 years: Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), a highly pathogenic human coronavirus that emerged in 2019, caused a large-scale disease, COVID-19, which the World Health Organization (WHO) eventually declared a pandemic came to make a declaration. About half (40-45%) of infected people are asymptomatic, and contagiousness usually peaks before the onset of symptoms, making it difficult to contain the spread of the disease. Covid-19 can cause severe pneumonia because it can trigger an overreaction of the immune system. These symptoms are defined as a cytokine storm in which the immune system overreacts, resulting in severe respiratory distress and subsequent shutdown of multiple organs.

Considering the importance of immune regulation in the severity and progression of COVID-19, the intervention of probiotics has been considered as a potential treatment due to its immunomodulatory ability in SARS-CoV-2 infection. Probiotics are live microorganisms that, when administered in appropriate amounts, provide health benefits to the recipient. Activates the immune system and improves resistance to infection. In a randomized, double-blind, placebo-controlled trial involving 100 children with severe sepsis, when probiotics were administered for several days, the function of probiotics to provide immunomodulatory activity was to promote inflammation (IL-6, IL-12p70, IL-17 and TNF-α) were significantly reduced and anti-inflammatory (IL-10 and TGF-β1) cytokines were increased. This fact is further confirmed by a recent meta-analysis showing that protein administration reduced IL-6 and C-reactive protein levels in middle-aged and elderly adults with chronic low-grade inflammation. Probiotics can prevent deterioration of lung function by potentially balancing the hyperreactive infectious state caused by SARS-CoV-2 through the regulation of the innate and acquired immune systems.

The benefits of microbiome modulation using probiotics in viral diseases have been demonstrated in many studies, namely some species of Lactobacilli and Bifidobateria, against influenza virus, rhinovirus, It has been proven from the fact that it has a protective function against respiratory syncytial virus, adenovirus and pneumovirus. The use of probiotics as antiviral agents has been routine in the aquaculture, poultry and livestock industries for decades. In our previous study, Lactobacillus plantarum Probio-38 and L. salivarius Probio-37 showed antiviral activity against TGEV (Transmissible GastroEnteritis coronaVirus). It was found to have In a human clinical trial involving 70 COVID-19 positive patients, oral bacteriotherapy consisting of multiple species of lactic acid bacteria showed a statistically significant effect on the clinical condition of COVID-19 patients. Within 72 hours, almost all patients treated with bacteria showed remission of diarrheal disease and other symptoms compared to the majority of patients who did not receive treatment. The predicted risk of progressive respiratory failure was 8-fold lower in patients treated with oral bacteria. The prevalence of patients transferred to an intensive care unit (ICU) and deaths was higher in patients who did not receive oral bacterial treatment. The use of probiotics as antiviral treatments against respiratory and enteric viruses has been widely reviewed as a new concept in medicine, and new attention is being paid to the use of probiotics against SARS-CoV-2. This study has a primary objective to evaluate the efficacy of probiotic metabolites in viral replication and immune regulation using in-vitro and in-silico studies.

Most viruses, including coronaviruses, use similar mechanisms to take over host cells as replication machinery to produce progeny. These processes generally include attachment and entry, transcription of copies, and assembly of the replication-transcription complex followed by genome replication and transcription, transcription of structural proteins, and finally virion assembly and release. As a countermeasure, host cells also equip innate and adaptive immune responses to limit viral multiplication. It is the competition between host and virus that ultimately determines the outcome of disease progression.

Naturally, host cell defense mechanisms are initiated spontaneously with the activation of phagocytes upon detection of viral entry. Activation of phagocytes triggers cells to produce reactive oxygen species (ROS), and accumulating evidence has suggested that ROS play an important role in the pathogenesis of COVID-19. Although ROS are well known for their detrimental effects, they are also some of the important mediators essential for normal cellular function. The ability to control overproduction of ROS during viral infection can exacerbate disease severity. Elevated levels of ROS are also known to upregulate the expression of pro-oxidant cytokines such as tumor necrosis factor (TNF) and interleukin-1 cytokine, which will ultimately further increase ROS production. This repeated cycle eventually causes tissue damage and triggers a potentially life-threatening cytokine storm. Excessive ROS is also a major cause of lung damage in other respiratory viral infections such as influenza-enza virus infection. Therefore, it is worth noting that regulating ROS levels is one of the important processes leading to pathological host responses to viral infection.

In addition, it is important to note that suppressing viral replication within cells and then removing inflammatory factors can also prevent a cytokine storm, the most lethal factor for COVID-19 patients. Since it has long been known that probiotics exert immune modulatory, antibacterial and antiviral properties, it is not surprising that these live beneficial bacteria have been advocated as potential adjuvant therapy or prophylaxis against SARS-CoV-2 infection. In parallel with the relatively low COVID-19 mortality rate, especially in countries like South Korea, fermented cabbage products such as kimchi have gained attention as an important dietary habit that can help mitigate the severity of COVID-19. In addition to modulating the antiviral immune response in respiratory and gastrointestinal mucosal tissues, L. plantarum, one of the abundant species found in kimchi, acts as a bacteriocin as an antiviral compound against several respiratory pathogens. has been reported to produce According to another in-silico study, Plantaricin W, D and JLA-9 were found to have high inhibitory activity against the catalytic site of the RNA-dependent RNA polymerase of SARS-CoV-2, which indicates the replication cycle of the virus. plays a major role in

Increased ROS levels during viral infection have also been reported to activate the Extracellular signal-Regulated Kinase (ERK) pathway, which is important in the pathogenesis of human coronaviruses. In addition to ROS, viruses or their components can also activate kinases. Mizutani et al. reported that SARS-CoV-infected Vero E6 cells showed enhanced ERK phosphorylation. In contrast, another study by Chen showed that the SARS-CoV S protein or SARS-CoV virus-like particle could phosphorylate ERK in A549 cells. Similarly, MERS and HCoV-299E also modulate the ERK signaling pathway during infection. The ERK pathway consists of three signaling cascades (Raf/MEK/ERK) and is important for cell growth. Because pathway activation is highly dynamic, many viruses (herpes simplex type-1 virus, influenza virus, Ebola virus, flavivirus) have been (flavivirus) and other viruses).

In addition to serving a viral support role, the ERK pathway is also important for maintaining inflammation during infection. Initiation and regulation of the inflammatory response is one of the key events controlled by mitogen-activated protein kinases (MAPKs). The ERK pathway is one of three members of the MAPK family, along with p38 and JNK. MAPK is activated upon viral invasion to produce inflammatory cytokines and chemokines. In the case of SARS-CoV-2, which resembles a highly pathogenic virus, the dysregulated inflammatory response that occurs during pathogen attack is called a cytokine storm and is strongly controlled by the p38 and ERK pathways.

Inflammation during infection represents a protective mechanism to inhibit pathogen spread, and the first line of defense usually includes type 1 interferons (IFN-α and IFN-β) and the multifunctional cytokine IL-6. Many studies have shown that SARS-CoV-2 can attenuate the IFN response, but recent findings have shown that IFN production, although delayed, is actually stimulated at significant levels. The groups also reported that high amounts of viral transcripts were observed before IFN induction in cells infected with SARS-CoV-2. Therefore, it is hypothesized that a high viral load is required to initiate an IFN response. These findings are also consistent with the observation that high viral loads were detected in COVID-19 cases soon after the onset of symptoms. The present inventors came to complete the present invention starting from the hypothesis that the low expression of these genes could be due to a lower amount of virus propagation in cells after treatment with P88-CFS according to the present invention.

The problems to be solved in the present invention are to inhibit SARS-CoV-2 infection and replication, reduce inflammation and intracellular accumulation of reactive oxygen species (ROS), reduce extracellular signal-regulated kinase (ERK) activity, and antiviral activity It is to provide novel Lactobacillus plantarum Probio 88, extracts and compositions thereof that improve

The present invention relates to Lactobacillus plantarum Probio 88 strain (KCTC14482BP).

Here, the strain is characterized in that it is used to inhibit infection and replication of SARS-CoV-2.

The strain is characterized in that it is used to reduce inflammation.

The strain is characterized in that it is used to reduce the intracellular accumulation of reactive oxygen species (ROS).

The strain is characterized in that it is used to reduce extracellular signal-regulated kinase (ERK) activity.

The strain is characterized in that it is used to enhance antiviral activity.

In addition, the extract of the present invention is a cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 containing the Lactobacillus plantarum Probio 88 strain (KCTC14482BP).

Here, the extract is characterized in that it contains Plantarisin E (PlnE) and Plantalysin F (PlnF), and is used to inhibit infection and replication of SARS-CoV-2.

The extract is characterized in that it is used to reduce inflammation.

The extract is characterized in that it is used to reduce the intracellular accumulation of reactive oxygen species (ROS).

The extract is characterized in that it is used to reduce extracellular signal-regulated kinase (ERK) activity.

The extract is characterized in that it is used to enhance antiviral activity.

The extract is characterized in that it is used to enhance antiviral activity.

In addition, the composition of the present invention is characterized in that it is for treatment of coronavirus including Lactobacillus plantarum Probio 88 strain (KCTC14482BP).

Here, the composition may be used as a feed, feed additive, food, functional food, cosmetic composition, pharmaceutical composition or quasi-drug.

In addition, Lactobacillus plantarum according to the present invention ( Lactobacillus plantarum ) Plantarisin E (PlnE) produced by Probio 88 strain (KCTC14482BP); And plantarisin F (PlnF) is characterized in that it is used to inhibit infection and replication of SARS-CoV-2.

The present invention inhibits SARS-CoV-2 infection and replication, reduces inflammation and intracellular accumulation of reactive oxygen species (ROS), reduces extracellular signal-regulated kinase (ERK) activity, improves antiviral activity, It has the effect of the present invention to provide a novel Lactobacillus plantarum Probio 88, an extract and a composition thereof, which can be usefully used as a coronavirus treatment or adjuvant treatment.

1 is a graph showing the degree of inhibition of the P88-CFS extract of the present invention against SARS-CoV-2 infection.
Figure 2 is a graph showing the MTT assay results of (a) HCE cells, (b) HEK cells, and (c) HeLa cells for the P88-CFS extract of the present invention at different concentrations.
FIG. 3 (a) is a photograph of reactive oxygen species (ROS) staining by dichlorofluorescein (H2DCFDA) staining, and (b) is a graph showing quantitative fluorescence intensity measured by Image J.
4 is an immunofluorescence confocal micrograph showing the effect of P88-CFS on p-ERK protein in HEK293 cells after infection with SARS-CoV-2.
Figure 5 is a graph showing gene expression of inflammatory markers in HEK293 cells after infection with and without P88-CFS treatment according to the present invention, (a) IFN-α, (b) IFN-β, and (c) IL-6 Expression of was measured by qPCR and normalized to GAPDH.
6 is a structural diagram showing the secondary structure of Sars-CoV-2 helicase nsp13.
Figure 7 is a graphic showing the molecular docking of PlnE and PlnF, (a) is the 3D structure of SARS-CoV-2 helicase nsp 13 (PDB ID: 6ZSL) used in molecular docking studies, (b) is Planta It is a construction model of lysine (PlnE), (c) is a construction model for plantarisin F (PlnF), and (d) shows PlnE inserted into the cavity of ss-RNA and ATP binding sites, (e) shows that PlnF is inserted into the cavity of ss-RNA and ATP binding sites.
8 is a diagram showing the bonding structures of (a) PlnE and (b) PlnF, and the hydrogen bond cut-off was set to 4.0 Å.
9 is a Ligplot+ interaction diagram of (a) PlnE and (b) PlnF, where hydrogen bonds are indicated by green dotted lines and hydrophobic interactions are indicated by red.

The above-described objects, features and advantages of the present invention will become more apparent by describing preferred embodiments of the present invention in detail. The detailed description that follows is not to be described in a limiting sense, and the scope of the present invention is limited only by the appended claims, along with all equivalents as claimed by those claims.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and embodiments.

Example

1. Bacterial strains, media and growth conditions

The bacteria used in the present invention is Lactobacillus plantarum Probio 88, which was deposited with the Korean Collection for Type Cultures on March 4, 2021 under accession number KCTC 14482BP. Strains were previously extracted from kimchi. The strain contains 5 g/L of soy peptone, 5 g/L of sodium acetate, 20 g/L of yeast extract, 20 g/L of d(+)-glucose, and 2 g/L of ammonium sulfate. L, Dipotassium phosphate 2 g/L, magnesium sulfate 0.1 g/L, manganese sulfate 0.05 g/L, and Tween 80 1 g/L. -Rogosa-Sharpe) culture medium was used for 18 hours at 37°C. The cultured strain was stored in 25% glycerol (-45 ° C) and activated three times in succession using 1% inoculum before experimental analysis. Here, all chemicals were purchased from Sigma-Aldrich (St. Louis, Mo, USA) unless otherwise stated.

2. SARS-CoV-2 virus

The SARS-CoV-2 strain was obtained by isolation from oropharyngeal swabs of patients infected with coronavirus disease 2019 (COVID-19). All work on SARS-CoV-2 was conducted under Biosafety Level 3 (BSL-3) restricted conditions. Virus stocks were generated by culturing viruses in HEK293 cells at 90% confluency for 48 hours. The resulting cultures were then purified and stored at -80°C. Control samples were prepared in the same way using uninfected cells. Virus stocks were quantified by titration.

3. Preparation of CFS (P88-CFS) extract of Lactobacillus plantarum Probio 88

To generate Cell-Free Supernatant (CFS), Lactobacillus plantarum Priobio 88 was incubated in conditioned MRS broth at 37°C for 24 hours followed by 3,500 rpm for 15 minutes Centrifuged. The generated supernatant was obtained and neutralized to pH 7.0 dmfh, and filter sterilized using a syringe filter (pore size 0.22 μm). Then, the supernatant was lyophilized and reconstituted by supplying water to 10% of the original volume. The resulting extract was prepared as P88-CFS and stored at -20°C for analysis.

4. In vitro inhibition of SARS-CoV-2 replication

First, HEK293 cells were seeded in a 96-well plate (TPP, Trasadingen, Switzerland) at a density of 4 x 104 cells per well. After 24 hours, the cells were cultured in 100 μl culture medium (penicillin (100 U/mL) and streptomycin (100 μg/mL), P88-CFS (50, 100, 150, 200 or 250 mg/ml) and Cultured with SARS-CoV-2 virus (0.1 multiplicity of infection) After incubation at 37° C. for 2 hours, the cells were washed 3 times with PBS and maintained in 100 μl of the same culture medium without the presence of virus. Positive and negative control samples were inoculated in the same manner, except that the P88-CFS was replaced with DMSO and Phosphate-Buffered Saline (PBS), respectively.Virus yield was measured using a quantitative real-time PCR method.

5. Cell viability assay

Human corneal epithelial (HCE), HEK293 and Hela cell lines were cultured in Complete medium supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL). ) in a humid environment of 5% CO ₂ at 37°C. The cells were seeded in 96-well plates at a density of 4×10 4 cells per well prior to MTT assay. After 24 hours, the cells were treated with DMEM containing various concentrations of P88-CFS (50, 100, 150, 200 or 250mg/ml) for 48 hours at 37°C and 5% CO ₂ . For adjustment, PBS was used to replace P88-CFS in DMEM, at the end of culture the cells were washed with PBS, 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2, After treatment with 50 μl of 5-diphenyl-2H-tetrazolium bromide (MTT) solution, the cells were incubated at 37° C. for 45 minutes. The cell supernatant was discarded, and formazan crystals were dissolved in isopropanol. Then, absorbance was measured at 570 nm. All assays were performed in triplicate. Viability was defined as the relative absorbance of P88-CFS treated cells compared to control cells.

6. Determination of intracellular reactive oxygen species (ROS)

Cells were cultured in 6-well plates in complete medium and treated with SARS-CoV-2. SARS-CoV-2 was isolated from oropharyngeal swabs from patients with COVID-19. Viral stocks were generated by infecting HEK293 cells at 90% confluency for 48 hours. Thereafter, the culture medium was aliquoted and stored at -80°C. Control samples were prepared in the same way using uninfected cells. Viral stock was quantified by titration. When the cells reached 60-70% confluence, the level of ROS was measured with different concentrations of P88-CFS. Media was removed and cells were washed twice with DMEM only. Cells were then incubated with 10 μM 2'-7'-dichlorodihydrofluorscein diacetate (DCFH-DA, Sigma Aldrich) in DMEM at 37°C for 30 minutes. Then, the cells were washed twice with PBS, and the fluorescence intensity of the DCFDA-treated cells was measured under a fluorescence microscope.

7. Immunocytochemistry of Hela cells

HEK293 cells were cultured in complete culture medium and infected with SARS-CoV-2. Then, the cells were washed 3 times with PBS and fixed in 4% formaldehyde for 10 minutes. Cells were then permeabilized using 0.2% Triton X. Cells were then treated with a signal enhancer and then blocked using 5% normal goat serum diluted in PBS. Cells were incubated at 4°C with the primary antibody of pERK (1:100) (Cell Signaling) for 12 hours. Then, cells were washed 3 times with PBS and incubated with secondary antibody for 1 hour. Then, cells were washed with PBS, and nuclei were counterstained with DAPI (Sigma Aldrich, USA). Finally, cells were washed with PBS and pictures were taken using a fluorescence microscope equipped with a digital camera (Nikon, Japan).

8. Quantitative real-time PCR

Total RNA was extracted from HEK293 cells using triazole reagent (Invitrogen) according to the manufacturer's instructions. The RNA concentration was measured using a NanoDrop, and the RNA was stored in diethyl pyrocarbonate-treated water at -80°C. In addition, 1 μg of RNA was primed with a random primer (Promega) in a reaction mixture having a total volume of 25 μl, followed by reverse transcription at 72° C. for 5 minutes and at 37° C. for 60 minutes. Real-time PCR was performed in a 7500 real-time PCR system (Applied Biosystems) using 2 μl of cDNA, 10 pmol of each gene-specific primer, and Power SYBR® Green PCR Master Mix (Applied Biosystems, USA). Detailed information describing primer sequences for rt-PCR is shown in Table 1 below.

Target Primer Directions Sequences (5 -> 3) GAPDH Forward
Reverse CAC CAC CAA CTG CTT AGC AC
CCC TGT TGC TGT AGC CAA AT IFNα Forward
Reverse GAT GGC AAC CAG TTC CAG AAG
AAA GAG GTT GAA GAT CTG CTG GAT IFNβ ForwardReverse CTC CAC TAC AGC TCT TTC CAT
GTC AAA GTT CAT CCT GTC CTT IL-6 Forward
Reverse AAC TCC TTC TCC AGA AGC GCC
GTG GGG CGG CTA CAT CTT T

9. Antiviral activity of Lactobacillus plantarum probio 88 using in-silico molecular docking

BLASTP the primary sequence to form 3D structures for both Plantaricin E and Plantaricin F of L. planta-rum Probio-88 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for sequence identity, and to identify appropriate templates for homology modeling. Due to the high percentage sequence identity with the target sequences plantaricins E and F, a comparative modeling approach was adopted to use plantaricins E (PlnE) and F (PlnF) (PDB IDs: 2JUI and 2RLW) as templates. SWISS-MODEL, an automated protein structure prediction server, was used, and the model with the highest QMEAN score was selected. Structural evaluation of the formed model was performed using Ramachandran pliot analysis.

Placing from Lactobacillus plantarum probio88 to SARS-CoV-2 Helicase using HADDOCK2.4, an automated protein-to-protein docking server (https://wenmr.science.uu.nl/) Protein-protein interactions of lantharicins E and F were investigated. Helicase nsp13 from SARS-CoV-2 (PDB ID: 6ZSL) was selected. The interacting residues for the ATP and ss-RNA binding sites are Glu537, Arg567, Arg443, His290, Arg442, Asn265, Gly439, Lys288, Ser485, Lys146, Lys139, Tyr180, His230, Tyr198, Arg212, Pro335, Arg339 and Asn516. determined and set as interface residues for docking. The cluster with the lowest HADDOCK score was selected and further analyzed for binding using PRODIGY. Visual Molecular Dynamics (VMD) was used to visualize the binding action.

Experiment result

The inhibitory effect of P88-CFS was initially evaluated in HEK293 cells. Here, the cells were first infected with the SARS-COV-2 virus and then treated with P88-CFS at a concentration ranging from 50 to 250 mg/ml. 1 is a graph showing the degree of inhibition of the P88-CFS extract of the present invention against SARS-CoV-2 infection. As shown in Figure 1, it was shown that the inhibition of SARS-COV-2 virus infection increased as the throughput of P88-CFS increased. Here, data are expressed as the mean±SD of three independent experiments. Based on the viral RNA copies determined by qPCR, the amount of virus in the culture supernatant was reduced compared to the untreated SARS-COV-2 infected control (0 mg/ml P88-CFS). Virus growth was significantly inhibited from 48% to 97% with increasing concentrations of P88-CFS.

Figure 2 is a graph showing the MTT assay results of (a) HCE cells, (b) HEK cells, and (c) HeLa cells for the P88-CFS extract of the present invention at different concentrations. Here, each data is expressed as the mean ± SD for three independent experiments, *** P < 0.001 compared to control; **P < 0.01; *P<0.05, as shown in Figure 2, P88-CFS tested against HCE, HEK and HeLa cell lines using the MTT assay for cytotoxicity showed that the metabolite was well tolerated by human cells. According to the guidelines for the degree of cytotoxicity (ISO 10993-5), cell viability greater than 80% is considered non-cytotoxic, 80-60% is considered weak, and less than 40% is considered strong cytotoxicity. . (a) HCE cells showed the strongest resistance to the P88-CFS extract according to the present invention, (b) HeLa and (b) HEK cells also showed strong resistance to the P88-CFS extract. Cell viability greater than 80% was maintained in HCE and HeLa cells when treated with 200 mg/ml of P88-CFS extract. However, HEK cells showed a higher sensitivity to the P88-CFS extract as culture medium containing more than 100 mg/mL of P88-CFS resulted in less than 80% cell viability. The above results demonstrate that P88-CFS can inhibit the replication of highly contagious human coronaviruses without serious cytotoxic effects when administered at 100 mg/ml. Therefore, the same dose was used for all subsequent experiments.

FIG. 3 (a) is a photograph of reactive oxygen species (ROS) staining by dichlorofluorescein (H2DCFDA) staining, and (b) is a graph showing quantitative fluorescence intensity measured by Image J. In the photograph of FIG. 3 (a), the treated cells were stained with H2DCFDA dye for 30 minutes, washed with PBS, and fluorescence images were acquired with a confocal microscope (scale: 500 μm). The present inventors further investigated the effect of P88-CFS on the intracellular accumulation of reactive oxygen species (ROS). Of note, ROS play an important role in the pathogenesis of COVID-19, where excessive accumulation of ROS can exacerbate disease severity. As shown in FIG. 3, it was observed that the ROS level increased about 2.4-fold when the cells were infected with SARS-COV-2. However, when infected cells were incubated with 50, 100 and 150 mg/mL P88-CFS, ROS levels were significantly reduced by about 1.7-fold, 1.2-fold and 0.9-fold, respectively.

4 is an immunofluorescence confocal micrograph showing the effect of P88-CFS on p-ERK protein in HEK293 cells after infection with SARS-CoV-2. Here, the immunofluorescence experiment is performed for DMSO (0.1%), viral titer, viral titer + 50 mg / ml, viral titer + 100 mg / ml, and viral titer + 150 mg / ml, respectively, images of pERK, DAPI and Merge. expressed. Specifically, p-ERK expression in HEK293 cells infected with SARS-CoV-2 was treated with 50, 100 and 150 mg/ml of P88-CFS. DMSO (0.1%) was used as a solvent for P88-CFS in all treatment groups. Cells were fixed for 12 hours after infection and confocal images were collected. Cell nuclei are shown in blue, and p-ERK are shown in green (scale bar: 5 μm). The MAPK/ERK pathway is an important pathway used by many viruses during pathogenesis. Phosphorylated ERK (p-ERK) is often associated with the presence of viral infection. As shown in Figure 4, expression of p-ERK was widely observed in both the cell nucleus and cytoplasm in virus-infected HEK293 cells without any treatment, whereas in mock cells treated with 0.1% DMSO Almost not detected. Although 50 mg/ml P88-CFS did not block p-ERK expression, treatment with 100 and 150 mg/ml P88-CFS drastically reduced ERK phosphorylation in SARS-CoV-2 infected cells.

Figure 5 is a graph showing gene expression of inflammatory markers in HEK 293 cells after infection with and without P88-CFS treatment according to the present invention, (a) IFN-α, (b) IFN-β, and (c) IL- Expression of 6 was measured by qPCR and normalized to GAPDH. All experiments were repeated three times and statistical analysis was performed by t-test. Data are expressed as mean±SD of three independent experiments (*P<0.05 compared to virus-infected cells; #P<0.05 compared to control). Finally, to evaluate the correlation between viral infection and inflammation, IFN-α, IFN-β and IL-6 were used in this study as one of the proinflammatory cytokines. As shown in Figure 5, the mRNA expression of IFN-α, IFN-β and IL-6 in the SARS-CoV-2 infected cells was significantly increased compared to the control group. In cells co-infected with SARS-CoV-2 and treated with 10 mg/ml of P88-CFS, mRNA expression levels of these pro-inflammatory cytokines were significantly reduced. Compared to infected cells (Infected), P88-CFS treated cells (Infected +) showed significant reductions of IFN-α, IFN-β and IL-6 by 2.2-fold, 1.5-fold and 1.7-fold, respectively. These findings suggest that the P88-CFS extract according to the present invention not only has antiviral activity but also regulates the inflammatory response caused by viral penetration into cells.

6 is a structural diagram showing the secondary structure of Sars-CoV-2 helicase nsp13. Here, the interacting residues for ss-RNA and ATP binding are red (Ser485, Lys146, Lys139, Tyr180, His230, Tyr198, Arg212, Pro335, Arg339, Asn516) and purple (Glu537, Arg567, Arg443, His290, Arg442, Asn265, Gly439, Lys288). The antiviral activity of L. plantarum Probio88 of the present invention was further evaluated using an in-silico molecular docking approach. Based on the whole genome sequence, the strain was found to produce the plantarisins PlnE and PlnF with 33 and 34 amino acid residues, respectively. The constructed models of PlnE and PlnF are composed of single helix chains, and their backbone structure was evaluated using Ramachandran pliot by measuring the psi(Ψ) and phi(Φ) angles of the backbone. As can be seen in Figure 6 and Table 2 below, Ramachandran plot statistical analysis indicated that 84% of the residues from PlnE and 92.9% of the PlnF construction model were in the most preferred regions. Table 2 below is a comparison of Ramachandran plot analysis for Plantarisin E and F construction models and templates.

Region of Plot
Plantaricin E Plantaricin F Built model template
(PDB ID: 2JUI) Built model template
(PDB ID: 2RLW) No. of residues % No. of residues % No. of residues % No. of residues % Residue in most favorite regions 21 84.0 21 84.0 26 92.9 25 89.3 Residue in additional allowed region 4 16.0 4 16.0 One 3.6 2 7.1 Residue in generously allowed regions 0 0.0 0 0.0 One 3.6 One 3.6 Residue in disallowed regions 0 0.0 0 0.0 0 0.0 0 0.0 Total 25 100 25 100 28 100 34 100

The results reflected the correlation well and were of good quality compared to the template structures described using NMR methods, namely 2JUI and 2RLW. The binding affinity of plantaricins E and F to the Sars-CoV-2 helicase nsp13 was evaluated using a protein-protein docking approach. The main function of helicase involved the release of self-annealed ss-RNA using ATP as an energy source. Therefore, the peripheral residues and ATP binding sites of ss-RNA acting as active residues were applied as HADDOCK protein-protein docking binding site parameters.

Figure 7 is a graphic showing the molecular docking of PlnE and PlnF, (a) is the 3D structure of SARS-CoV-2 helicase nsp 13 (PDB ID: 6ZSL) used in molecular docking studies, (b) is Planta It is a construction model of lysine (PlnE), (c) is a construction model for plantarisin F (PlnF), and (d) shows PlnE inserted into the cavity of ss-RNA and ATP binding sites, (e) shows that PlnF is inserted into the cavity of ss-RNA and ATP binding sites. As can be seen in FIG. 7 and Table 3 below, the binding of PlnE and F to the SARS-CoV-2 helicase nsp13 is -17.4 kcal/mol and -15.6 kcal, respectively, due to the long extended helical structures of PlnE and PlnF. It was distributed across ss-RNA and ATP binding sites with a binding affinity of /mol. Table 3 below is a summary of molecular docking results showing PlanE and the number of interactions involved in PlanE.

Analysis Types of Plantaricin E F Binding Interaction G, (kcal/mol) -17.4 -15.6 K _d (M) at 37℃ 5.8E-13 1.0E-11 Number of interactions Hydrogen Bonding 15 12 Hydrophobics 16 23

The docking results showed that the N-termini of PlnE and PlnF were bound to the ATP binding site, whereas the C-termini of PlnE and PlnF were attached to the ss-RNA binding site of helicase nsp13.

8 is a diagram showing the bonding structures of (a) PlnE and (b) PlnF, and the hydrogen bond cut-off was set to 4.0 Å. As shown in FIG. 8, PlnE such as Val35, Gly32, and Phe31 formed several hydrogen bonds with Gln537, Arg443, and Lys 288 at the ATP binding site of SARS-CoV-2 nsp 13. Gln537 and Arg443 have recently been reported by Newman and co-workers as major sites of ATP hydrolysis. In addition, Gly43 located at the N-terminus of PlnE formed a hydrogen bond interaction with one of the ss-RNA interface residues, that is, Arg212 of SARS-CoV-2 nsp 13. Similarly, in the case of PlnF, hydrogen bonds were formed between Asn30 with His290, Lys288 of SARS-CoV-2 nsp13, Ser24 with Arg442, and Asn265 of SARS-CoV-2 nsp13. The ss-RNA binding site of SARS-CoV-2 nsp13 was occupied by Gly48 and Phe49 at the N-terminus of PlnF by forming two hydrogen bonds with Arg212 and Asn516.

9 is a Ligplot+ interaction diagram of (a) PlnE and (b) PlnF, where hydrogen bonds are indicated by green dotted lines and hydrophobic interactions are indicated by red. As shown in FIG. 9, it was confirmed that hydrophobic interactions, which are known to contribute primarily to protein-protein interactions, dominate both PlnE and F interactions with SARS-CoV-2 nsp13 helicase.

From the above, it was confirmed that when SARS-CoV-2 infected cells are treated with P88-CFS according to the present invention, most viruses are eradicated after infection, and the effect becomes better as the concentration of the P88-CFS extract increases. Concomitant with the reduction of viral replication, P88-CFS reduced inflammation and ERK activity, which are important stepwise manifestations of viral invasion. Gene expressions of IFN-α, IFN-β and IL-6 were significantly increased when infected with SARS-CoV-2, and the expression of these genes was lower in the presence of P88-CFS. It has been shown that SARS-CoV-2 activates ERK upon infecting HeLa cells. In addition, L. plantarum Probio88 according to the present invention demonstrated antiviral activity using both PlnE and PlnF in an in-silico molecular docking approach. The high binding affinity and formation of hydrogen bonds showed that the binding of PlnE and PlnF to helicase could act as a blocker by preventing the binding of ss-RNA to helicase. In this regard, the use of ATP by helicases for the unwinding process may fail because the ATP binding site is occupied by the C-terminus of plantalysin. In addition, hydrophobic interactions, one of the major contributors to protein-protein interactions, were found to be dominant in both PlnE and PlnF interactions with helicases. This gives the possibility of maintaining protein binding stability, although the binding of plantarisin to helicase cannot be directly inferred. The infectivity of coronaviruses is highly dependent on spike proteins that promote viral attachment and host cell entry. However, there is great variability in the structure of the spike protein between coronavirus species, especially in the multiple variants of SARS-CoV-2 itself due to genetic mutations. Conversely, helicase nsp13 is a highly conserved non-structural protein among all coronavirus species. This means that therapeutics targeting this enzyme will be more applicable against SARS-CoV-2, its variants and closely related coronavirus species such as SARS-CoV and MERS-CoV. This proves that Lactobacillus plantarum Probio88 according to the present invention is capable of protecting against mutant strains of SARS-CoV-2.

The SARS-CoV-2 inhibitory activity demonstrated with P88-CFS according to the present invention demonstrates that probiotics can be used as an integrated therapeutic approach with vaccines and other antiviral agents to inhibit the spread of highly infectious pathogens. Importantly, natural supplements such as probiotics have been proven safe for decades, along with many other promising health benefits. Probiotics may not have distinct antiviral mechanisms like commercial drugs. However, the results of this study support the valuable insight that probiotics are promising candidates for preventing exacerbation of COVID-19 infection. Therefore, Lactobacillus plantarum Probio 88 according to the present invention is used as an adjuvant for the treatment of SARS-CoV-2 by mixing it with grain flour used as a raw material in feed, or by mixing carriers or additives to produce medicines such as tablets, capsules, It can be used as a probiotic for food, or by mixing a certain amount with raw materials used in cosmetics, it can be used in various fields such as pharmaceuticals, food, feed, and cosmetics according to conventional methods in the art.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present application can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it will be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of protection of the present application should be construed as including all changes or modifications that can be derived from the meaning, scope and equivalent concept of the claims to be described later rather than the above detailed description included in the scope of the present application.

sequence listing

P88 16S

AGAGTTTGAT CCTGGCTCAG GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAAC GAGCTCTGGT

ATTGATTGGT GCTTGCATCA TGATTTACAT TTGAGTGAGT GGCGAACTGG TGAGTAACAC GTGGGAAACC

TGCCCAGGAG CGGGGATAA CACCTGGAAA CAGATGCTAA TACCGCATAA CAACTTGGAC CGCATGGTCC

GAGCTTGAAA GATGGCTTCG GCTATCACTT TTGGATGGTC CCGCGGCGTA TTAGCTAGAT GGTGGGGTAA

CGGCTCACCA TGGCAATGAT ACGTAGCCGA CCTGAGAGGG TAATCGGCCA CATTGGGACT GAGACACGGC

CCAAACTCCT ACGGGAGGCA GCAGTAGGGA ATCTTCCACA ATGGACGAAA GTCTGATGGA GCAACGCCGC

GTGAGTGAAG AAGGGTTTCG GCTCGTAAAA CTCTGTTGTT AAAGAAGAAC ATATCTGAGA GTAACTGTTC

AGGTATTGAC GGTATTTAAC CAGAAAGCCA CGGCTAACTA CGTGCCAGCA GCCGCGGTAA TACGTAGGTG

GCAAGCGTTG TCCGGATTTA TTGGGCGTAA AGCGAGCGCA GGCGGTTTTTT TAAGTCTGAT GTGAAAGCCT

TCGGCTCAAC CGAAGAAGTG CATCGGAAAC TGGAAAACTT GAGTGCAGAA GAGGACAGTG GAACTCCATG

TGTAGCGGTG AAATGCGTAG ATATATGGAA GAACACCAGT GGCGAAGGCG GCTGTCTGGT CTGTAACTGA

CGCTGAGGCT CGAAAGTATG GGTAGCAAAC AGGATTAGAT ACCCTGGTAG TCCATACCGT AAACGATGAA

TGCTAAGTGT TGGAGGGTTT CCGCCCTTCA GTGCTGCAGC TAACGCATTA AGCATTCCGC CTGGGGAGTA

CGGCCGCAAG GCTGAAACTC AAAGGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC

GAAGCTACGC GAAGAACCTT ACCAGGTCTT GACATACTAT GCAAATCTAA GAGATTAGAC GTTCCCTTCG

GGGACATGGA TACAGGTGGT GCATGGTTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA

CGAGCGCAAC CCTTATTATC AGTTGCCAGC ATTAAGTTGG GCACTCTGGT GAGACTGCCG GTGACAAACC

GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTA TGACCTGGGC TACACACGTG CTACAATGGA

TGGTACAACG AGTTGCGAAC TCGCGAGAGT AAGCTAATCT CTTAAAGCCA TTCTCAGTTC GGATGTAGG

CTGCAACTCG CCTACATGAA GTCGGAATCG CTAGTAATCG CGGATCAGCA TGCCGCGGTG AATACGTTCC

CGGGCCTTGT ACACACCGCC CGTCACACCA TGAGAGTTTG TAACACCCAA AGTCGGTGGG GTAACCTTTT

AGGAACCAGC CGCCTAAGGT GGGACAGATG ATTAGGGTGA AGTCGTAACA AGGTAGCCGT AGGAGAACCT

GCGGCTGGAT CACCT

Korea Center for Biological Resources (KCTC) KCTC14482BP 20210304

Claims (17)

Lactobacillus plantarum ( Lactobacillus plantarum ) Probio 88 strain (KCTC14482BP). The strain according to claim 1 , wherein the strain is used to inhibit infection and replication of SARS-CoV-2. According to claim 1, wherein the strain is Lactobacillus plantarum characterized in that used to reduce inflammation ( Lactobacillus plantarum ) Probio 88 strain. The Lactobacillus plantarum Probio 88 strain according to claim 1, wherein the strain is used to reduce the intracellular accumulation of reactive oxygen species (ROS). The Lactobacillus plantarum Probio 88 strain according to claim 1, wherein the strain is used to reduce extracellular signal-regulated kinase (ERK) activity. The Lactobacillus plantarum Probio 88 strain according to claim 1, wherein the strain is used to enhance antiviral activity. Lactobacillus plantarum ( Lactobacillus plantarum ) Cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 containing Probio 88 strain (KCTC14482BP). The method of claim 7, wherein the extract contains plantarisin E (PlnE) and plantalysin F (PlnF) and is used to inhibit infection and replication of SARS-CoV-2. Cell-free supernatant (P88-CFS) extract of Lantharum Probio 88. The cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 according to claim 7, wherein the extract is used to reduce inflammation. The cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 according to claim 7, wherein the extract is used to reduce the intracellular accumulation of reactive oxygen species (ROS). The cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 according to claim 7, wherein the extract is used to reduce extracellular signal-regulated kinase (ERK) activity. The cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 according to claim 7, wherein the extract is used to enhance antiviral activity. The cell-free supernatant (P88-CFS) extract of Lactobacillus plantarum Probio 88 according to claim 7, wherein the extract is used to enhance antiviral activity. A composition comprising Lactobacillus plantarum Probio 88 strain (KCTC14482BP) for treatment of coronavirus. The composition according to claim 14, wherein the composition is a feed, feed additive, food, functional food, cosmetic composition, pharmaceutical composition or quasi-drug. Plantaricin E (PlnE) produced by Lactobacillus plantarum Probio 88 strain (KCTC14482BP), characterized in that it is used to inhibit infection and replication of SARS-CoV-2. Plantarisin F (PlnF) produced by Lactobacillus plantarum Probio 88 strain (KCTC14482BP), characterized in that it is used to inhibit infection and replication of SARS-CoV-2.

Images