CN116875484A

CN116875484A - Probiotic double-bacterium combined preparation and application thereof in antagonizing influenza

Info

Publication number: CN116875484A
Application number: CN202310626050.XA
Authority: CN
Inventors: 李艳; 韩美晴; 李卫芬; 乐敏
Original assignee: Nanjing Kangyou Biotechnology Co ltd; Zhejiang University ZJU
Current assignee: Nanjing Kangyou Biotechnology Co ltd; Zhejiang University ZJU
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-10-13

Abstract

The invention belongs to the field of microbial technology and biological agents for resisting influenza virus infection, and particularly relates to a probiotic double-bacterium combination with an influenza antagonizing capability and application thereof, namely influenza antagonizing. The invention discloses a probiotic double-bacteria combined preparation which consists of lactobacillus plantarum (Lactobacillus plantarum) Lac16 and lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) P118 with equal amounts. The invention also provides application of the probiotic double-bacterium combined preparation in preparing medicines for treating or preventing avian influenza virus infection. The probiotic double bacteria combination (Lac16+P118) can effectively improve the body weight reduction caused by influenza infection, reduce the pulmonary virus titer and has obvious effect of antagonizing influenza A.

Description

Probiotic double-bacterium combined preparation and application thereof in antagonizing influenza

Technical Field

The invention belongs to the field of microbial technology and biological agents for resisting influenza virus infection, and particularly relates to a probiotic double-bacterium combination with an influenza antagonizing capability and application thereof, namely influenza antagonizing.

Background

Influenza (influza) is an infectious respiratory disease caused by influenza virus (IFV). Influenza a, b and c viruses can be classified according to antigenicity of influenza virus internal Nucleoprotein (NP) and matrix protein (M1), wherein influenza a is the most predominant subtype causing human influenza disease (Noda et al, 2006). As early as mid-century, influenza virus has entered human vision (Shi Y et al, 2014). To date 1918, five influenza pandemics have historically been shared in the world, with the influenza subtypes responsible for the pandemics being predominantly H1N1, H2N2 and H3N2, several of which are caused by influenza viruses of animal origin infecting humans across the host barrier (Wang Dayan et al, 2018). In addition to pandemic, animal-derived influenza (especially avian influenza) continues to cross host restrictions, continually causing sporadic or small-scale human-borne epidemic (Yuan, 2015). In animal farming, both human and avian influenza viruses infect pigs, which are equivalent to the "mixer" vehicle for influenza virus, and similarly swine influenza virus (swine influenza virus, SIV) can be passed back to birds and humans (Yang Shuai et al, 2013;Wang Z et al,2021).

The current influenza prevention and treatment methods mainly include early vaccination prevention and treatment with related drugs. There are three classes of drugs approved by the U.S. food and drug administration (Food and Drug Administration, FDA) for use in the treatment of human influenza: neuraminidase inhibitors (e.g., oseltamivir), M2 ion channel blockers (e.g., amantadine), and polymerase inhibitors. Although vaccines are the cornerstone for controlling influenza virus infection, there are many challenges and uncertainties therein, the immune response spectrum generated by vaccines is relatively narrow and of short duration (KIM H et al, 2017). On the one hand, there is a constant antigen drift and sporadic antigen transfer in the viral surface glycoproteins, so influenza vaccines must be updated periodically; on the other hand, due to the increasing emergence of more resistant viral varieties, the mass production of highly effective influenza vaccines is a great challenge, and new drugs or epidemic prevention methods are urgently needed to reduce and control influenza outbreaks (Rodriguez L et al,2017;Farrukee R et al,2017).

Relevant evidence can be found on different animal models, different strains of influenza virus and different probiotic types to demonstrate the role of probiotics in antagonizing influenza virus. For example: qiang Zhang et al (2020) found that bifidobacterium animalis was able to significantly reduce the severity of H7N9 subtype influenza infection; naoyoshi Maeda (2009) believes that high temperature inactivated Lactobacillus plantarum L-137 can significantly reduce pulmonary virus titer of mice after infection with H1N1 subtype, stimulate secretion of macrophages/dendritic cells, and thereby generate helper T cells to protect the body; alexander Yitbarek et al (2018) demonstrate that a combination of five lactobacilli (Lactobacillus salivarius, l.johnsonii, l.reuteri, l.cristatus and l.gasser) can modulate the innate immune response of chickens to infection by H9N2 subtype and possibly promote homeostasis by modulating the gut microbiota by assessing the cloacal virus titres and the expression of interferon- α (IFN- α), interferon- β (IFN- β) and interleukin-22 (IL-22) in the trachea, lungs, ileum and cecum tonsils. Probiotics have been studied for decades for use in the prevention or treatment of various diseases, including allergy and various intestinal diseases, and for the treatment of gastrointestinal, respiratory and even cancer.

The invention of CN106011001A informs a fowl and livestock intestinal canal ecological restoration microbial inoculum for resisting virus and bacteria to replace antibiotics, which comprises Saccharomyces cerevisiae ACCC20037, candida utilis ACCC20060, bacillus subtilis (Bacillus subtilis) ACCC10114, bacillus licheniformis ACCC10146, rhodopseudomonas palustris (Rhodopseudomonas palustris) ACCC10649, lactobacillus plantarum (Lactobacillus plantarum) ACCC10141, lactobacillus rhamnosus (Lactobacillus rhamnosus) ACCC10534 and the like, and is mixed with water extract of lemon and astragalus. The purposes of adjusting the ecological balance of the intestinal flora of livestock and poultry, improving the immunity of the organism, resisting viruses and bacteria are achieved by utilizing the polysaccharide in the astragalus, the limonin and the polysaccharide in the lemon and the extracellular polysaccharide produced by each probiotics.

The invention of CN107075460A (clostridium butyricum strain with immunity enhancing and antiviral activity and application thereof) reports that: clostridium butyricum Fb5-3 strain (KCTC 12753 BP) or clostridium butyricum S-45-5 strain (KCTC 12754 BP), fb5-3 strain was antiviral against influenza virus (PR 8) (1.0 MOI).

The invention of CN105861399A (Lactobacillus plantarum for preventing chicken necrotic enteritis) and application thereof) informs a Lactobacillus plantarum Lac16 with the preservation number of: cctccc NO: m201659. It can prevent necrotic enteritis of chicken; inhibiting clostridium perfringens growth, thereby relieving the influence on the production performance reduction of broiler chickens and the damage of intestinal mucosa structures after clostridium perfringens attacks toxin; can also improve the macrophage immune function of chickens and promote the growth of broiler chickens.

Disclosure of Invention

The invention aims to solve the technical problem of providing a probiotic double-bacterium combined preparation and application thereof in antagonizing influenza, namely prevention of animal influenza virus.

In order to solve the technical problems, the invention provides a probiotic double bacteria combined preparation which consists of lactobacillus plantarum (Lactobacillus plantarum) Lac16 and lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) P118 with equal amounts.

Specifically: a probiotic double bacteria combination preparation, which is obtained by suspending lactobacillus plantarum Lac16 and lactobacillus rhamnosus P118 in sterile PBS in equal amounts (equal bacteria amount);

in the probiotic double-bacterium combined preparation, the total bacterial concentration of lactobacillus plantarum Lac16 and lactobacillus rhamnosus P118 is more than or equal to 1 multiplied by 10 ⁹ CFU/ml。

That is, the optimal ratio of the effective bacterial concentration of lactobacillus plantarum Lac16 (Lactobacillus plantarum Lac) to that of lactobacillus rhamnosus P118 (Lactobacillus rhamnosus P) is 1:1, and the total effective bacterial concentration after mixing the two is 1:1

≥1×10 ⁹ CFU/ml。

The preservation number of lactobacillus plantarum (Lactobacillus plantarum) Lac16 is CCTCC NO: m201659, lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) P118 with a collection number of cctccc NO: m20221065.

Description: lactobacillus plantarum Lac16 (collection number CCTCC NO: M201659) is reported in CN 105861399A.

The preservation information of lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) P118 is as follows:

the preservation name is: lactobacillus rhamnosus P118 Lacticaseibacillus rhamnosus P, deposit unit: china center for type culture collection, preservation address: university of martial arts in chinese; preservation date: 2022, 7 and 8, accession number: cctccc NO: m20221065.

As an improvement of the probiotic double bacteria combination preparation of the invention:

the lactobacillus plantarum Lac16 and lactobacillus rhamnosus P118 are both subjected to anaerobic culture in MRS liquid culture medium, wherein the culture temperature is 37+/-1 ℃, and the culture time is 24+/-12 hours.

The invention also provides application of the probiotic double-bacterium combined preparation in preparing medicines for treating or preventing avian influenza virus infection.

Influenza virus is preferably A/Mink/China/01/2014 (H9N 2), A/California/04/2009 (H1N 1).

Compared with the traditional method for preventing influenza infection, the invention has the following technical advantages:

1. the probiotic double bacteria combination (Lac16+P118) can effectively improve the body weight reduction caused by influenza infection, reduce the pulmonary virus titer and has obvious effect of antagonizing influenza A.

2. The probiotic double bacteria combination (Lac16+P118) can quickly regulate the immune response of a host, improve the number of NK cells, granulocytes and monocytes in the body, and can maintain for a certain time to continuously protect the subsequent body infection.

3. The probiotic double bacteria combination (Lac16+P118) in the invention can not only excite organism immune response, but also reduce the expression of certain immune cytokines in the middle stage of influenza infection, reduce the reaction degree of inflammation to a certain extent, and alleviate the influenza symptom aggravated by cytokine storm caused by excessive immunity.

4. The probiotic double bacteria combination (Lac16+P118) can regulate the richness of intestinal microorganisms in a host body, promote the intestinal environment to be balanced, and indirectly protect the host against influenza.

Drawings

FIG. 1 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on body weight of SPF mice prior to H9N2 infection.

FIG. 2 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on body weight of SPF mice after H9N2 infection.

FIG. 3 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the clinical status score of SPF mice after H9N2 infection.

FIG. 4 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the viral load of SPF mouse rhinowash after H9N2 infection.

FIG. 5 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the pulmonary viral load of SPF mice after H9N2 infection.

FIG. 6 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on lung pathology in SPF mice after H9N2 infection.

FIG. 7 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on T cell, NK cell, monocyte, granulocyte changes in blood of SPF mice after H9N2 infection.

FIG. 8 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the changes in the immunocytokines interleukin-1 (IL-1β), interleukin-6 (IL-6) in the lung of SPF mice after H9N2 infection.

FIG. 9 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the change of the immunocytokine interferon-gamma (IFN-gamma), interferon-L2 &3 (IFN-L2 & 3) tumor necrosis factor-alpha (TNF-alpha) in the lung of SPF mice after H9N2 infection.

FIG. 10 is a graph showing the variation of gastric lavage double bacteria (Lac16+P118) on intestinal microbiota diversity and flora structure of SPF mice.

FIG. 11 is a graph showing the variation of intestinal microbial diversity and flora structure of SPF mice by gastric lavage double bacteria combination (Lac16+P118).

FIG. 12 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on body weight of SPF mice prior to H1N1 infection.

FIG. 13 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on body weight of SPF mice after H1N1 infection.

FIG. 14 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the clinical status score of SPF mice body weight after H1N1 infection.

FIG. 15 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on lung pathology in SPF mice after H1N1 infection.

FIG. 16 is the effect of the gastric lavage double bacteria combination (Lac16+P118) on the pulmonary viral load of SPF mice after H1N1 infection.

FIG. 17 is the effect of individual doses of intragastric Lac16 and P118 on body weight of SPF mice prior to H9N2 infection, respectively.

FIG. 18 is the effect of individual doses of gastric lavage Lac16 and P118, respectively, on body weight of SPF mice after H9N2 infection.

FIG. 19 is the effect of individual doses of gastric lavage Lac16 and P118, respectively, on the viral load of SPF mouse rhinowash after H9N2 infection.

FIG. 20 is the effect of individual doses of intragastric Lac16 and P118 on pulmonary viral load of SPF mice after H9N2 infection, respectively.

Detailed Description

The test methods and conditions in the examples of the present invention are conventional methods unless otherwise specified. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. The technical scheme of the invention is a conventional scheme in the field if no special description exists; the reagents or materials described are commercially available unless otherwise specified.

The invention takes avian influenza viruses H9N2 and H1N1 as an example to illustrate the application of lactobacillus-lactobacillus plantarum Lac16 (Lactobacillus plantarum Lac) and lactobacillus rhamnosus P118 (Lactobacillus rhamnosus P118) in preparing medicaments for preventing influenza virus infection.

The sterile PBS was PBS buffer pH7.4 (137 mM NaCl,2.7mM KCl,10mM Na ₂ HPO ₄ ,2mM KH ₂ PO ₄ ) After filtration (0.22 μm), sterile PBS was obtained.

Example 1: preparation method of probiotic double-bacterium combined preparation

1. Lactobacillus plantarum Lac16 (CCTCC NO: M201659) is informed in the invention of CN105861399A, lactobacillus plantarum for preventing necrotic enteritis of chickens and application thereof.

2. Rhamnose bacillus P118 (CCTCC NO: M20221065).

3. Rhamnose bacillus P118 and lactobacillus plantarum Lac16, which are respectively treated as follows:

the stock solutions are all stored in a refrigerator at the temperature of minus 80 ℃ and are revived at room temperature when needed, bacterial solutions are picked up by an inoculating loop and are streaked on an MRS flat plate culture dish for reviving, and the stock solutions are cultured for 36 to 48 hours under the anaerobic condition at the temperature of 37 ℃. The recovered single colony is selected and cultured in 1ml MRS liquid culture medium for 24 hours under the anaerobic condition at 37 ℃, and then inoculated into 10ml MRS liquid culture medium for expansion culture according to the volume ratio of 1:100, and the culture is continued for 12 hours under the anaerobic condition at 37 ℃. The concentration of the bacterial liquid in the expansion culture is adjusted to obtain the concentration of 5 multiplied by 10 ⁸ CFU/ml bacterial suspension. The concentrations were 5X 10 ⁸ CFU/ml of rhamnose bacillus P118 bacterial suspension and lactobacillus plantarum Lac16 bacterial suspension are mixed according to a ratio of 1:1 (v/v) to prepare composite bacterial suspension, and therefore the concentration of two bacteria in the composite bacterial suspension is 5 multiplied by 10 ⁸ CFU/ml, total concentration of 1X 10 ⁹ CFU/ml。

Centrifuging the composite bacterial suspension at 4deg.C and 5000rpm for 10 min, discarding supernatant, washing with sterile PBS once, and allowing it to finally reach 1×10 ⁹ The total bacterial amount of CFU/ml is resuspended in sterile PBS, and a probiotic double bacteria combination preparation (i.e., probiotic double bacteria combination preparation, abbreviated as lactobacillus preparation) is obtained.

Experiment 1: effect of the probiotic double bacteria combination preparation prepared in example 1 on body weight and clinical manifestation of mice before and after infection with influenza virus.

1. Effect of lactobacillus plantarum Lac16, lactobacillus rhamnosus P118 and probiotic double bacteria combined preparation (lac16+p118) on weight change in mice:

the mice used in this example were purchased from Shanghai Laek and were SPF-rated female C57BL/6 mice of 4 weeks old, for a total of 22. Mice were randomly divided into 2 groups, PBS control group (11) and double bacteria combined Mix group (11). All SPF mice were bred in a manner conforming to the conventional regulations.

Mice after acclimation, each mice of the experimental group were perfused every other day with 100 μl of the probiotic double bacteria combination preparation (1×10 ⁹ CFU/ml of bacterial fluid), each mouse of the control group was perfused with 100 μl of sterile PBS and the mouse body weight was recorded.

Nasal drop infection with influenza A/Mink/China/01/2014 (H9N 2) (virus concentration 1X 10) ⁷ TCID50/ml, 20 μl/dose, only once for nasal drip infection), infection when the day was recorded as day0, mice were observed for status 14 consecutive days after challenge, body weights were measured, and clinical manifestations were recorded (tables 1, 2, FIGS. 1, 2).

TABLE 1 Effect of gastric lavage of probiotic double bacteria on mice weight gain

Note that: days are days before the mice are challenged, the number of days before the mice are challenged is 0, and the starting date of stomach infusion is-14.

From the weight results, after the probiotic double-bacteria combined preparation was intragastrically, the weight gain was greater and the trend of rapid growth was maintained until the day of challenge (fig. 1).

TABLE 2 influence of probiotic double bacteria combination preparations on body weight of mice after influenza virus infection

Note that: days are days after the mice are challenged, the challenge current day is 0, and the ending date is 14.

From the weight change of mice after virus challenge, the weight of the mice reaches the lowest point on the 6 th day of development of influenza virus, the mice start to enter a recovery period after one week of virus infection, and the weight of the mice is greatly increased, which follows the development progress of influenza. The average body weight of the mice in the experimental group is not only higher than that of the mice in the control group, but also basically no reduction, so that the gastric lavage probiotic double bacteria combined preparation can obviously improve the reduction condition of the body weight of the mice (figure 2).

2. Effect of probiotic double bacteria combination formulation (Lac16+P118) on clinical score of mice

In addition to monitoring body weight, clinical performance of post-challenge mice was scored as follows: 5, the method comprises the following steps: health (no clinical symptoms), score 4: mild (slightly wrinkled fur); 3, the method comprises the following steps: moderate (moderate fold of fur, red nose), 2 points: severe (pulmonary breath sound, passive Mao Lingluan) and severe 1 score (no response to stimulus). The clinical status monitoring period was 14 days, starting the score from the day of challenge.

TABLE 3 Effect of probiotic double bacteria combination formulations on clinical scoring of mice after influenza infection

The clinical scores showed that clinical symptoms and body weight trends remained approximately consistent, mice recovered substantially normal after 12 days of virus infection, and the mice status of the probiotic group was better than the control group (table 3, fig. 3).

Experiment 2: effect of the probiotic double bacteria combination preparation prepared in example 1 on pulmonary virus titer of mice after influenza virus infection.

Based on the foregoing experimental results, a further explanation of how the probiotic double bacteria combination preparation (Lac16+P118) affects influenza infection follows. SPF mice are divided into two groups, a control group is filled with gastric PBS, and an experimental group is filled with a gastric probiotic double-bacteria combined preparation. Treatment group setup mode reference experiment 1.

Mice were sacrificed on day 3 and day 7 of challenge, and lung tissue and nasal washes were removed from the mice, respectively. The lung tissue was homogenized, the homogenized lung tissue solution was centrifuged at 3500rpm at 4℃for 10-15min, and both the supernatant and the nasal wash were stored in MEM medium (Gibco| Thermo Fisher Scientific) containing 2% BSA, and then the lung tissue was subjected to influenza virus content measurement using MDCK cells, and the titer measurement method was referred to Naoyoshi Maeda et al (2009).

The results of the test showed that the overall trend of the change in viral titer in the respiratory tract of mice was higher in early stage of infection (day 3) than in late stage of infection (day 7). Compared with PBS group, the lung virus titer of mice treated with lactobacillus double bacteria combination was significantly reduced (tables 4, 5, FIGS. 4, 5), and the lung virus titer results were consistent with those of rhinovirus titer results. The results show that the double-bacterium combination of the gastric lavage lactobacillus can effectively reduce the pulmonary virus content of mice, and the effect is more obvious in the early stage of influenza infection.

TABLE 4 mouse rhino-wash virus titer changes after lactobacillus parainfluenza infection

Note that: the unit of lung influenza virus content is lgTCID50/ml.

TABLE 5 changes in pulmonary viral titers in mice following infection with lactic acid bacteria

Note that: the unit of lung influenza virus content is lgTCID50/ml.

Description: lactobacillus plantarum (Lactobacillus plantarum) ACCC10141 is used for replacing lactobacillus plantarum Lac16 of the invention, lactobacillus rhamnosus (Lactobacillus rhamnosus) ACCC10534 is used for replacing lactobacillus rhamnosus P118 of the invention, and the rest are equivalent; namely, the probiotic double bacteria combined preparation (Lac16+P118) is changed to be prepared from Lactobacillus plantarum (Lactobacillus plantarum) ACCC10141 and Lactobacillus rhamnosus (Lactobacillus r)hamnosus) ACCC10534, and the concentration of the two bacteria in the double bacteria combination preparation a is 5×10 ⁸ CFU/ml. The results show that: no significant differences in viral titers were observed in the lungs of mice treated with the dual bacterial combination formulation a compared to the PBS group, either 3 days post-infection or 7 days post-infection.

Experiment 3: effect of the probiotic double-bacteria combined preparation prepared in example 1 on pathological changes in lung sections of mice after influenza virus infection.

Treatment group setup mode reference experiment 1.

Immediately after the mice were sacrificed on day0 and day 7 of challenge, the lung tissues of the mice were removed, fixed in 4% paraformaldehyde for more than 48 hours, and after removal of the prepared pathological paraffin sections and hematoxylin-eosin (HE) staining, the sections were finally observed under a microscope (fig. 6).

When the groups are compared, when the virus is not infected, the clear and complete alveolus outline, the trachea outline and the blood vessel outline of the two groups of lung sections can be seen, and symptoms such as inflammatory cell infiltration and the like are avoided. On day 7, the change of the lung condition of the control group is obviously seen, mainly including large-scale inflammatory cell infiltration of the lung, increased necrotic lesions, pulmonary congestion, substantial disappearance of alveoli and no clear visible lung structure; while the probiotic double bacteria combined preparation group has few lesions. The lung section staining result is consistent with the lung titer result, and the lactobacillus double-bacterium combination can be used for protecting the lung and reducing the occurrence of lung inflammation diseases in the process of infecting mice with influenza.

Experiment 4: effect of the probiotic double bacteria combination preparation prepared in example 1 on immune cells and pulmonary immune cytokines in mice after influenza virus infection.

Treatment group setup mode reference experiment 1.

And (3) a step of: influence of probiotic double-bacteria combined preparation on immune cells in mice after influenza virus infection

On day0, day 3, and day 7 of challenge, the mice were bled by orbital bleeding, and the collected blood was anticoagulated (EDTA). The peripheral blood is firstly subjected to erythrocyte lysis, is washed after the erythrocyte lysis is completed, is stained by adding an antibody, is then subjected to dead cell staining, and finally is subjected to on-machine detection. To maintain cell viability, the entire treatment process was performed in a low temperature environment at 4 ℃.

The results of flow cytometry show that after 2 weeks of feeding with the lactobacillus double bacteria combination, T cells in the mouse body are activated in advance, at CD45 ⁺ The ratio in cells was shown to be significantly higher relative to the control group and remained higher at the early stage of influenza infection (day 3) and fallen back at the late stage of infection (day 7) (fig. 7). Compared with a control group, NK cells can always keep a higher level in the body of the mouse (figure 7) as main killer cells in the virus infection process, which shows that the probiotic double-bacteria combined preparation can effectively excite the activity of immune cells in the body of the mouse, so that the body still keeps better immune defensive ability in the influenza infection process.

And II: effect of the probiotic double bacteria combined preparation on the mouse lung immunocytokine after H9N2 influenza virus infection.

On day 3 of challenge, after the mice were sacrificed by cervical scission, the mice lung tissue was removed intact, 1ml of sterile PBS was added to a 2ml EP tube, and homogenization conditions were: 55HZ, run time 25s, pause time 8s, run times 40 times. Centrifuging after homogenizing, wherein the centrifuging conditions are as follows: at 3500rpm,4℃for 10-15min, and the supernatant was discarded after centrifugation. The lung tissue was subjected to RNA extraction using Bioteke kit and fluorescent quantitative PCR (RT-qPCR) was used to determine whether the treatment with the lactobacillus combination during influenza infection induced a change in immunocytokines in vivo.

Six cytokines were tested this time, including: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interferon-stimulating factor-L (IFN-L2 & 3) and interferon-stimulating factor-gamma (IFN-gamma), and the related primer sequences are shown in Table 6.

TABLE 6 upstream and downstream primer sequences for immune cytokines related

The results of the measurement of cytokine expression levels in the lungs of mice showed that, on day 3 after infection with influenza virus, the cytokine content in vivo was somewhat reduced in mice treated with the lactobacillus double bacteria combination compared to the PBS control group, but was not significantly significant (p.gtoreq.0.05) in view of individual differences. Compared with the control group, the average value of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) of the lactobacillus bifidus group is reduced, and the average value of the whole group of interferon stimulating factors-L (IFN-L2 & 3), interferon stimulating factors-lambda (IFN-lambda) and tumor necrosis factor-alpha (TNF-alpha) is higher than that of the control group. In combination with the results of lung sectioning in example 4, it was readily found that a reduction in cytokine levels inhibited local inflammation and mediated antiviral effects required for influenza virus regression.

Therefore, the probiotic double bacteria combined preparation fed in the invention can assist in mobilizing the immune response of the organism so as to resist the invasion of influenza.

Experiment 5: effect of the probiotic double-bacteria combined preparation prepared in example 1 on the richness of the intestinal flora of mice before and after infection with H9N2 influenza virus.

Treatment group setup mode reference experiment 1.

On days 0, 3 and 7 of challenge, fresh cecal contents of mice were collected, stored in a-80 ℃ freezer, and sent to the relevant detection company for 16S measurement. The method comprises the steps of DNA extraction and quality inspection, PCR amplification and quality inspection, library construction and quality inspection, on-machine sequencing and the like from the step of receiving samples to the step of obtaining final sequencing data. Sequencing was performed using the PE250/PE150/PE300 mode of the Illumina Novaseq 6000/Miseq high throughput sequencing platform.

The returned data was analyzed based on the Qiime2 platform. FIGS. 10 and 11 are weighted-uniferac-empor analysis charts. Influenza infection reduces the richness of the mouse microbial flora, which is more pronounced as the infection progresses.

In order to classify the intestinal microbiota on Class (fig. 10), the species of the intestinal microbiota of mice was most abundant after 14 days of feeding the lactobacillus bifidus group (Day 0) compared to the control group, and the clostridium (Clostridia) of the lactobacillus bifidus group (LP) was significantly more than that of the control group, and the rhodobacter (coriobacteria), actinomycetes (actionobacteria) and Bacilli (bacili) were correspondingly more.

From the classification of the intestinal flora onto the Family (fig. 11), the abundance of Lachnospiraceae, corynebacteriaceae, eggerthellaceae and Micrococcaceae in the lac16+p118 group increased significantly. The abundance of Akkermansiaceae, clostridia _UCG-014 and Oscillospiraceae decreased on day 0. When mice were infected with influenza virus, the composition of the intestinal flora was changed compared to uninfected mice, showing a decrease in abundance on day 3 post infection. The original dominant families Lachnospiraceae, corynebacteriaceae, eggerthellaceae and the like all show descending trends, and Akkermansiaceae increases remarkably. The comprehensive intestinal flora structural change can show that the lactobacillus double-bacteria combination can regulate the intestinal flora richness of the organism before the infection of influenza, which proves that the probiotics in the invention can indirectly improve the intestinal flora richness in the organism.

After influenza infection, the number of probiotics in the intestinal canal of the mice in the control group is obviously reduced, the whole intestinal flora structure is disordered, the risk of intestinal diseases is increased, and the development of the organism disease is not facilitated. The dry prognosis of the lactobacillus and double bacteria combination obviously increases the abundance of intestinal probiotics, reduces the colonization of harmful bacteria, maintains the balance of intestinal bacteria, and improves the environment of the intestinal bacteria to indirectly play a role in antagonizing influenza.

Experiment 6: protection of mice after H1N1 influenza virus infection by the probiotic double bacteria combination preparation prepared in example 1.

And (3) a step of: effect of Lactobacillus bifidus combination on mice weight change and clinical State score

The mice used in this example were purchased from Shanghai Laek and were SPF-rated female BALB/c 4 weeks old. Mice were randomly divided into 2 groups, PBS control group (10) and lactobacillus double bacteria combination group (10) respectively. All SPF mice were kept in accordance with the conventional method of laboratory animal management at university of Zhejiang.

After the mice had stabilized in state, 100. Mu.l of the probiotic double bacteria combination preparation (1X 10) was administered to each mouse of the experimental group every other day for 2 weeks before challenge ⁹ CFU/ml bacterial liquid), each mouse of the control group was perfused with 100 μl of sterile PBS and subjected to nasal drip infection (virus concentration of 1×10) with influenza virus a/California/04/2009 (H1N 1) ⁵ TCID50/ml, 20 μl/dose, only once for nasal drip infection), when the infection was recorded as Day0, mice were observed for 14 consecutive days after challenge, body weights were measured and clinical manifestations were recorded (tables 7, 8, 9, FIGS. 12, 13), and clinical manifestation scoring criteria were the same as in experiment 1.

TABLE 7 Effect of Lactobacillus double combinations on mouse body weight after H1N1 influenza Virus infection

The mice reached their minimum body weight at day 7 post-infection, and gradually increased. The dual-strain combination of the gastric lavage lactobacillus can obviously improve the weight loss condition of Balb/c mice infected with H1N1 influenza virus, and the recovery is faster and better.

TABLE 8 Effect of lactic acid bacteria on clinical score of mice post H9N2 influenza virus infection

The clinical symptoms of BALB/c mice were more pronounced (fig. 14), and on day 7 post challenge, the control group was Mao Lingluan, disordered, listlessness, general weakness, redder nose with mucus attached. The mice with the probiotics double bacteria group are smooth, have better spirit, have obvious reaction actions after being stimulated, and have almost no symptoms of runny nose. Thus, the lactobacillus double-bacterium combination has the effect of improving the clinical symptoms of the BALB/c mice after influenza.

And II: influence of lactobacillus double-bacterium combination on pathological changes of lung sections of mice

Mice were sacrificed at day 7 post infection with cervical breaks, immediately removed from their lung tissue, fixed in 4% paraformaldehyde, after which pathological paraffin sections were prepared and hematoxylin-eosin (HE) stained and finally the sections were observed under a microscope (fig. 15).

Although there was also a different degree of inflammatory cell infiltration in the lactobacillus bifidus group (lac16+p118), there was more severe bleeding and inflammatory cell infiltration than in the PBS group, and normal alveolar tissue could not be seen. The results of this example are consistent with those of example 4, which demonstrates that the lactobacillus double bacteria combination of the present invention still has the effect of protecting the symptoms of pulmonary infection in mice infected with influenza after model replacement.

Thirdly,: influence of Lactobacillus double-bacteria combination on pulmonary viral titer of mice

Mice were sacrificed on day 3 of challenge and the whole lung tissue of the mice was removed. Lung tissue was homogenized, and lung tissue supernatant after homogenization was stored in MEM medium containing 2% BSA, and lung tissue influenza virus content was measured using MDCK cells, and the titer measurement method was the same as in example 3.

The pulmonary virus titer results (table 9, fig. 16) showed that the pulmonary virus titer of mice fed the lactobacillus bifidus combination was about 10-fold lower on the third day after infection compared to the control group, which also has a certain agreement with the results of example 3.

TABLE 9 changes in pulmonary viral titre in mice following infection with influenza virus by lactic acid bacteria

Note that: the unit of lung influenza virus content is lgTCID50/ml.

Therefore, all results of experiment 6 are consistent with the experimental results of C57BL/6 mice infected with H9N2, which shows that the lactobacillus double-strain combination still has the effect of preventing influenza virus infection after the model is replaced, and the lactobacillus double-strain combination has wider application significance.

Comparative experiment 1

The concentration obtained in example 1 was 1X 10 ⁹ CFU/ml of the rhamnose P118 bacterial suspension was centrifuged at 5000rpm at 4℃for 10 min, the supernatant was discarded, washed once with sterile PBS and finally brought to 1X 10 ⁹ The CFU/ml bacterial amount is resuspended in sterile PBS to obtain the rhamnose bacteria P118 bacterial agent.

The concentration obtained in example 1 was 1X 10 ⁹ CFU/ml Lactobacillus plantarum Lac16 bacterial suspension was centrifuged at 4℃and 5000rpm for 10 min, the supernatant was discarded, washed once with sterile PBS and finally 1X 10 ⁹ The CFU/ml bacterial amount is resuspended in sterile PBS to obtain the Lactobacillus plantarum Lac16 bacterial agent.

The probiotic double-bacteria combined preparation in the experiment 1-2 is changed into the rhamnose bacillus P118 single-bacteria preparation and the lactobacillus plantarum Lac16 single-bacteria preparation, and the rest refers to the experiment mode, and the comparison between the obtained result and the probiotic double-bacteria combined preparation is as follows:

and (3) a step of: effects of the rhamnose P118 single agent and the lactobacillus plantarum Lac16 single agent on the weight change and clinical status score of mice, respectively.

The mice used in this example were purchased from Shanghai Laek and were SPF-rated 4 week old female C57BL/6 mice. Mice were randomly divided into 3 groups, PBS control group (10), rhamnose bacillus P118 single bacterial group (10), lactobacillus plantarum Lac16 single bacterial group (10). All SPF mice were kept in accordance with the conventional method of laboratory animal management at university of Zhejiang.

After the mice had stabilized in state, 100. Mu.l of the Lactobacillus rhamnosus P118 monobacteria preparation, lactobacillus plantarum Lac16 monobacteria preparation (1×10) were administered to each mouse of the experimental group every other day for 2 weeks before challenge ⁹ CFU/ml of bacterial fluid), each mouse of the control group was perfused with 100 μl of PBS.

Nasal drop infection with influenza A/Mink/China/01/2014 (H9N 2) (virus concentration 1X 10) ⁷ TCID50/ml, 20 μl/dose, only once for nasal drip infection), infection when Day0 was recorded, mice status was observed 14 days after challenge, body weight was measured and clinical manifestations were recorded (Table 10, FIGS. 18, 19), clinical tablesThe scoring criteria are the same as in experiment 1.

TABLE 10 influence of P118, lac16 Single inoculum on body weight of mice after H9N2 influenza Virus infection

From the weight results, the Lac16 single bacterial agent and the P118 single bacterial agent with the same dosage are effective in promoting growth and resisting influenza. After 14 days of gastric lavage, the weight of the P118 single-strain mice increased more than that of the Lac16 single-strain mice, but the weights of the two single-strain mice increased more than that of the PBS group, but the mice were not significantly different from the PBS group (P > 0.05), which indicates that the single-strain growth-promoting effect is not as good as that of the lactobacillus mixed bacteria. After infection with H9N2 influenza virus, changes in mice body weight were monitored and found that the mice in both Lac16 and P118 monobacteria had a less severe body weight loss than PBS group, but remained insignificant (P > 0.05). After 8 days of challenge (Day 8), the mice body weight concluded that the recovery trend was greatly rising, indicating better prognosis. Therefore, it can be obtained that Lac16 single-bacterial agent and P118 single-bacterial agent, although having a certain protective effect on influenza-infected mice, are far less effective than the two-bacterial-mixed agent.

It should be noted that: the "SPF-class 4-week-old female C57BL/6 mice" used in this comparative experiment 1 was not the same lot as the "SPF-class 4-week-old female C57BL/6 mice" used in the above experiment 1, and thus PBS as a control was different from the above experimental results.

And II: the lactobacillus rhamnosus P118 single agent and the lactobacillus plantarum Lac16 single agent respectively affect the pulmonary virus titer of mice.

Treatment group setup was as described in experiments 1 and 2.

TABLE 11 changes in mouse rhinowash virus titer following P118, lac16 single agent convection virus infection

Note that: the unit of lung influenza virus content is lgTCID50/ml.

TABLE 12 pulmonary viral titer change in mice after P118, lac16 single agent convection virus infection

Note that: the unit of lung influenza virus content is lgTCID50/ml.

Nasal washes and lung tissue were collected from mice at day 3 and day 7 post infection and titers were determined. The results show that on day 3 after the challenge, the nasal wash and pulmonary virus titer of mice with the group of gastric lavage Lac16 single agent and P118 single agent are slightly reduced, the nasal wash titer is reduced by a factor of 0.5 (lg-0.3) and the pulmonary tissue titer is reduced by a factor of 0.5 without significance. On day 7 after challenge, lac16 single agent could still maintain virus titer levels lower than control (PBS), but the mouse virus titer of P118 single agent had not been much different from control (PBS).

Finally, it should be noted that the above list is only a few specific embodiments of the invention, and that many variations of the invention are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.

Claims

1. The probiotic double-bacteria combined preparation is characterized in that: consists of equal amounts of Lactobacillus plantarum (Lactobacillus plantarum) Lac16 and Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) P118.

2. The probiotic double bacteria combination preparation of claim 1 wherein: is prepared by suspending lactobacillus plantarum Lac16 and lactobacillus rhamnosus P118 in sterile PBS;

3. The probiotic double bacteria combination preparation of claim 2 wherein:

4. Use of a probiotic double bacteria combination preparation according to claims 1-3 for the preparation of a medicament for the treatment or prevention of avian influenza virus infection.