Application of lactococcus cremoris D2022 in prevention and treatment of respiratory system infectious diseases

Abstract

The invention relates to the technical field of medicines, and discloses application of lactococcus cremoris D2022 in prevention and treatment of respiratory system infectious diseases. The lactococcus cremoris D2022 is a lactococcus lactis subsp. Cremoris strain, the study on the strain finds that the lactococcus cremoris D2022 is very strong in capacity of decomposing quercetin in vitro, animal model experiments verify that the lactococcus cremoris D2022 has relatively obvious prevention and treatment effects on influenza virus infection, and particularly, the lactococcus cremoris D2022 has relatively obvious prevention and treatment effects on influenza virus infection. The anti-influenza virus action mechanism of the strain comprises the steps that the strain decomposes intestinal food to generate DAT substances to enhance IFN-beta expression so as to achieve the anti-virus effect, and excessive inflammatory response is inhibited by lowering inflammatory pathways. In conclusion, the lactococcus cremoris D2022 is expected to become a new weapon for prevention and clinical adjuvant therapy of the influenza virus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of Lactococcus cremoris D2022 in preventing and treating respiratory infectious diseases. Background Art

[0002] Influenza is one of the pathogens that most easily causes respiratory diseases and has caused multiple global pandemics, which has aroused serious public health concerns. Infectious diseases caused by influenza viruses have always been a major challenge to global public health. The high variability and rapid spread of influenza viruses have posed a series of challenges to traditional prevention and control measures, such as vaccines and antiviral drugs. In this context, it is particularly urgent to find new and innovative prevention and control strategies.

[0003] Although there are currently small molecule drugs that inhibit influenza virus replication, such as oseltamivir, due to the high variability of influenza viruses and the wide variety of influenza viruses, there is still a need to provide more drugs that can be applied to different influenza viruses. Based on previous studies, it is of great significance to focus on the antiviral mechanism, immunomodulatory function and interaction of existing drugs used to treat influenza viruses in the host immune system, thereby providing new ideas and solutions for the treatment of influenza viruses for the scientific and medical fields.

[0004] With the continuous deepening of microbiological research, the search for microbial preparations with broad application potential to combat diseases such as influenza has become one of the focuses of scientific attention. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a use of Lactococcus cremoris D2022 in preventing and treating respiratory infectious diseases.

[0006] The specific technical scheme of the present invention is: In one aspect, the present invention provides the use of Lactococcus cremoris D2022 in the preparation of a medicament for preventing and / or treating respiratory infectious diseases.

[0007] Lactococcus lactis D2022 is a strain of Lactococcus lactis, its preservation number is CGMCC NO.25934, the preservation date is October 19, 2022, the preservation classification name is Lactococcus lactis subsp. cremoris D2022, and the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0008] Lactococcus cremoris D2022 is a strain of Lactococcus lactis subspecies cremoris obtained by the researchers of the present invention through prior application screening. The present invention found in the continued study of the strain that Lactococcus cremoris D2022 has a strong ability to decompose quercetin in vitro, and animal experiments have shown that it has a relatively obvious preventive and therapeutic effect on influenza virus infection. At the same time, experiments have proved that Lactococcus cremoris D2022 is harmless to animals. Therefore, it is proved that Lactococcus cremoris D2022 is expected to become a new weapon for the prevention and clinical auxiliary treatment of influenza virus.

[0009] Preferably, Lactococcus cremoris D2022 is used in the preparation of a medicament for preventing and / or treating viral pneumonia.

[0010] As another preferred embodiment, Lactococcus cremoris D2022 is used in the preparation of a medicament for preventing and / or treating influenza virus infection.

[0011] Lactococcus lactis subsp.cremoris D2022 (L.cremoris D2022) can effectively reduce lung pathology and inflammatory damage, protect colon length, and prolong the survival rate of mice, showing excellent application prospects in the preparation of influenza treatment and / or prevention drugs.

[0012] As another preferred embodiment, Lactococcus cremoris D2022 is used in the preparation of a medicament for preventing and / or treating influenza virus A / PR / 8 (H1N1) infection.

[0013] As a preferred embodiment of the above application, the dosage form of the drug includes granules, ordinary tablets, dispersible tablets, effervescent tablets, orally disintegrating tablets, lozenges, chewable tablets, capsules, soft capsules, microcapsules, pills, powders, pellets, sustained-release preparations, controlled-release preparations, oral liquid preparations, injections, lyophilized powders and bacterial liquid preparations.

[0014] On the other hand, the present invention provides a composition for treating respiratory infectious diseases, characterized in that the active ingredient of the composition is Lactococcus creamerii D2022.

[0015] As a preferred embodiment of the above composition, the respiratory infectious disease is viral pneumonia or influenza virus infection.

[0016] More preferably, the respiratory infectious disease is influenza virus A / PR / 8 (H1N1) infectious disease.

[0017] Preferably, the above composition comprises one or more pharmaceutically acceptable excipients.

[0018] Compared with the prior art, the present invention has the following technical effects: Lactococcus cremoris D2022 is a strain of Lactococcus lactis subspecies cremoris. The present invention has found in the study of this strain that Lactococcus cremoris D2022 has a strong ability to decompose quercetin in vitro. Animal model experiments have verified that it has a relatively obvious preventive and therapeutic effect on influenza virus infection. Specifically, its anti-influenza virus mechanism includes that the bacteria decompose intestinal food to produce DAT substances to enhance IFN-β expression and thus have an antiviral effect, and by reducing inflammatory pathways, it inhibits excessive inflammatory responses. In summary, it is proved that Lactococcus cremoris D2022 has the potential to become a new weapon for the prevention and clinical auxiliary treatment of influenza viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the survival curve of each experimental group in the influenza infection mouse model; Figure 2 This is the weight change curve of each experimental group in the influenza infection mouse model; Figure 3 is the lung index% of mice in each experimental group of the influenza infection mouse model; Figure 4 The length of the colon of mice in each experimental group of the influenza infection mouse model; Figure 5 The viral load of lung tissue in each experimental group of influenza infection mouse model on the third day; Figure 6 This is the viral load in lung tissue of each experimental group in the influenza infection mouse model on the seventh day; Figure 7 This is the light microscopic image of lung tissue of mice in the blank control group; Figure 8 Light microscopic images of lung pathological damage in the influenza infection mouse model group; Fig. 9 Light microscopic images of lung pathological damage in the Lactococcus cremoris intervention group of the influenza-infected mouse model; Fig.10 This is a comparison chart of lung tissue pathology scores in the normal control group and each experimental group of the influenza infection mouse model; Fig.11 This is a comparison chart of IL-1β values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.12 This is a comparison chart of IL-6 values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.13 This is a comparison chart of TNF-α values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.14 This is a comparison chart of KC values ​​of lung tissues in the normal control group and each experimental group of the influenza-infected mouse model at three and seven days; Fig.15This is a comparison chart of IL-10 values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.16 This is a comparison chart of IL-4 values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.17 This is a comparison chart of IFN-γ values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model at three and seven days; Fig.18 This is a comparison chart of IL-12 values ​​in lung tissue of the normal control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig.19 This is a comparison chart of serum IL-1β values ​​in the normal mouse control group and each experimental group of the influenza-infected mouse model on days 3 and 7; Fig. 20 Comparison chart of serum IL-6 values ​​of normal control group and each experimental group of influenza infection mouse model on day 3 and day 7; Fig.21 This is a comparison chart of serum TNF-α values ​​in the normal control group and each experimental group of the influenza-infected mouse model at three and seven days; Fig. 22 The comparison chart of serum KC values ​​of normal control group and each experimental group of influenza infection mouse model on day 3 and day 7; Fig.23 Comparison chart of serum IL-10 values ​​of normal control group and each experimental group of influenza infection mouse model on day 3 and day 7; Fig.24 Comparison chart of serum IL-4 values ​​of normal control group and each experimental group of influenza infection mouse model on day 3 and day 7; Fig.25 This is a comparison chart of serum IFN-γ values ​​in the normal control group and each experimental group of the influenza-infected mouse model at three and seven days; Fig.26 Comparison chart of serum IL-12 values ​​of normal control group and each experimental group of influenza infection mouse model on day 3 and day 7; Fig. 27 The differentially expressed genes in each group of mice on three days are shown; Fig.28 The differentially expressed genes in each group of mice at seven days; Fig.29 Kegg enrichment bubble diagram for each group of mice on three days; Fig.30 This is the Kegg enrichment bubble chart for each group of mice over seven days. DETAILED DESCRIPTION

[0020] Lactococcus lactis D2022 is a Lactococcus lactis strain with probiotic properties obtained by researchers of the present invention from Beijing soy milk. It is a strain of Lactococcus lactis subsp. cremoris, with a preservation number of CGMCC NO.25934 and a preservation date of October 19, 2022. The preservation classification is named Lactococcus lactis subsp. cremoris D2022, and the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration. Lactococcus lactis D2022 has been disclosed in the prior application CN117004510A of this unit and is a public strain.

[0021] The present invention has found in the continued study of the strain that Lactococcus cremoris D2022 has a strong ability to decompose quercetin in vitro, and animal experiments have shown that it has a relatively obvious preventive and therapeutic effect on influenza virus infection. At the same time, experiments have proved that Lactococcus cremoris D2022 is harmless to animals. Therefore, it is proved that Lactococcus cremoris D2022 is expected to become a new weapon for the prevention and clinical auxiliary treatment of influenza virus.

[0022] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0023] Example 1 Isolation, preservation and identification of Lactococcus cremoris D2022 strain 1.1 Isolation of D2022 strain (1) Take 100 μL of raw bean juice sample and add it to an EP tube pre-filled with 900 μL of sterile PBS. Perform gradient dilution on the sample in sequence until the concentration of the sample extract is diluted to 10-6 times; (2) 100 μL of samples of different dilutions were spread on BHI solid culture medium containing 5% defatted sheep blood (hereinafter referred to as BHI blood plate) and placed in an incubator; (3) Culture at 37°C, 0.5% CO2 for 48 h; (4) Remove the culture dish, pick colonies with different morphological characteristics with a sterile inoculation loop, transfer to a new BHI blood plate for purification, and culture anaerobically at 37°C for 48 h. Transfer three times in a row, and culture the purified strains in liquid MRS at pH 3.5. Screen strains with excellent acid-resistant growth for use in experiments or frozen storage.

[0024] 1.2 Strain preservation This laboratory uses BHI medium containing 25% glycerol as the bacterial culture medium for frozen preservation of bacterial strains. The method is as follows: (1) Sterilize a 2 mL sterilization tube by autoclaving at 121°C for 15 min for use; (2) After the bacteria have been transferred to the solid medium three times, add 1.5 mL of sterile bacterial preservation solution to the culture dish; (3) Use an L-rod to scrape the culture dish to ensure that the colonies are fully integrated into the bacterial preservation solution; (4) Transfer the bacterial solution to a bacterial preservation tube, mix well, and store at -80°C.

[0025] 1.3 Observation of colony appearance and bacterial morphology Lactococcus cremoris is a facultative anaerobic bacterium. It grows well under anaerobic conditions, and its colonies are milky white and smooth. Under aerobic conditions, Lactococcus cremoris grows poorly, with smaller colonies and transparent to light white color. Under microscopic observation, Lactococcus cremoris is a Gram-positive coccus.

[0026] 1.4 Extraction of total bacterial DNA A single colony was inoculated on a BHI blood plate and cultured anaerobically overnight at 37°C. The DNA was extracted using a bacterial genomic DNA extraction kit (Nanjing Novozyme). DNA was extracted according to the instructions of DNA extraction kit.

[0027] 1.5 Accurately identify strains by comparing ANI and DDH with model strains The genome of the strain was extracted and draft sequenced. The strain was further accurately identified by genome correlation analysis (digital DNA-DNA hybridization (dDDH); dDDH < 70%) and average nucleotide identity (Average Nucleotide Identity, ANI; ANI < 95%)) by comparison with the model strain. Both methods are the gold standard for determining prokaryotic species and are completed online (dDDH, http: / / ggdc.dsmz.de; ANI, http: / / enve-omics.ce.gatech.edu / ani / ).

[0028] 1.5.1 Genome extraction and draft submission This study used the DNA extraction kit produced by Nanjing Novozyme Biotechnology Co., Ltd. for gene extraction. The specific operation steps are as follows: (1) Since the bacteria are Gram-positive, lysozyme solution needs to be added to the bacterial precipitate and treated at 37°C for 2 h to ensure that the cell wall is fully destroyed.

[0029] (2) Add 20 μL of proteinase K solution to the above mixture and shake to remove protein impurities; then add 200 μL of buffer BCL and shake to mix; then incubate in a 55°C water bath for 10 min and mix by inversion to ensure that there is no impurity precipitation in the solution.

[0030] (3) Add 150 μL of anhydrous ethanol and shake to mix. During this process, flocculent precipitates will be generated. Transfer all the liquid and floccules in the EP tube to the adsorption column FastPure gDNA Mini Columns II, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0031] (4) Place the adsorption column in a waste liquid collection tube, add 500 μL of buffer WA to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0032] (5) Add 600 μL of buffer WB, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, put it into a new collection tube, and centrifuge at 12,000 rpm for 2 min. This step is repeated twice to ensure that the DNA is clean.

[0033] (6) Place the adsorption column in a collection tube, open the cover and leave it at room temperature for 3 to 5 minutes to dry, in order to allow the remaining rinse solution (mainly ethanol) in the adsorption column to evaporate.

[0034] (7) Transfer the adsorption column to a new EP tube, add 200 μL of Elution Buffer (preheated to 55°C) to the adsorption column membrane, place at room temperature for 2-5 minutes, and centrifuge at 12000 rpm for 1 minute to obtain pure bacterial genomic DNA. Transport the cold chain to Beijing Deaoping Biotechnology Co., Ltd. to complete the draft sequencing of the genome.

[0035] 1.5.2 Genome correlation analysis (1) Basic characteristics of the genome The draft results showed that the genome size of strain D2022 was 2634034 bp and the GC content was 35.42%.

[0036] (2) dDDH and ANI The dDDH and ANI values ​​of the model strain of Lactococcus lactis subsp.cremoris DSM 20069 and the LPP strain were compared. The results showed that the dDDH and ANI values ​​between the model strain DSM 20314T and the LPP strain were 85.00% and 98.38%, respectively, supporting that the two strains were the same bacterial species.

[0037] The preservation information of this strain is as follows: the strain preservation number is CGMCC NO.25934, the preservation date is October 19, 2022, the preservation classification name is Lactococcus lactis subsp.cremoris D2022 strain, the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0038] Example 2 Experiment on decomposition of quercetin by L.cremoris D2022 Weigh 10 mg of quercetin and dissolve it in 6 mL DMSO. Add 47 mL of BHI liquid medium to 3 mL of quercetin to prepare BHI liquid medium containing 1 mg / 10 mL quercetin. Add it to a 15 mL bacterial culture tube. In order to save the culture medium, use 4.5 mL of culture medium + 0.5 mL of bacteria (concentration is 2×10 10 ) was cultured in an anaerobic incubator at 37°C for 20 hours, and the color change was observed. After centrifugation at 8000rmp for 3 minutes, 100μL of the supernatant was reacted with an equal amount of 1% AlCl3 (dissolved in methanol) for 5 minutes, and the OD value was measured at a wavelength of 405nm. The standard strain Clostridium orbiscindens (purchased from ATCC) was used as the positive control, and PBS was used as the negative control. The standard curve was drawn after multiple dilution with the standard substance (1×PBS), and the decomposition rate of quercetin of each sample was calculated according to the standard curve. The results showed that the decomposition rate of quercetin of the positive control was 87.5%, and the decomposition rate of quercetin of Lactococcus lactis D2022 was 70.9%, which had the best performance among the screened strains. The decomposition rate of quercetin of Bacteroides thetaiotaomicron was 51.2%, and the decomposition rate of quercetin of Lactobacillus plantarum was 65.6%.

[0039] Example 3 Study on the anti-influenza function of L.cremoris D2022 3.1 Research methods: 48 6-8 week old female SPF C57BL / 6J mice weighing 13-15g were randomly divided into 3 groups, each with 16 mice. The 3 groups were: Control group (blank group, also known as blank control group, not infected with influenza virus), Model group (model group, 100 μL 1×PBS was given intragastric administration for 7 consecutive days on the second day after influenza virus infection), L.cremoris group (Lactococcus cremoris group, 100 μL Lactococcus cremoris was given intragastric administration for 7 consecutive days on the second day after influenza virus infection). The research process is as follows: 1% sodium pentobarbital solution (50 mg / kg) was prepared and injected intraperitoneally to anesthetize mice. After anesthesia, mice in the two infection groups (model group and Lactococcus cremoris group) were given 40 μL influenza virus A / PR / 8 (H1N1) strain for nasal inoculation, 20 μL in each left and right nose, and the inoculation dose was 600 PFU. The blank group was given nasal inoculation with an equal volume of PBS. After nasal drops, the general condition of the mice was observed daily, and the body weight and feed consumption were recorded. Samples were collected on the 3rd and 7th days after infection. On the 3rd day after infection, some mice were killed (6 mice in each group), and the whole lung homogenate was taken to detect the viral load. On the 7th day after infection, each group of mice (10 mice in each group) was weighed, and the mice were killed by eye removal and bleeding. The eye blood of the mice was collected, and after being stored at 4°C overnight, the supernatant was taken at 8000rpm for 10 minutes. The colorectum from the ileocolic junction to the anus was separated from each group, and the colon length (cm) was measured; the whole lung was weighed in each group, and the lung index was calculated. The right lung was stored at -80°C, and the left lung was fixed in 4% paraformaldehyde. The colon was taken to measure the length and weight, and the middle 1cm was taken and fixed in 4% paraformaldehyde. Among them, lung index = lung weight (g) / body weight (g) × 100%.

[0040] Three other groups of mice were selected, with 10 mice in each group. The mice in the two infection groups (model group and Lactococcus cremoris group) were intranasally injected with 40 μL of influenza virus A / PR / 8 (H1N1) strain, 20 μL in each nostril, with an infection dose of 1200 PFU. Other steps were handled in the same way. The mice were observed for two weeks after infection, and the mouse survival curve was analyzed.

[0041] 3.2 Evaluation indicators The effects of L.cremoris D2022 intervention on the body weight, colon length, influenza virus load in lung tissue, and levels of multiple cytokines in serum and lung tissue of mice induced by influenza A virus were investigated. Lung transcriptome analysis and lung histopathological examination were performed on each group of mice.

[0042] The pathological condition was evaluated in three aspects. The higher the score, the more serious the pathological condition, and the full score was 4. The specific pathological scoring criteria are shown in Table 1.

[0043] Table 1 score Scoring Criteria 0 The alveolar walls are intact without thickening, inflammatory infiltration, or congestion. 1 Slight diffuse inflammatory cell infiltration (neutrophils) of the alveolar wall, no obvious thickening of the alveolar wall 2 Obvious and extensive inflammatory cell infiltration (neutrophils and monocytes), mild thickening of alveolar walls (1-2 times) 3 Severe inflammatory cell infiltration, significant thickening of alveolar walls, and 25%-50% of lung tissue consolidation 4 Severe inflammatory cell infiltration, significant thickening of alveolar walls, >50% lung tissue consolidation 3.3 Research Results: 3.3.1 Evaluation of the efficacy of L. cremoris D2022 against influenza virus infection (see 2.1 for details of the groups) (1) The influenza virus PR8 strain that is pathogenic to mice was used to infect C57BL / 6J mice to establish a mouse influenza virus lung infection model. After influenza virus infection, PBS and live L. cremoris D2022 bacteria (2×10 9 CFU / mouse), body weight was monitored daily, and 6 mice in each group were killed on the 3rd day, and the whole lung homogenate was taken to detect the viral load. The remaining 10 mice in each group were killed on the 7th day, and the lung index, lung tissue viral load, and multiple factors in the lung and serum were tested. The results are shown in Figure 1-Figure 6 ,in: Figure 1 is the survival curve after influenza infection; Figure 2 Weight changes after influenza infection; Figure 3 is the lung index % of mice in each group; Figure 4 is the colon length of mice in each group; Figure 5 The viral load in lung tissue was measured on the third day after infection; Figure 6 This is the viral load in lung tissue on the seventh day after infection.

[0044] The results showed that the treatment with L.cremoris D2022 live bacteria had a significant difference in weight change, and could effectively reduce the lung index, increase colon length, protect lung and colon damage, and significantly reduce the viral load of lung tissue on the third day. It can be seen that L.cremoris D2022 live bacteria treatment has a significant protective effect on the death of influenza mice.

[0045] (2) Pathological and electron microscopic observation of lung tissue on day 7 after virus infection. Figure 7 to Figure 9 .

[0046] Depend on Figure 7 It can be seen that the alveolar structure of the blank group mice was clear, there was no exudate in the lumen, the blood vessels were not dilated and congested, and there was no obvious inflammatory cell infiltration; Figure 8 It can be seen that the alveolar tissue structure of the mice in the model group was unclear, the alveolar wall was significantly thickened (blue arrow), a large number of inflammatory cells infiltrated in the pulmonary interstitium and alveoli (black arrow), pulmonary microvessels were congested and hemorrhaged, and some had features of pulmonary consolidation (red arrow); Fig. 9 It can be seen that the alveolar structure in the Lactococcus lactis group was clear, and the inflammatory cell infiltration was less than that in the model group (black arrow).

[0047] Scoring was performed according to the lung tissue pathology scoring criteria. Fig.10 .Depend on Fig.10It can be seen that the pathological score of the model group was higher than that of the blank control group (P<0.01), and the pathological score of the Lactococcus lactis group was lower than that of the model group (*p<0.05, **p<0.01).

[0048] 3.3.2 Effects of local and systemic inflammatory factors on mice infected with L. cremoris D2022 influenza virus (1) On the 3rd and 7th days, multiple cytokines were detected in the lung tissues of each group of mice using liquid phase chips. The results are as follows: Figure 11-18 As shown, among which: IL-1β content results are shown in Fig.11 , IL-6 content results are shown in Fig.12 TNF-α content results are shown in Fig.13 The results of chemokine KC content are shown in Fig.14 , IL-10 content results are shown in Fig.15 , IL-4 content results are shown in Fig.16 , IFN-γ content results are shown in Fig.17 , IL-12 content results are shown in Fig.18 .

[0049] The results showed that 7 days after infection, the levels of IL-1β, IL-6, TNF-α, chemokine KC, IL-10, IL-4, IFN-γ, and IL-12 in the model group were significantly higher than those in the control group; 7 days after infection, Lactococcus creamer intervention could significantly reduce the levels of IL-6, IL-10 and KC, and significantly increase the levels of IL-4 and IL-12, but there was no statistical difference in the effects on other detected factors.

[0050] At the same time, the results showed that there were no significant changes in cytokines and chemokines in lung tissue among the groups 3 days after infection.

[0051] (2) On the 3rd and 7th days, the peripheral serum of each group of mice was tested for multiple cytokines using a liquid phase chip, such as Figure 19-26 The results of IL-1β content in peripheral blood are shown in Fig.19 The results of IL-6 content in peripheral blood are shown in Fig. 20 The results of peripheral blood TNF-α content are shown in Fig.21 The results of peripheral blood KC content are shown in Fig. 22 The results of IL-10 content in peripheral blood are shown in Fig.23 The results of peripheral blood IFN-γ content are shown in Fig.24 .

[0052] The results showed that there was no significant change in the cytokines and chemokines in the lung tissues among the groups 3 days after infection; 7 days after infection, the levels of IL-1β, IL-6, TNF-α, KC, IL-10, and IFN-γ in the peripheral blood of the model group were significantly higher than those of the control group. Lactococcus lactis intervention significantly reduced the levels of IL-1β, TNF-α, and chemokine KC, and significantly increased the levels of IL-4 and IL-12. There was no statistical difference in the effects on other detected factors.

[0053] Combination Figures 11 to 26 The multifactorial detection results show that intervention with Lactococcus cremoris can alleviate the immune inflammatory damage caused by influenza virus.

[0054] On the 3rd and 7th days, the number of genes in each group of mice was counted, and the results are shown in Fig. 27 and Fig.28 ,in: Fig. 27 The differentially expressed genes in each group of mice on three days are shown; Fig.28 Figure 1 shows the differentially expressed genes in each group of mice over seven days. On the third and seventh days, the Kegg pathways in each group of mice were enriched. Fig.29 and Fig.30 ,in: Fig.29 Kegg enrichment bubble diagram for each group of mice on three days; Fig.30 This is the Kegg enrichment bubble chart for each group of mice over seven days.

[0055] The results of Kegg pathway enrichment showed that compared with the model group, there were 391 differentially expressed genes increased in the Lactococcus cremoris group within three days and 104 differentially expressed genes decreased, while there were 268 differentially expressed genes increased in the Lactococcus cremoris group within seven days and 359 differentially expressed genes decreased; from the perspective of enriched pathways, the inflammatory pathway was not obvious within three days, and the inflammatory pathway within seven days included the interaction between viral proteins, cytokines and cytokine receptors, the IL-17 signaling pathway and the AMPK signaling pathway, and the pathways were mainly down-regulated.

[0056] In summary, this Example 3 established a mouse influenza virus infection model and confirmed that Lactococcus creminis D2022 has a relatively obvious effect in preventing and treating influenza virus infection. Its possible mechanism of action includes that the bacteria decompose intestinal food to produce DAT-like substances to enhance IFN-β expression and thus have an antiviral effect, and inhibit excessive inflammatory response by decreasing inflammatory pathways.

[0057] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of Lactococcus cremoris D2022 in the preparation of medicines for preventing and / or treating respiratory infectious diseases.

2. The use according to claim 1, characterized in that: The dosage forms of the drug include granules, ordinary tablets, dispersible tablets, effervescent tablets, orally disintegrating tablets, lozenges, chewable tablets, capsules, soft capsules, microcapsules, pills, powders, drop pills, sustained-release preparations, controlled-release preparations, oral liquid preparations and injections.

3. Use of Lactococcus creamerii D2022 in the preparation of drugs for preventing and / or treating viral pneumonia.

4. Use of Lactococcus creamerii D2022 in the preparation of drugs for preventing and / or treating influenza virus infection diseases.

5. Use of Lactococcus cremoris D2022 in the preparation of drugs for preventing and / or treating influenza virus A / PR / 8 (H1N1) infection diseases.

6. A composition for treating respiratory infectious diseases, characterized in that: The active ingredient of the composition is Lactococcus cremoris D2022.

7. The composition according to claim 6, characterized in that: The respiratory infectious disease is viral pneumonia or influenza virus infection.

8. The composition according to claim 7, characterized in that: The respiratory infectious disease is an influenza virus A / PR / 8 (H1N1) infectious disease.

9. The composition according to claim 6, characterized in that: The composition includes one or more pharmaceutically acceptable excipients.

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