WO2020133805A1 - 一种具有抗流感能力的益生菌混合制剂及其应用 - Google Patents

一种具有抗流感能力的益生菌混合制剂及其应用 Download PDF

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WO2020133805A1
WO2020133805A1 PCT/CN2019/082656 CN2019082656W WO2020133805A1 WO 2020133805 A1 WO2020133805 A1 WO 2020133805A1 CN 2019082656 W CN2019082656 W CN 2019082656W WO 2020133805 A1 WO2020133805 A1 WO 2020133805A1
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influenza
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bifidobacterium breve
ccfm1026
ccfm1025
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French (fr)
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陈卫
陆文伟
翟齐啸
刘馨阳
赵建新
杭锋
张灏
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/519—Breve
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the invention relates to a probiotic mixed preparation with anti-influenza ability and its application, belonging to the technical field of microbes and the technical field of medicine.
  • Influenza is often pandemic in autumn and winter, mainly caused by influenza virus.
  • Influenza viruses can be divided into three types: influenza A virus, influenza B virus, and influenza C virus.
  • Human influenza is mainly caused by influenza A virus and influenza B virus.
  • animal influenza viruses do not infect humans, and human influenza viruses do not infect animals, but pigs are the exception.
  • Pigs can be infected with avian influenza virus as well as human influenza virus, but they are still mainly infected with avian influenza virus. However, once they are infected with avian influenza virus, they can be easily transmitted to people and cause human influenza pandemic, for example, 1819-1920 The "Spanish flu" that occurred in 2000.
  • “Spanish flu” is the most severe pandemic in the history of the world, involving a wide range, with a clinical incidence of up to 40%. It is accompanied by various types of pneumonia complications, resulting in the death of 20-40 million people, the death toll is far Beyond World War I. Due to the limitations of scientific and technological conditions, people were unable to isolate the causative agent of "Spanish flu” at that time. Until 1997, American scientists published in Science that the 1918 flu virus was very similar to the swine flu virus. A virus closely related to influenza A virus (H1N1).
  • pandemic influenzas still broke out worldwide, resulting in different levels of human suffering and economic losses. It can be said that since the emergence of influenza, it has been unable to fully control, showing an intermittent outbreak.
  • influenza vaccines that have been put on the market are inactivated influenza vaccine (TIV) and attenuated live vaccine (LAIV). They are composed of three viruses, including two influenza A virus strains and one influenza B virus Strain. There are two main types of western medicine for the treatment of related influenza infections. One is neuraminidase inhibitors such as oseltamivir, zanamivir, and paramivir. The mechanism is through the glycoprotein nerve that acts on the surface of the virus.
  • Aminase prevents virus particles from invading human cells; the other is M2 ion channel blockers such as amantadine and amantadine. These drugs act on the proton channel M2 protein by blocking its protein ion channel to Inhibit the replication of influenza A virus.
  • vaccine injection cannot effectively protect the body from virus infection for a long time, and drug treatment has side effects on the central nervous system while killing the virus. Therefore, there is still a need for a drug or treatment that can effectively protect the body from influenza virus infection for a long time, and can also relieve some clinical symptoms of influenza, and at the same time, will not bring side effects to the central nervous system of patients.
  • intestinal microbes have an important role in maintaining human health.
  • the health effects of probiotics in human intervention studies include improving children's acute diarrhea, alleviating children's milk allergies, atopic dermatitis, and alleviating people.
  • Irritable bowel syndrome, and probiotics may affect the intestinal mucosa, balance local microbiota by inhibiting the growth of pathogenic microorganisms, thereby enhancing local and systemic immune responses.
  • probiotics may also affect the intestinal contents.
  • the composition and activity of the microbiome it has also been reported that influenza caused by viruses can affect the structure of the intestinal flora, and specific probiotics can effectively reduce the duration and severity of acute rotavirus gastroenteritis. Therefore, it may be possible to start with intestinal microbes and try to find new drugs or new methods for preventing and treating influenza to overcome the shortcomings of existing treatment drugs and treatment methods with obvious side effects.
  • the present invention provides a probiotic mixed preparation containing Lactobacillus mucosae CCFM1025 and Bifidobacterium breve CCFM1026.
  • This probiotic mixed preparation has an anti-influenza effect, specifically embodied in: (1) significantly improve the weight loss of influenza mice; (2) significantly improve the blood indicators of influenza mice; (3) significantly improve the respiratory infection inflammation of influenza mice Status; (4) Significantly reduce the viral load of the lungs of influenza mice (ie significantly inhibit the replication and value-added of influenza virus in influenza mice); (5) significantly increase the expression of antiviral protein MxA in the lungs of influenza mice, Therefore, this probiotic mixed preparation has great application prospects in the preparation of products for the prevention and/or treatment of atopic dermatitis and even the prevention and/or treatment of influenza.
  • the invention provides a probiotic mixed preparation with anti-influenza ability, the probiotic mixed preparation containing Lactobacillus mucosae CCFM1025 and Bifidobacterium breve CCFM1026;
  • the Lactobacillus mucosae CCFM1025 was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 11, 2018.
  • the deposit number is GDMCC No. 60460, and the deposit address is Building 59, Courtyard 100, Xianlie Middle Road, Guangzhou 5th Floor;
  • the Bifidobacterium breve CCFM1026 has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 11, 2018.
  • the deposit number is GDMCC No. 60459, and the deposit address is No. 59, Courtyard 100, Xianlie Middle Road, Guangzhou Floor 5th floor.
  • the Lactobacillus mucosae CCFM1025 was isolated from human feces. The strain was sequenced and analyzed. Its 16S rRNA sequence is shown in SEQ ID NO.1. The sequence was compared in GenBank and the results showed The strain is Lactobacillus mucosa, named Lactobacillus mucosae CCFM1025.
  • Lactobacillus mucosae CCFM1025 are short rod-shaped; the colonies are round, rough, and transparent; they grow at 45°C and do not grow at 15°C; they are grown in MRS liquid medium at 37°C for 12 hours During the stable period, heterogeneous fermentation produces acid and gas from glucose.
  • the Bifidobacterium breve CCFM1026 was isolated from human fecal samples. The strain was sequenced and analyzed. Its 16S rRNA sequence is shown in SEQ ID NO. 2, and the sequence was aligned in GenBank. The results showed that the coverage rate between the strain and Bifidobacterium breve was 100%, and the similarity (Ident) was 99%. Therefore, the strain was determined to be Bifidobacterium breve and named as Bifidobacterium breve CCFM1026.
  • the cells of the Bifidobacterium breve CCFM1026 are short rod-shaped, with positive Gram staining, irregular methylene blue staining, no spores, flagella and capsules, and no movement; colonies are round white; at 37°C Incubation for 30 h under anaerobic conditions can reach a stable period, and glucose is used for atypical hetero-lactic fermentation.
  • the number of viable bacteria in Lactobacillus mucosae CCFM1025 in the probiotic mixed preparation is not less than 1 ⁇ 10 6 CFU/mL; in the probiotic mixed preparation, short bifid
  • the number of viable bacteria of Bifidobacterium breve CCFM1026 is not less than 1 ⁇ 10 6 CFU/mL.
  • the invention provides the application of the probiotic mixed preparation in the preparation of products for preventing and/or treating influenza.
  • the number of viable bacteria of Lactobacillus mucosae CCFM1025 is not less than 1 ⁇ 10 6 CFU/mL, and the viable bacteria of Bifidobacterium breve CCFM1026 The number is not less than 1 ⁇ 10 6 CFU/mL.
  • the product comprises food, medicine or health food.
  • the medicine contains a probiotic mixed preparation, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
  • the food includes dairy products, soy products, or fruit and vegetable products produced using a starter containing a probiotic mixed preparation; or the food includes a solid beverage containing a probiotic mixed preparation.
  • the present invention provides a product for preventing and/or treating influenza, which contains the above-mentioned probiotic mixed preparation with anti-influenza capability.
  • the number of viable bacteria of Lactobacillus mucosae CCFM1025 is not less than 1 ⁇ 10 6 CFU/mL, and the viable bacteria of Bifidobacterium breve CCFM1026 The number is not less than 1 ⁇ 10 6 CFU/mL.
  • the product comprises food, medicine or health food.
  • the medicine contains a probiotic mixed preparation, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
  • the food includes dairy products, soy products, or fruit and vegetable products produced using a starter containing a probiotic mixed preparation; or the food includes a solid beverage containing a probiotic mixed preparation.
  • the preparation method of the starter is to inoculate (Bifidobacterium breve) CCFM1026 into the medium at an inoculation amount of 5-8% of the total mass of the medium, in an anaerobic environment at 37°C Incubate for 30 hours to obtain the culture solution; centrifuge the culture solution to obtain the bacterial cells; wash the bacterial cells with phosphate buffer pH 7.2 for 2 to 4 times, and then resuspend them with a trehalose lyoprotectant containing 100g/L. Resuspended liquid was obtained; the resuspended liquid was freeze-dried by vacuum freezing to obtain (Bifidobacteriumbreve) CCFM1026 powder;
  • Lactobacillus mucosae CCFM1025 was inoculated into the medium at an inoculation amount of 5-8% of the total mass of the medium, and cultured at 37°C for 18h to obtain a culture solution; the culture solution was centrifuged to obtain bacterial cells; The body is washed with phosphate buffer pH 7.2 for 2 to 4 times and then resuspended with a trehalose lyoprotectant containing 100 g/L to obtain a resuspended solution; the resuspended solution is freeze-dried in a vacuum freeze method to obtain Lactobacillus mucosa (Lactobacillus mucosae) CCFM1025 bacterial powder;
  • the mass ratio of the freeze-dried protective agent to the bacterial cells is 2:1.
  • the pH of the culture medium is 6.8.
  • the protective agent comprises 100 g/L skim milk powder, 150 g/L trehalose, and 10 g/L sodium L-glutamate.
  • the present invention provides a probiotic mixed preparation containing Lactobacillus mucosae CCFM1025 and Bifidobacterium breve CCFM1026.
  • the probiotic mixed preparation has an anti-influenza effect, which is specifically embodied in:
  • this probiotic mixed preparation has great application prospects in the preparation of products for the prevention and/or treatment of atopic dermatitis and even the prevention and/or treatment of influenza.
  • the deposit number is GDMCC No. 60460, and the deposit address is Guangzhou Martyr. 5th Floor, Building 59, Courtyard 100, Middle Road.
  • Figure 1 Comparison of body weight changes of influenza mice in different groups (Lactobacillus mucosa).
  • Figure 2 Comparison of blood test indexes (neutrophils) of influenza mice of different groups (Lactobacillus mucosa).
  • FIG. 3 Comparison of blood test indexes (lymphocytes) of influenza mice in different groups (Lactobacillus mucosa).
  • Figure 4 Comparison of lung histopathological sections of influenza mice of different groups (Lactobacillus mucosa).
  • FIG. 5 Comparison of lung viral load in different groups of influenza mice (Lactobacillus mucosa).
  • Figure 6 Comparison of body weight changes of influenza mice in different groups (Bifidobacterium breve).
  • Figure 7 Comparison of blood test indexes (neutrophils) of influenza mice of different groups (Bifidobacterium breve).
  • Figure 8 Comparison of blood test indexes (lymphocytes) of influenza mice of different groups (Bifidobacterium breve).
  • Figure 9 Comparison of lung histopathological sections of influenza mice of different groups (Bifidobacterium breve).
  • Figure 10 Comparison of the expression levels of antiviral protein MxA in the lungs of influenza mice of different groups (Bifidobacterium breve).
  • Figure 11 Comparison of body weight changes of different groups of influenza mice (mixed bacteria).
  • Figure 12 Comparison of blood test indexes (neutrophils) of influenza mice in different groups (mixed bacteria).
  • FIG. 13 Comparison of blood test indexes (lymphocytes) of influenza mice in different groups (mixed bacteria).
  • Figure 14 Comparison of lung histopathological sections of different groups of influenza mice (mixed bacteria).
  • Figure 15 Comparison of lung viral load in different groups of influenza mice (mixed bacteria).
  • Figure 16 Comparison of the expression levels of antiviral protein MxA in the lungs of influenza mice of different groups (mixed bacteria).
  • MRS plate (g/L): peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium citrate 2g/L, K 2 PO 4 ⁇ 3H 2 O 2.6g/L, MgSO 4 ⁇ 7H 2 00.1g/L, MnSO 4 0.05g/L, Tween 801ml/L, agar 20g/L, cysteine amino acid salt 0.5g/L.
  • Example 1-1 Screening and identification of Lactobacillus mucosa
  • the sample is pretreated and stored in a refrigerator at -80°C in about 20% glycerol. After taking out the thaw, mix the sample and draw 0.5mL of the sample to 4.5mL to contain 0.05% cysteine 0.9% physiological saline for gradient dilution, select the appropriate gradient dilution and spread on MRS plate supplemented with 0.05% cysteine, incubate at 37°C for 48h, pick typical colonies to streak on MRS plate for purification, select Take a single colony and transfer to liquid MRS medium (containing 0.05% cysteine) to enrich the bacteria and store it with 30% glycerol to obtain strain CCFM1025 and strain F1.
  • liquid MRS medium containing 0.05% cysteine
  • CCFM1025 and F1 The genomes of CCFM1025 and F1 were extracted, and the 16S rDNA of CCFM1025 and F1 were amplified and sequenced (Shanghai Biotech Engineering Co., Ltd.), and the sequences were compared in GenBank. The results showed that the strain was Lactobacillus mucosa, named Lactobacillus mucosae CCFM1025 and Lactobacillus mucosae F1.
  • Example 1-2 Culture of Lactobacillus mucosa
  • Lactobacillus mucosae CCFM1025 was inserted into MRS medium (containing 0.05% cysteine) and cultured at 37°C for 48h to produce a growth curve. It was found that it was cultured at 37°C for 12h to reach a stable period. It is heterogeneous fermentation, which can produce acid and gas from glucose.
  • Lactobacillus mucosae CCFM1025 was inserted into the MRS medium (containing 0.05% cysteine) and cultured at 10, 15, 20, 25, 30, 35, 40, 45, and 50°C for 48 hours and observed.
  • the growth condition is good at 20 ⁇ 35°C. It can still grow at 45°C, but it hardly grows at or below 15°C or 50°C.
  • Example 1-3 Effect of Lactobacillus mucosa on body weight of influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups.
  • the 5 groups were named as: blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) , Lactobacillus mucosa intervention group (CCFM1025), intragastric administration of Lactobacillus mucosa CCFM1025, Lactobacillus mucosa intervention group (F1), intragastric administration of Lactobacillus mucosa F1, 8 in each group.
  • Control blank control group
  • Model influenza model group
  • Tuatment ribavirin-administered treatment group
  • F1 Lactobacillus mucosa intervention group
  • F1 intragastric administration of Lactobacillus mucosa F1, 8 in each group.
  • the L. mucosa intervention group (CCFM1025) was gavaged daily with 10 9 CFU of Lactobacillus mucosa CCFM1025 bacterial suspension dilution, and the L. mucosa F1 intervention group (F1) was gavaged with the same amount of L. mucosa F1 Bacterial suspension dilution, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the same amount was given by L. mucosa F1 intervention group (F1)
  • the L. mucosa F1 bacterial suspension dilution, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • Lactobacillus mucosa CCFM1025 of the present invention can significantly improve the weight loss symptoms of influenza mice.
  • Example 1-4 Effect of Lactobacillus mucosa on blood indexes of influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups.
  • the 5 groups were named as: blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) 8.
  • Lactobacillus mucosa intervention group (CCFM1025), intragastric administration of Lactobacillus mucosa, Lactobacillus mucosa intervention group (F1), intragastric administration of Lactobacillus mucosa F1, 8 in each group.
  • the L. mucosa intervention group (CCFM) was intragastrically fed with 10 9 CFU of Lactobacillus mucosa bacterial suspension dilution
  • the L. mucosa F1 intervention group (F1) was intragastrically fed with the same amount of L. mucosa F1 bacteria Suspend the diluent, and the remaining groups (Control, Model, Treatment) are intragastrically fed with 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Lactobacillus mucosa intervention group (CCFM) was gavaged with 10 9 CFU of Lactobacillus mucosa suspension suspension, and the L. mucosa F1 intervention group (F1) was gavaged with the same amount. Lactobacillus mucosa F1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • mice On the 5th day after the challenge, the mice were sacrificed after blood collection. The blood of the removed mice was placed in an anticoagulant tube and gently shaken to make the blood fully contact with the anticoagulant, and then sent to the animal hospital for routine blood analysis and detection (In the early stage of influenza virus infection, a large number of natural immune cells, such as neutrophils and lymphocytes, will participate in the defense process. Therefore, blood routine analysis and detection focuses on the changes of neutrophils and lymphocytes). The results are shown in Figure 2-3. .
  • Lactobacillus mucosa CCFM1025 of the present invention can exert the same immune regulation function on the body.
  • Example 1-5 Effect of Lactobacillus mucosa on inflammation of respiratory tract infection in influenza mice
  • mice weighing 20-24g were randomly divided into 4 groups.
  • the 4 groups were named as: blank control group (Control), influenza model group (Model), drug treatment group (Reatment) administered with ribavirin ), L. mucosa intervention group (CCFM), L. mucosa intragastric administration, L. mucosa intervention group (F1), L. mucosa intragastric administration F1, 8 per group.
  • the L. mucosa intervention group (CCFM) was intragastrically fed with 10 9 CFU of Lactobacillus mucosa bacterial suspension dilution
  • the L. mucosa F1 intervention group (F1) was intragastrically fed with the same amount of L. mucosa F1 bacteria Suspend the diluent, and the remaining groups (Control, Model, Treatment) are intragastrically fed with 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Lactobacillus mucosa intervention group (CCFM) was gavaged with 10 9 CFU of Lactobacillus mucosa suspension suspension, and the L. mucosa F1 intervention group (F1) was gavaged with the same amount.
  • Lactobacillus mucosa F1 suspension suspension, blank control group and influenza model group (Control, Model) were intragastrically administered with 0.2 mL of normal saline.
  • the lung tissue of the blank group (Control) mice is relatively complete and there is no infiltration of inflammatory cells; the model group (Model) mice have large-scale inflammation characterization and even hyperemia; the treatment group (Treatment) and mucosal milk
  • the lungs of mice in the Bacillus intervention group (CCFM1025) group were slightly infiltrated by inflammatory cells, which occurred near the bronchus.
  • influenza infection can cause mice to suffer from influenza pneumonia.
  • the tissue structure is destroyed and a large amount of inflammation and infiltration occur; and the Lactobacillus mucosa CCFM1025 of the present invention can relieve lung inflammation in mice
  • the condition, alleviating its pneumonia, has the same effect as the ribavirin drug treatment group commonly used for influenza.
  • Example 1-6 Effect of Lactobacillus mucosa on lung viral load in influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups.
  • the 5 groups were named as: blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) 8.
  • Lactobacillus mucosa intervention group (CCFM1025), intragastric administration of Lactobacillus mucosa, Lactobacillus mucosa intervention group (F1), intragastric administration of Lactobacillus mucosa F1, 8 in each group.
  • the L. mucosa intervention group (CCFM) was intragastrically fed with 10 9 CFU of Lactobacillus mucosa bacterial suspension dilution
  • the L. mucosa F1 intervention group (F1) was intragastrically fed with the same amount of L. mucosa F1 bacteria Suspend the diluent, and the remaining groups (Control, Model, Treatment) are intragastrically fed with 0.2 mL of normal saline every day.
  • Lactobacillus mucosa intervention group (CCFM) was gavaged with 10 9 CFU of Lactobacillus mucosa suspension suspension, and the L. mucosa F1 intervention group (F1) was gavaged with the same amount. Lactobacillus mucosa F1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • mice On the 5th day after the challenge, blood was taken to kill the mice. After the mice were sacrificed, the right lung tissues of the mice were placed in 1 mL of TRIZOL, frozen and stored in a refrigerator at -80°C. The right lung tissue samples were placed on ice during extraction After the upper melt, use a DEPC-treated sterile grinder to grind, add 200 ⁇ L of chloroform, mix thoroughly, centrifuge at 4°C, 12000rpm for 10min, remove the supernatant 500 ⁇ L, add an equal volume of isopropanol, mix , Centrifuge at 4°C and 12000rpm for 10min, remove the supernatant, add 75% ethanol to wash the RNA once, and centrifuge at 4°C and 12000rpm for 10min. After removing the supernatant, wait for the alcohol to evaporate and dry, add 40 ⁇ L of DEPC treated water to dissolve RNA, and the extracted RNA is stored in the refrigerator at -80°C for later use.
  • the viral load was determined by qPCR method, GAPDH was used as internal reference, the classic 2- ⁇ t calculation method was used, and the model group was used as the comparative processing data. The results are shown in Figure 5.
  • the viral load in the lungs of the mice in the drug treatment group is 38.03% of the model group, while the viral load in the lungs of the mice in the intervention group (CCFM1025) of the present invention is only the model group. 16.30%.
  • Example 2-1 Screening and identification of Bifidobacterium breve
  • the sample is pretreated and stored in a refrigerator at -80°C in about 20% glycerol. After taking out the thaw, mix the sample and draw 0.5mL of the sample to 4.5mL to contain 0.05% cysteine 0.9% physiological saline for gradient dilution, select the appropriate gradient dilution and spread on MRS plate supplemented with 0.05% cysteine, incubate at 37°C for 48h, pick typical colonies to streak on MRS plate for purification, select Take a single colony and transfer to liquid MRS medium (containing 0.05% cysteine) to enrich the bacteria, and save it with 30% glycerol to obtain strain CCFM1026 and strain B1.
  • the genomes of CCFM1026 and B1 were extracted, and the 16S rDNA of CCFM1026 and B1 were amplified and sequenced (Shanghai Biotech Engineering Co., Ltd.). The sequences were compared in GenBank. The results showed that the strains were covered with Bifidobacterium breve Query cover is 100% and similarity (Ident) is 99%, so the strain is determined to be Bifidobacterium breve, named Bifidobacterium breve CCFM1026 and Bifidobacterium breve B1 .
  • Example 2-2 Cultivation of Bifidobacterium breve
  • Bifidobacterium breve CCFM1026 was inserted into MRS solid medium (containing 0.05% cysteine) and cultured at 37°C for 48h. The colonies were observed and found to be round white;
  • the Bifidobacterium breve CCFM1026 was inserted into MRS liquid medium (containing 0.05% cysteine) and cultured at 37°C for 48h. The growth curve was made, and it was found that the culture was stable under anaerobic conditions at 37°C for 30h. In this period, glucose was used for atypical hetero-lactic fermentation.
  • Example 2-3 Effect of Bifidobacterium breve on body weight of influenza mice
  • mice weighing 20-24g were randomly divided into 4 groups.
  • the 4 groups were named as: blank control group (Control), influenza model group (Model), drug treatment group (Reatment) administered with ribavirin ), Gavage Bifidobacterium breve CCFM1026 intervention group (CCFM1026), Gavage Bifidobacterium breve B1 intervention group (group B1), 8 per group.
  • the Bifidobacterium breve intervention group (CCFM1026) was gavaged with 10 9 CFU of Bifidobacterium breve suspension suspension every day, and the Bifidobacterium breve B1 intervention group (B1) was gavaged with the same number of short pairs Bifidobacterium B1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Bifidobacterium breve (CCFM1026) was gavaged with 10 9 CFU of Bifidobacterium breve CCFM1026 suspension suspension, and the Bifidobacterium breve B1 intervention group (B1) was also gavaged. Amounts of Bifidobacterium breve B1 suspension suspension, and the remaining groups (Control, Model, Treatment) were intragastrically administered with 0.2 mL of normal saline every day.
  • Example 2-4 The effect of Bifidobacterium breve on blood indexes of influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups.
  • the 5 groups were named as: blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) , Gavage Bifidobacterium breve CCFM1026 intervention group (CCFM1026), Gavage Bifidobacterium breve B1 short intervention group (group B1), 8 in each group.
  • the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was fed with 10 9 CFU of Bifidobacterium breve suspension suspension every day, and the Bifidobacterium breve B1 intervention group (B1) was given the same amount of short Bifidobacterium B1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Bifidobacterium breve (CCFM1026) was gavaged with 10 9 CFU of Bifidobacterium breve CCFM1026 suspension suspension, and the Bifidobacterium breve B1 intervention group (B1) was also gavaged. Amounts of Bifidobacterium breve B1 suspension suspension, and the remaining groups (Control, Model, Treatment) were intragastrically administered with 0.2 mL of normal saline every day.
  • the Bifidobacterium breve of the present invention has the same effect as the ribavirin medicine, and can actively participate in the influenza-related immune regulation of mice to relieve the flu symptoms of mice.
  • Example 2-5 Effect of Bifidobacterium breve on respiratory tract infection and inflammation in influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups, and the 5 groups were named as blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment).
  • Control blank control group
  • Model influenza model group
  • Tuatment ribavirin-administered treatment group
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026
  • Bifidobacterium breve B1 short intervention group B1 group
  • the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was fed with 10 9 CFU of Bifidobacterium breve suspension suspension every day, and the Bifidobacterium breve B1 intervention group (B1) was given the same amount of short Bifidobacterium B1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Bifidobacterium breve (CCFM1026) was gavaged with 10 9 CFU of Bifidobacterium breve CCFM1026 suspension suspension, and the Bifidobacterium breve B1 intervention group (B1) was also gavaged. Amounts of Bifidobacterium breve B1 suspension suspension, and the remaining groups (Control, Model, Treatment) were intragastrically administered with 0.2 mL of normal saline every day.
  • FIG. 9 the lung tissue of the control group mice is relatively complete without infiltration of inflammatory cells; the model group mice have severe inflammatory infiltration and even local bleeding; Bifidobacterium breve B1 intervention Moderate inflammation infiltration occurred near the bronchi in group (B1), while the lung inflammation in the treatment group (Treatment) and Bifidobacterium breve intervention group (CCFM1026) was relatively mild.
  • influenza infection can cause mice to suffer from influenza pneumonia.
  • the tissue structure is destroyed and a large amount of inflammation and infiltration occurs; and the bifidobacterium brevis of the present invention can relieve lung inflammation in mice
  • the condition, alleviating its pneumonia, has the same effect as the ribavirin drug treatment group commonly used for influenza.
  • Example 2-6 Effect of Bifidobacterium breve on lung viral load in influenza mice
  • mice weighing 20-24g were randomly divided into 5 groups.
  • the 5 groups were named as: blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) , Gavage Bifidobacterium breve CCFM1026 intervention group (CCFM1026), Gavage Bifidobacterium breve B1 short intervention group (group B1), 8 in each group.
  • the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was fed with 10 9 CFU of Bifidobacterium breve suspension suspension every day, and the Bifidobacterium breve B1 intervention group (B1) was given the same amount of short Bifidobacterium B1 suspension suspension, the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Bifidobacterium breve (CCFM1026) was gavaged with 10 9 CFU of Bifidobacterium breve CCFM1026 suspension suspension, and the Bifidobacterium breve B1 intervention group (B1) was also gavaged. Amounts of Bifidobacterium breve B1 suspension suspension, and the remaining groups (Control, Model, Treatment) were intragastrically administered with 0.2 mL of normal saline every day. On the 5th day after the challenge, blood was taken and the mice were sacrificed.
  • mice After the mice were sacrificed, the right lung tissues of the mice were placed in 1ml of TRIZOL, frozen and stored in a refrigerator at -80°C, and the right lung tissue samples were placed on ice during extraction
  • a sterile grinder treated with DEPC add 200ul of chloroform, mix thoroughly, centrifuge at 4°C, 12000rpm for 10min, remove 500 ⁇ L of supernatant, add an equal volume of isopropanol, mix well , Centrifuge at 4°C, 12000rpm for 10min, remove the supernatant, add 75% ethanol to wash the RNA once, and centrifuge at 4°C, 12000rpm for 10min.
  • After removing the supernatant after the alcohol has evaporated to dryness, add 40 ⁇ L of DEPC treated water to dissolve RNA, and the extracted RNA is stored in the refrigerator at -80°C for later use.
  • the MxA expression in the lungs of the mice in the medication group (Treatment) is 2.62 times that of the model group (Model)
  • the MxA expression in the lungs of the mice in the Bifidobacterium breve B1 intervention group is 2.67 in the model group (Model)
  • CCFM1026 the expression of MxA in the lungs of mice in the Bifidobacterium breve intervention group (CCFM1026) was 3.46 times that of the model group (Model), which could significantly increase the expression of MxA (p value 0.0292).
  • the above experiments show that the Bifidobacterium breve CCFM1026 of the present invention can significantly enhance the immune ability of influenza mice, and promote the increase of its MxA antiviral protein expression, thereby combating the replication of the virus, helping the body to recover, the respiratory tract inflammation is significantly reduced, and the effect is even superior Used for medical treatment.
  • Example 3-1 Effect of mixed bacteria on the body weight of influenza mice
  • mice weighing 20-24g were randomly divided into 6 groups, and the 6 groups were named as: blank control group (Control), influenza model group (Model), treatment group administered with ribavirin (Treatment) , Lactobacillus mucosa intragastric administration group (CCFM1025), Bifidobacterium breve CCFM1026 intervention group (CCFM1026), and probiotic mixed preparation group intragastric administration group (CCFM1025+1026), each group 8 pcs.
  • Control blank control group
  • Model influenza model group
  • Treatment group administered with ribavirin Treatment group administered with ribavirin
  • Tratment Treatment group administered with ribavirin
  • CCFM1025 Lactobacillus mucosa intragastric administration group
  • CCFM1026 intervention group CCFM1026 intervention group
  • probiotic mixed preparation group intragastric administration group CCFM1025+1026
  • CCFM1025 Lactobacillus mucosa intervention group
  • CCFM1026 intervention group CCFM1026
  • the probiotic intervention group (CCFM1025+1026) of the probiotic mixed preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacteria
  • the amount of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • CCFM1025 gavage of 10 9 CFU bacterial suspension of Lactobacillus mucosa CCFM1025 bacterial suspension
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026 gavage of 10 9 CFU bacterial suspension of Bifidobacterium breve CCFM1026 bacterial suspension, mixed with probiotics
  • the bacteria intervention group (CCFM1025+1026) of the preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacterial amount of L.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Gavage was continued 4 days after challenge, in which the Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the Bifidobacterium breve CCFM1026 intervention group (CCFM1026) was gavaged 10 9 Bifidobacterium breve CCFM1026 suspension suspension of CFU bacteria, probiotic mixed preparation group (CCFM1025+1026) gavage 10 9 Probiotics mixed preparation of CFU bacteria (probiotic mixed preparation contains 5 ⁇ 10 8 CFU Lactobacillus bacteria amount mucosa CCFM1025,5 ⁇ 10 8 CFU Bifidobacterium breve strain amount CCFM1026), the remaining group (Control, Model, Treatment) 0.2mL of saline per day orally.
  • the Lactobacillus mucosa intervention group CCFM1025
  • Example 3-2 Effect of mixed bacteria on blood indexes of influenza mice
  • mice weighing 20-24g were randomly divided into 6 groups, and the 6 groups were named as: blank control group (Control), influenza model group (Model), treatment group administered with ribavirin (Treatment) , Lactobacillus mucosa intragastric administration group (CCFM1025), Bifidobacterium breve CCFM1026 intervention group (CCFM1026), and probiotic mixed preparation group intragastric administration group (CCFM1025+1026), each group 8 pcs.
  • Control blank control group
  • Model influenza model group
  • Treatment group administered with ribavirin Treatment group administered with ribavirin
  • Tratment Treatment group administered with ribavirin
  • CCFM1025 Lactobacillus mucosa intragastric administration group
  • CCFM1026 intervention group CCFM1026 intervention group
  • probiotic mixed preparation group intragastric administration group CCFM1025+1026
  • CCFM1025 Lactobacillus mucosa intervention group
  • CCFM1026 intervention group CCFM1026
  • the probiotic intervention group (CCFM1025+1026) of the probiotic mixed preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacteria
  • the amount of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • CCFM1025 gavage of 10 9 CFU bacterial suspension of Lactobacillus mucosa CCFM1025 bacterial suspension
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026 gavage of 10 9 CFU bacterial suspension of Bifidobacterium breve CCFM1026 bacterial suspension, mixed with probiotics
  • the bacteria intervention group (CCFM1025+1026) of the preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacterial amount of L.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Gavage was continued 4 days after challenge, in which the Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the Bifidobacterium breve CCFM1026 intervention group (CCFM1026) was gavaged 10 9 Bifidobacterium breve CCFM1026 suspension suspension of CFU bacteria, probiotic mixed preparation group (CCFM1025+1026) gavage 10 9 Probiotics mixed preparation of CFU bacteria (probiotic mixed preparation contains 5 ⁇ 10 8 CFU Lactobacillus bacteria amount mucosa CCFM1025,5 ⁇ 10 8 CFU Bifidobacterium breve strain amount CCFM1026), the remaining group (Control, Model, Treatment) 0.2mL of saline per day orally.
  • the Lactobacillus mucosa intervention group CCFM1025
  • mice On the 5th day after the challenge, the mice were sacrificed after blood collection. The blood of the removed mice was placed in an anticoagulant tube and gently shaken to make the blood fully contact with the anticoagulant, and then sent to the animal hospital for routine blood analysis and detection (In the early stage of influenza virus infection, a large number of natural immune cells, such as neutrophils and lymphocytes, will participate in the defense process. Therefore, blood routine analysis and detection will focus on the changes of neutrophils and lymphocytes. The results are shown in Figure 12-13. .
  • the probiotic mixed preparation group (CCFM1025+1026) tends to be slightly stronger than the blank control group (Control) than the two single bacteria (CCFM1025, CCFM1026)
  • the group shows that the probiotics ingested by the mice can participate in the immune regulation of the body, assist the body to jointly resist the invasion of influenza virus, and maintain the health of the body, and the effect of the mixed bacteria preparation is better than that of the single bacteria.
  • Example 3-3 Effects of mixed bacteria on respiratory tract infection and inflammation in influenza mice
  • mice weighing 20-24g were randomly divided into 6 groups, which were named as blank control group (Control), influenza model group (Model), and ribavirin-administered treatment group (Treatment) , Lactobacillus mucosa intragastric administration group (CCFM1025), Bifidobacterium breve CCFM1026 intervention group (CCFM1026), probiotic mixed gavage group bacterial intervention group (CCFM1025+1026), each group 8 pcs.
  • Control blank control group
  • Model influenza model group
  • Tuatment ribavirin-administered treatment group
  • CCFM1025 Lactobacillus mucosa intragastric administration group
  • CCFM1026 intervention group CCFM1026 intervention group
  • probiotic mixed gavage group bacterial intervention group CCFM1025+1026
  • CCFM1025 Lactobacillus mucosa intervention group
  • CCFM1026 intervention group CCFM1026
  • the probiotic intervention group (CCFM1025+1026) of the probiotic mixed preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacteria
  • the amount of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • CCFM1025 gavage of 10 9 CFU bacterial suspension of Lactobacillus mucosa CCFM1025 bacterial suspension
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026 gavage of 10 9 CFU bacterial suspension of Bifidobacterium breve CCFM1026 bacterial suspension, mixed with probiotics
  • the bacteria intervention group (CCFM1025+1026) of the preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacterial amount of L.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Gavage was continued 4 days after challenge, in which the Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the Bifidobacterium breve CCFM1026 intervention group (CCFM1026) was gavaged 10 9 CFU bacterial amount of Bifidobacterium breve CCFM1026 suspension suspension, probiotic mixed preparation intervention group (CCFM1025+1026) was intragastrically fed 0.2 mL of 10 9 CFU bacterial amount of probiotic mixed preparation (probiotic mixed preparation contains 5 ⁇ 10 8 CFU bacterial volume of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU bacterial volume of Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • the Lactobacillus mucosa intervention group
  • the lung tissue of the blank group (Control) mice is relatively complete and there is no infiltration of inflammatory cells; the model group (Model) mice show large-scale inflammation characterization and even hyperemia; the treatment group (Treatment) and probiotics
  • the mixed preparation intervention group CCFM1025+1026
  • some moderate and mild inflammation can also be observed in the lungs of mice, but the bronchial structure and other structures are still intact, and the degree of pathology has been relieved to a certain extent.
  • the mixed bacterial preparation intervention group CCFM1025+1026)
  • the bronchial villi are more complete and the surrounding inflammation is less.
  • Example 3-4 Effect of mixed bacteria on viral load in the lungs of influenza mice
  • mice weighing 20-24g were randomly divided into 6 groups, and the 6 groups were named as: blank control group (Control), influenza model group (Model), treatment group administered with ribavirin (Treatment) , Lactobacillus mucosa intragastric administration group (CCFM1025), Bifidobacterium breve CCFM1026 intervention group (CCFM1026), and probiotic mixed preparation group intragastric administration group (CCFM1025+1026), each group 8 pcs.
  • Control blank control group
  • Model influenza model group
  • Treatment group administered with ribavirin Treatment group administered with ribavirin
  • Tratment Treatment group administered with ribavirin
  • CCFM1025 Lactobacillus mucosa intragastric administration group
  • CCFM1026 intervention group CCFM1026 intervention group
  • probiotic mixed preparation group intragastric administration group CCFM1025+1026
  • CCFM1025 Lactobacillus mucosa intervention group
  • CCFM1026 intervention group CCFM1026
  • the probiotic intervention group (CCFM1025+1026) of the probiotic mixed preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacteria
  • the amount of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • CCFM1025 gavage of 10 9 CFU bacterial suspension of Lactobacillus mucosa CCFM1025 bacterial suspension
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026 gavage of 10 9 CFU bacterial suspension of Bifidobacterium breve CCFM1026 bacterial suspension, mixed with probiotics
  • the bacteria intervention group (CCFM1025+1026) of the preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacterial amount of L.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Gavage was continued 4 days after challenge, in which the Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the Bifidobacterium breve CCFM1026 intervention group (CCFM1026) was gavaged 10 9 Bifidobacterium breve CCFM1026 suspension suspension of CFU bacteria, probiotic mixed preparation group (CCFM1025+1026) gavage 10 9 Probiotics mixed preparation of CFU bacteria (probiotic mixed preparation contains 5 ⁇ 10 8 CFU Lactobacillus bacteria amount mucosa CCFM1025,5 ⁇ 10 8 CFU Bifidobacterium breve strain amount CCFM1026), the remaining group (Control, Model, Treatment) 0.2mL of saline per day orally.
  • the Lactobacillus mucosa intervention group CCFM1025
  • mice On the 5th day after the challenge, blood was taken and the mice were sacrificed. After the mice were sacrificed, the right lung tissues of the mice were placed in 1ml of TRIZOL, frozen and stored in a refrigerator at -80°C, and the right lung tissue samples were placed on ice during extraction After the upper melt, use a sterile grinder treated with DEPC, add 200ul of chloroform, mix thoroughly, centrifuge at 4°C, 12000rpm for 10min, remove 500 ⁇ L of supernatant, add an equal volume of isopropanol, mix well , Centrifuge at 4°C, 12000rpm for 10min, remove the supernatant, add 75% ethanol to wash the RNA once, and centrifuge at 4°C, 12000rpm for 10min. After removing the supernatant, after the alcohol has evaporated to dryness, add 40 ⁇ L of DEPC treated water to dissolve RNA, and the extracted RNA is stored in the refrigerator at -80°C for later use.
  • the viral load was determined by qPCR method, GAPDH was used as internal reference, the classic 2- ⁇ t calculation method was used, and the model group was used as comparative processing data. The results are shown in Figure 15.
  • Example 3-5 Effect of mixed bacteria on viral load in the lungs of influenza mice
  • mice weighing 20-24g were randomly divided into 6 groups, and the 6 groups were named as: blank control group (Control), influenza model group (Model), treatment group administered with ribavirin (Treatment) , Lactobacillus mucosa intragastric administration group (CCFM1025), Bifidobacterium breve CCFM1026 intervention group (CCFM1026), and probiotic mixed preparation group intragastric administration group (CCFM1025+1026), each group 8 pcs.
  • Control blank control group
  • Model influenza model group
  • Treatment group administered with ribavirin Treatment group administered with ribavirin
  • Tratment Treatment group administered with ribavirin
  • CCFM1025 Lactobacillus mucosa intragastric administration group
  • CCFM1026 intervention group CCFM1026 intervention group
  • probiotic mixed preparation group intragastric administration group CCFM1025+1026
  • CCFM1025 Lactobacillus mucosa intervention group
  • CCFM1026 intervention group CCFM1026
  • the probiotic intervention group (CCFM1025+1026) of the probiotic mixed preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacteria
  • the amount of Lactobacillus mucosa CCFM1025, 5 ⁇ 10 8 CFU Bifidobacterium breve CCFM1026), and the remaining groups (Control, Model, Treatment) were intragastrically fed 0.2 mL of normal saline every day.
  • CCFM1025 gavage of 10 9 CFU bacterial suspension of Lactobacillus mucosa CCFM1025 bacterial suspension
  • Bifidobacterium breve CCFM1026 intervention group CCFM1026 gavage of 10 9 CFU bacterial suspension of Bifidobacterium breve CCFM1026 bacterial suspension, mixed with probiotics
  • the bacteria intervention group (CCFM1025+1026) of the preparation group was intragastrically fed with a probiotic mixed preparation of 10 9 CFU bacteria (the probiotic mixed preparation contained 5 ⁇ 10 8 CFU bacterial amount of L.
  • the ribavirin drug treatment group (Treatment) was intraperitoneally injected with the drug ribavirin for treatment.
  • Gavage was continued 4 days after challenge, in which the Lactobacillus mucosa intervention group (CCFM1025) was gavaged with 10 9 CFU bacterial suspension dilution of Lactobacillus mucosa CCFM1025, and the Bifidobacterium breve CCFM1026 intervention group (CCFM1026) was gavaged 10 9 Bifidobacterium breve CCFM1026 suspension suspension of CFU bacteria, probiotic mixed preparation group (CCFM1025+1026) gavage 10 9 Probiotics mixed preparation of CFU bacteria (probiotic mixed preparation contains 5 ⁇ 10 8 CFU Lactobacillus bacteria amount mucosa CCFM1025,5 ⁇ 10 8 CFU Bifidobacterium breve strain amount CCFM1026), the remaining group (Control, Model, Treatment) 0.2mL of saline per day orally.
  • the Lactobacillus mucosa intervention group CCFM1025
  • mice On the 5th day after the challenge, blood was taken and the mice were sacrificed. After the mice were sacrificed, the right lung tissues of the mice were placed in 1ml of TRIZOL, frozen and stored in a refrigerator at -80°C, and the right lung tissue samples were placed on ice during extraction After the upper melt, use a sterile grinder treated with DEPC, add 200ul of chloroform, mix thoroughly, centrifuge at 4°C, 12000rpm for 10min, remove 500 ⁇ L of supernatant, add an equal volume of isopropanol, mix well , Centrifuge at 4°C, 12000rpm for 10min, remove the supernatant, add 75% ethanol to wash the RNA once, and centrifuge at 4°C, 12000rpm for 10min. After removing the supernatant, after the alcohol has evaporated to dryness, add 40 ⁇ L of DEPC treated water to dissolve RNA, the extracted RNA is stored in the refrigerator at -80 °C for use.
  • the anti-viral protein MxA expression in the lungs of mice in the bacterial intervention group (CCFM1025+1026) is the highest, which can reach 5.311 times that of the model group, indicating that the probiotic mixed preparation of the present invention can effectively participate in the body’s fight against influenza virus Process, enhance the expression of its antiviral protein MxA, and jointly help the body to inhibit viral replication, and the Bifidobacterium breve intervention group (CCFM1026) and L.
  • CCFM1025 can also increase the expression of the antiviral protein MxA to a certain extent (respectively) (3.464, 3.074 times of the model group), the expression level is more than the drug treatment group (Treatment), but not as good as the mixed bacterial agent intervention group (CCFM1025+1026).
  • Example 3-6 Preparation of solid beverages containing mixed bacteria
  • Lactobacillus mucosae CCFM1025 was inoculated into the medium at an inoculation amount of 5-8% of the total mass of the medium, and cultured at 37°C for 18h to obtain a culture solution; the culture solution was centrifuged to obtain bacterial cells; The body is washed with phosphate buffer solution with pH 7.2 for 2 to 4 times, and then resuspended with a trehalose-containing lyoprotectant containing 100 g/L of trehalose (the mass ratio of lyoprotectant and bacteria is 2:1) to obtain a re-suspension. Solution; freeze-drying the resuspended solution in vacuum to obtain Lactobacillus mucosae CCFM1025 powder;
  • Lactobacillus mucosae CCFM1025 powder and Bifidobacterium breve CCFM1026 powder containing 10 10 CFU were mixed with maltodextrin 1g to obtain Lactobacillus mucosae CCFM1025 and short Bifidobacterium (Bifidobacterium breve) CCFM1026 solid beverage.

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Abstract

本发明公开了一种具有抗流感能力的益生菌混合制剂及其应用，属于微生物技术领域以及医药技术领域。此益生菌混合制剂具有抗流感的作用，具体体现在：(1)显著改善流感小鼠体重下降程度；(2)显著改善流感小鼠血液指标；(3)显著改善流感小鼠的呼吸道感染炎症状况；(4)显著降低流感小鼠肺部病毒载量(即显著抑制流感病毒在流感小鼠体内的复制、增值)；(5)显著增强流感小鼠肺部抗病毒蛋白MxA的表达量，因此，此益生菌混合制剂在制备预防和/或治疗特应性皮炎乃至预防和/或治疗流感的产品中，具有巨大的应用前景。

Description

一种具有抗流感能力的益生菌混合制剂及其应用 技术领域
本发明涉及一种具有抗流感能力的益生菌混合制剂及其应用，属于微生物技术领域以及医药技术领域。
背景技术
流感常在秋冬季时大流行，主要由流感病毒诱发。流感病毒可分为甲型流感病毒、乙型流感病毒、丙型流感病毒三种，人所患的流感主要是由甲型流感病毒和乙型流感病毒引起的。一般动物流感病毒不会感染人，人流感病毒也不会感染动物，但是猪比较例外。猪既可以感染禽流感病毒，也可以感染人流感病毒，但主要还是感染禽流感病毒，不过，它们一旦感染禽流感病毒，就极易传染给人，引起人流感大流行，例如，1819-1920年发生的“西班牙流感”。
“西班牙流感”作为世界历史上最严重的一次流感大流行，涉及范围广，临床发病率高达40％，并伴随多种类型的肺炎并发症，造成2000-4000万人的死亡，死亡人数远远超出第一次世界大战。因受科学技术条件的限制，当时人们未能分离出“西班牙流感”的致病原，直到1997年，美国科学家在《Science》上发表称1918年的流感病毒与猪流感病毒十分相似，是一种与甲型流感病毒(H1N1)密切相关的病毒。
而随后的近一百年时间内，世界范围内仍爆发了多次流感大流行，造成不同程度的人员受难及经济损失。可以说，从流感出现以来，就一直无法完全控制，呈现间断式爆发。
流感无法完全控制的一个主要原因就在于，虽然，多数流感病毒不耐热，于56℃下加热30分钟即可灭活，在室温下传染性亦很快会丧失，但是，流感病毒的变异度极高，其中，变异最频繁的应属甲型流感病毒，每隔十几年便会发生一个抗原大变异，产生一个新的毒株，这种变化称作抗原转变/抗原的质变；在流感病毒亚型内也会发生抗原的小变异，主要是抗原氨基酸序列的点突变，称作抗原漂移/抗原的量变，从而使得人们无法拥有长期有效预防流感的疫苗。
医药领域对流感病毒的致病机理研究早已开展。动物实验研究表明，有多种致病通路可诱发呼吸道的感染，继而引起严重的呼吸系统疾病，而呼吸系统疾病引发的炎症主要集中在动物肺部，表现为肺炎形式，例如，组织病理学检查呼吸道感染小鼠肺部可发现小鼠肺泡结构被破坏，肺间隔断裂，肺泡上皮细胞坏死、脱落等病变；少部分小鼠肺组织可见肺间隔增宽；小鼠局部病变肺组织可见上皮细胞增生。
目前，世界卫生组织认为每年在流感流行高峰前期接种疫苗是最有效的预防手段。现阶段，已经上市的三价流感疫苗有灭活流感疫苗(TIV)和减毒活疫苗(LAIV)两种，由3种病毒组成，包含2种甲型流感病毒株和1种乙型流感病毒株。而相关流感感染的药物治疗主要有两类西药，一类是神经氨酸酶抑制剂如奥司他韦、扎那米韦、帕拉米韦，其机理是通过作用在病毒表面的糖蛋白神经氨酸酶，使病毒颗粒无法侵害人体细胞；另一类是M2离子通道阻滞剂如金刚烷胺和金刚乙胺，此类药物是作用于质子通道M2蛋白，通过阻扰其蛋白离子通道以抑制甲型流感病毒的复制。
但是，疫苗注射不能长久有效保护机体不受病毒侵染，而药物治疗在杀死病毒的同时对中枢神经系统有副作用。因此，仍然需要一种药物或治疗方式，既能够长久有效保护机体不受流感病毒侵染，也能够缓解流感的一些临床病症，同时，不会给患者中枢神经系统带来副作用。
近年，大量的研究表明，肠道微生物对于维持人体健康有着重要的作用，同时，益生菌在人类干预研究中对健康的影响包括改善儿童急性腹泻，缓解儿童牛奶过敏、特应性皮炎以及缓解人肠易激综合征，并且，益生菌可能通过肠道粘膜产生影响，通过抑制病原微生物的生长来平衡局部微生物群，进而增强局部和全身免疫反应，此外，益生菌还可能影响肠道内容物中微生物群的组成和活性；也已有报道指出，病毒引起的流感可影响肠道菌群结构，且特定益生菌可有效减轻急性轮状病毒胃肠炎的持续时间和严重程度。因此，或许可从肠道微 生物入手，尝试找到预防以及治疗流感的新药物或新方法，以克服现有治疗药物以及治疗方法副作用明显等的缺陷。
发明内容
为解决上述问题，本发明提供了一种含有粘膜乳杆菌(Lactobacillus mucosae)CCFM1025以及短双歧杆菌(Bifidobacterium breve)CCFM1026的益生菌混合制剂。此益生菌混合制剂具有抗流感的作用，具体体现在：(1)显著改善流感小鼠体重下降程度；(2)显著改善流感小鼠血液指标；(3)显著改善流感小鼠的呼吸道感染炎症状况；(4)显著降低流感小鼠肺部病毒载量(即显著抑制流感病毒在流感小鼠体内的复制、增值)；(5)显著增强流感小鼠肺部抗病毒蛋白MxA的表达量，因此，此益生菌混合制剂在制备预防和/或治疗特应性皮炎乃至预防和/或治疗流感的产品中，具有巨大的应用前景。
本发明的技术方案如下：
本发明提供了一种具有抗流感能力的益生菌混合制剂，所述益生菌混合制剂含有粘膜乳杆菌(Lactobacillus mucosae)CCFM1025以及短双歧杆菌(Bifidobacterium breve)CCFM1026；
所述粘膜乳杆菌(Lactobacillus mucosae)CCFM1025已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60460，保藏地址为广州市先烈中路100号大院59号楼5楼；
所述短双歧杆菌(Bifidobacterium breve)CCFM1026已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60459，保藏地址为广州市先烈中路100号大院59号楼5楼。
所述粘膜乳杆菌(Lactobacillus mucosae)CCFM1025是从人体粪便中分离得到的，该菌株经测序分析，其16S rRNA序列如SEQ ID NO.1所示，将该序列在GenBank中进行比对，结果显示菌株为粘膜乳杆菌，命名为粘膜乳杆菌(Lactobacillus mucosae)CCFM1025。
所述粘膜乳杆菌(Lactobacillus mucosae)CCFM1025的菌体呈短杆状；菌落为圆形粗糙、透明；于45℃生长，15℃不生长；37℃下在MRS液体培养基中培养12小时即达到稳定期，异型发酵，从葡萄糖产酸产气。
所述短双歧杆菌(Bifidobacterium breve)CCFM1026是从人体粪便样本中分离得到的，该菌株经测序分析，其16S rRNA序列如SEQ ID NO.2所示，将该序列在GenBank中进行比对，结果显示菌株与短双歧杆菌的覆盖率(Query cover)为100％、相似度(Ident)为99％，故判定菌株为短双歧杆菌，命名为短双歧杆菌(Bifidobacterium breve)CCFM1026。
所述短双歧杆菌(Bifidobacterium breve)CCFM1026的菌体呈短棒状，革兰氏染色阳性，亚甲基蓝染色着色不规则，无芽孢、鞭毛和荚膜，不运动；菌落为圆形白色；于37℃厌氧条件下培养30h可达稳定期，利用葡萄糖进行非典型异性乳酸发酵途径。
在本发明的一种实施方式中，所述益生菌混合制剂中粘膜乳杆菌(Lactobacillus mucosae)CCFM1025的活菌数不低于1×10 6CFU/mL；所述益生菌混合制剂中短双歧杆菌(Bifidobacterium breve)CCFM1026的活菌数不低于1×10 6CFU/mL。
本发明提供了上述益生菌混合制剂在制备预防和/或治疗流感的产品中的应用。
在本发明的一种实施方式中，所述产品中，粘膜乳杆菌(Lactobacillus mucosae)CCFM1025的活菌数不低于1×10 6CFU/mL、短双歧杆菌(Bifidobacterium breve)CCFM1026的活菌数不低于1×10 6CFU/mL。
在本发明的一种实施方式中，所述产品包含食品、药品或保健食品。
在本发明的一种实施方式中，所述药品含有益生菌混合制剂、药物载体和/或药用辅料。
在本发明的一种实施方式中，所述食品包含使用含有益生菌混合制剂的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品包含含有益生菌混合制剂的固体饮料。
本发明提供了一种用于预防和/或治疗流感的产品，所述产品含有上述一种具有抗流感能力的益生菌混合制剂。
在本发明的一种实施方式中，所述产品中，粘膜乳杆菌(Lactobacillus mucosae)CCFM1025 的活菌数不低于1×10 6CFU/mL、短双歧杆菌(Bifidobacterium breve)CCFM1026的活菌数不低于1×10 6CFU/mL。
在本发明的一种实施方式中，所述产品包含食品、药品或保健食品。
在本发明的一种实施方式中，所述药品含有益生菌混合制剂、药物载体和/或药用辅料。
在本发明的一种实施方式中，所述食品包含使用含有益生菌混合制剂的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品包含含有益生菌混合制剂的固体饮料。
在本发明的一种实施方式中，所述发酵剂的制备方法为将(Bifidobacterium breve)CCFM1026按照占培养基总质量5～8％的接种量接种到培养基中，于37℃的厌氧环境下培养30h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬，得到重悬液；将重悬液真空冷冻法冻干，得到(Bifidobacterium breve)CCFM1026菌粉；
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025按照占培养基总质量5～8％的接种量接种到培养基中，于37℃下培养18h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬，得到重悬液；将重悬液真空冷冻法冻干，得到粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉；
将得到的粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉与短双歧杆菌(Bifidobacterium breve)CCFM1026菌粉混合，得到发酵剂；
所述冻干保护剂和菌体的质量比为2:1。
在本发明的一种实施方式中，所述培养基的pH为6.8。
在本发明的一种实施方式中，所述保护剂包含100g/L的脱脂奶粉、150g/L的海藻糖以及10g/L的L-谷氨酸钠。
有益效果：
本发明提供了一种含有粘膜乳杆菌(Lactobacillus mucosae)CCFM1025以及短双歧杆菌(Bifidobacterium breve)CCFM1026的益生菌混合制剂，此益生菌混合制剂具有抗流感的作用，具体体现在：
(1)显著改善流感小鼠体重下降程度；
(2)显著改善流感小鼠血液指标；
(3)显著改善流感小鼠的呼吸道感染炎症状况；
(4)显著降低流感小鼠肺部病毒载量(即显著抑制流感病毒在流感小鼠体内的复制、增值)；
(5)显著增强流感小鼠肺部抗病毒蛋白MxA的表达量，
因此，此益生菌混合制剂在制备预防和/或治疗特应性皮炎乃至预防和/或治疗流感的产品中，具有巨大的应用前景。
生物材料保藏
一株粘膜乳杆菌(Lactobacillus mucosae)CCFM1025，分类学命名为Lactobacillus mucosae，已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60460，保藏地址为广州市先烈中路100号大院59号楼5楼。
一株短双歧杆菌(Bifidobacterium breve)CCFM1026，分类学命名为Bifidobacterium breve，已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60459，保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1：不同组别流感小鼠的体重变化对比(粘膜乳杆菌)。
图2：不同组别流感小鼠的血液检测指标(中性粒细胞)对比(粘膜乳杆菌)。
图3：不同组别流感小鼠的血液检测指标(淋巴细胞)对比(粘膜乳杆菌)。
图4：不同组别流感小鼠的肺部组织病理学切片对比(粘膜乳杆菌)。
图5：不同组别流感小鼠的肺部病毒载量对比(粘膜乳杆菌)。
图6：不同组别流感小鼠的体重变化对比(短双歧杆菌)。
图7：不同组别流感小鼠的血液检测指标(中性粒细胞)对比(短双歧杆菌)。
图8：不同组别流感小鼠的血液检测指标(淋巴细胞)对比(短双歧杆菌)。
图9：不同组别流感小鼠的肺部组织病理学切片对比(短双歧杆菌)。
图10：不同组别流感小鼠的肺部抗病毒蛋白MxA的表达量对比(短双歧杆菌)。
图11：不同组别流感小鼠的体重变化对比(混菌)。
图12：不同组别流感小鼠的血液检测指标(中性粒细胞)对比(混菌)。
图13：不同组别流感小鼠的血液检测指标(淋巴细胞)对比(混菌)。
图14：不同组别流感小鼠的肺部组织病理学切片对比(混菌)。
图15：不同组别流感小鼠的肺部病毒载量对比(混菌)。
图16：不同组别流感小鼠的肺部抗病毒蛋白MxA的表达量对比(混菌)。
具体实施方式
下述实施例中涉及的培养基如下：
MRS平板(g/L)：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K 2PO 4·3H 2O 2.6g/L、MgSO 4·7H 200.1g/L、MnSO 40.05g/L、吐温801ml/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L。
实施例1-1：粘膜乳杆菌的筛选及鉴定
1、筛选
以人体粪便为样本，将样本经预处理后，在20％左右甘油中保存于-80℃冰箱，取出解冻后，混匀样本吸取0.5mL样本加到4.5mL，以含有0.05％半胱氨酸的0.9％生理盐水进行梯度稀释，选择合适的梯度稀释液涂布在加了0.05％半胱氨酸的MRS平板上，于37℃培养48h，挑取典型菌落至MRS平板上划线纯化，挑取单菌落转接至液体MRS培养基(含0.05％半胱氨酸)增菌，30％甘油保藏，得到菌株CCFM1025和菌株F1。
2、鉴定
提取CCFM1025、F1的基因组，将CCFM1025、F1的16S rDNA进行扩增和测序(上海生工生物工程股份有限公司)，将该序列在GenBank中进行比对，结果显示菌株为粘膜乳杆菌，命名为粘膜乳杆菌(Lactobacillus mucosae)CCFM1025和粘膜乳杆菌(Lactobacillus mucosae)F1。
实施例1-2：粘膜乳杆菌的培养
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025接入MRS固体培养基(含0.05％半胱氨酸)中于37℃培养48h后，观察其菌落并在显微镜下对其菌体进行观察，发现其菌落为圆形粗糙、透明，其菌体为短杆状。
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025接入MRS培养基(含0.05％半胱氨酸)中于37℃培养48h，制作生长曲线，发现其于37℃下培养12h达到稳定期，同时，观察发现其为异型发酵，可从葡萄糖产酸产气。
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025接入MRS培养基(含0.05％半胱氨酸)中分别于10、15、20、25、30、35、40、45、50℃下培养48h后观察其生长状况，20～35℃生长良好，45℃条件下依旧能够生长，但于15℃或低于15℃或50℃几乎不生长。
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025接入MRS培养基(含0.05％半胱氨酸)中于37℃培养48h后，转入新鲜的MRS培养基(含0.05％半胱氨酸)中，同样条件培养30h，6000g离心菌体15min，0.9％生理盐水洗涤菌体后6000g再次离心10min，得到菌体，用30％蔗糖溶液重悬，冻存在-80℃待用。
实施例1-3：粘膜乳杆菌对流感小鼠体重的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳 杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃粘膜乳杆菌F1的粘膜乳杆菌干预组(F1)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余4组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验的5天中，每天灌胃前称重，连续记录五日体重变化，结果如图1所示。
由图1可知，攻毒后的第3天小鼠体重开始出现下降，第4天时体重下降最为明显(P<0.05)，与对照组(Control)相比，模型组(Model)体重下降超过10％，药物治疗组(Treatment)体重下降约5％，粘膜乳杆菌F1干预组体重下降5.5％左右，而粘膜乳杆菌CCFM1025干预组体重下降仅为3％。
说明本发明的粘膜乳杆菌CCFM1025可显著改善流感小鼠体重减轻症状。
实施例1-4：粘膜乳杆菌对流感小鼠血液指标的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌的粘膜乳杆菌干预组(CCFM1025)、灌胃粘膜乳杆菌F1的粘膜乳杆菌干预组(F1)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余3组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，将取出的小鼠血液放置在抗凝管中，轻轻晃动，使血液于抗凝剂充分接触，随后送至动物医院进行血常规分析检测(流感病毒感染初期，大量天然免疫细胞，如中性粒细胞和淋巴细胞会参与防御过程，因此，血常规分析检测重点检测中性粒细胞和淋巴细胞变化)，结果如图2-3所示。
由图2-3可知，与空白组(Control)相比，模型组(Model)中性粒细胞显著升高，淋巴细胞显著降低，而其余四组则无显著变化。
说明本发明的粘膜乳杆菌CCFM1025同利巴韦林药物一样，对机体可起到相同的免疫调节功能。
实施例1-5：粘膜乳杆菌对流感小鼠呼吸道感染炎症的影响
取体重20-24g的健康ICR雌鼠32只，随机分成4组，4组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林的药物治疗组(Treatment)、灌胃粘膜 乳杆菌的粘膜乳杆菌干预组(CCFM)、灌胃粘膜乳杆菌F1的粘膜乳杆菌干预组(F1)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余3组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，空白对照组以及流感模型组(Control、Model)灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，立即取出小鼠左肺置于4％多聚甲醛中固定，固定后进行组织病理学切片，切片后对小鼠肺部组织病理学切片进行苏木精-伊红染色，通过职业技术人员对小鼠肺部组织病理学切片进行组织病理评分，结果如图4所示。
由图4可知，空白组(Control)小鼠肺部组织结构较完整，无炎症细胞浸润；模型组(Model)小鼠则出现大规模炎症表征，甚至充血情况；治疗组(Treatment)和粘膜乳杆菌干预组(CCFM1025)组小鼠肺部则轻度炎症细胞浸润，发生在支气管附近。
以上动物实验表明，流感感染可使小鼠患流感性肺炎，肺部被病毒侵染后，其组织结构被破坏，出现大量炎症浸润；而本发明的粘膜乳杆菌CCFM1025可缓解小鼠肺部炎症状况，减轻其肺炎病症，同流感常用药利巴韦林药物治疗组的效果相当。
实施例1-6：粘膜乳杆菌对流感小鼠肺部病毒载量的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌的粘膜乳杆菌干预组(CCFM1025)、灌胃粘膜乳杆菌F1的粘膜乳杆菌干预组(F1)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余3组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM)灌胃10 9CFU菌量的粘膜乳杆菌菌悬稀释液，粘膜乳杆菌F1干预组(F1)灌胃同样数量的粘膜乳杆菌F1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，处死小鼠后，取小鼠右肺组织置于1mL的TRIZOL中，冻存于-80℃冰箱备用，提取时将右肺组织样品置冰上融化后，采用经DEPC处理的无菌研磨器研磨，加入200μL的氯仿，充分混匀后，于4℃、12000rpm下离心10min，去上清500μL，加入等体积的异丙醇，混匀后，于4℃、12000rpm下离心10min，去上清，加入75％的乙醇洗涤RNA一次，于4℃、12000rpm下离心10min，去上清后，待酒精挥发干，加入40μL的DEPC处理水，溶解RNA，提取的RNA冻存于-80℃冰箱备用。
利用qPCR法测定病毒载量，以GAPDH为内参，采用经典的2 -ΔΔt计算方法，以模型组作为对比处理数据，结果如图5所示。
由图5可知，药物治疗组(Treatment)小鼠肺部病毒载量为模型组的38.03％，而本发明 的粘膜乳杆菌组干预组(CCFM1025)小鼠肺部病毒载量仅为模型组的16.30％。
以上实验表明，本发明发的粘膜乳杆菌CCFM1025可明显减低流感感染小鼠肺部病毒载量，存在显著性差异(P＝0.0022)，粘膜乳杆菌F1组和药物治疗组(Treatment)小鼠肺部病毒载量降低均存在显著性(P值分别为0.0309和0.0170)，这足以说明本发明的粘膜乳杆菌CCFM1025在抵抗病毒侵染方面效果优于流感药物治疗作用。
实施例2-1：短双歧杆菌的筛选及鉴定
1、筛选
以人体粪便为样本，将样本经预处理后，在20％左右甘油中保存于-80℃冰箱，取出解冻后，混匀样本吸取0.5mL样本加到4.5mL，以含有0.05％半胱氨酸的0.9％生理盐水进行梯度稀释，选择合适的梯度稀释液涂布在加了0.05％半胱氨酸的MRS平板上，于37℃培养48h，挑取典型菌落至MRS平板上划线纯化，挑取单菌落转接至液体MRS培养基(含0.05％半胱氨酸)增菌，30％甘油保藏，得到菌株CCFM1026和菌株B1。
2、鉴定
提取CCFM1026、B1的基因组，将CCFM1026、B1的16S rDNA进行扩增和测序(上海生工生物工程股份有限公司)，将该序列在GenBank中进行比对，结果显示菌株与短双歧杆菌的覆盖率(Query cover)均为100％、相似度(Ident)均为99％，故判定菌株为短双歧杆菌，命名为短双歧杆菌(Bifidobacterium breve)CCFM1026和短双歧杆菌(Bifidobacterium breve)B1。
实施例2-2：短双歧杆菌的培养
将短双歧杆菌(Bifidobacterium breve)CCFM1026接入MRS固体培养基(含0.05％半胱氨酸)中于37℃培养48h后，观察其菌落，发现其菌落为圆形白色；
在显微镜下对其菌体进行观察并对其进行染色，发现其菌体呈短棒状，革兰氏染色阳性，亚甲基蓝染色着色不规则，无芽孢、鞭毛和荚膜，不运动。
将短双歧杆菌(Bifidobacterium breve)CCFM1026接入MRS液体培养基(含0.05％半胱氨酸)中于37℃培养48h，制作生长曲线，发现其于37℃厌氧条件下培养30h可达稳定期，利用葡萄糖进行非典型异性乳酸发酵途径。
将短双歧杆菌(Bifidobacterium breve)CCFM1026于MRS培养基(含0.05％半胱氨酸)中于37℃培养48h后，转入新鲜的MRS培养基(含0.05％半胱氨酸)中，同样条件培养30h，6000g离心菌体15min，0.9％生理盐水洗涤菌体后6000g再次离心10min，得到菌体，用30％蔗糖溶液重悬，冻存在-80℃待用。
实施例2-3：短双歧杆菌对流感小鼠体重的影响
取体重20-24g的健康ICR雌鼠32只，随机分成4组，4组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林的药物治疗组(Treatment)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃短双歧杆菌B1的短双歧干预组(B1组)，每组8只。
实验前2周，每天给短双歧杆菌干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余4组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，短双歧杆菌干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，短双歧杆菌(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验的5天中，每天灌胃前称重，连续记录五日体重变化，结果如图6所示。
由图6可知，攻毒后的第3天小鼠体重开始出现下降，第4天时体重下降最为明显(P<0.05)，与对照组(Control)相比，模型组(Model)体重下降超过10％，药物治疗组(Treatment)体重下降约5％，而短双歧杆菌干预组(CCFM1026)和短双歧杆菌B1组体重下降也仅为5％。
说明本发明的短双歧杆菌CCFM1026和B1均可显著改善流感小鼠体重减轻症状。
实施例2-4：短双歧杆菌对流感小鼠血液指标的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃短双歧杆菌B1的短双歧干预组(B1组)，每组8只。
实验前2周，每天给短双歧杆菌CCFM1206干预组(CCFM1206)灌胃10 9CFU菌量的短双歧杆菌菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余4组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，短双歧杆菌干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，短双歧杆菌(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，将取出的小鼠血液放置在抗凝管中，轻轻晃动，使血液于抗凝剂充分接触，随后送至动物医院进行血常规分析检测(流感病毒感染初期，大量天然免疫细胞，如中性粒细胞和淋巴细胞会参与防御过程，因此，血常规分析检测重点检测中性粒细胞和淋巴细胞变化)，结果如图7-8所示。
由图7-8可知，与空白组(Control)相比，模型组(Model)小鼠中性粒细胞和淋巴细胞百分比明显出现异常，显著异于空白组(Control)(p值分别为0.0015和0.0011)，然而短双歧杆菌干预组(CCFM1026)和药物治疗组(Treatment)小鼠的中性粒细胞和淋巴细胞百分比趋向空白对照组(Control)，且无显著性差异。
说明本发明的短双歧杆菌同利巴韦林药物效果一样，可积极参与到小鼠的流感相关免疫调节中，缓解小鼠流感病症。
实施例2-5：短双歧杆菌对流感小鼠呼吸道感染炎症的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃短双歧杆菌B1的短双歧干预组(B1组)，每组8只。
实验前2周，每天给短双歧杆菌CCFM1206干预组(CCFM1206)灌胃10 9CFU菌量的短双歧杆菌菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余4组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，短双歧杆菌干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，短双歧杆菌(CCFM1026)灌胃10 9CFU菌量的短双歧 杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，立即取出小鼠左肺置于4％多聚甲醛中固定，固定后进行组织病理学切片，切片后对小鼠肺部组织病理学切片进行苏木精-伊红染色，通过职业技术人员对小鼠肺部组织病理学切片进行组织病理评分，结果如图9所示。
由图9可知，空白组(Control)小鼠肺部组织结构较完整，无炎症细胞浸润；模型组(Model)小鼠则出现严重炎症浸润现象，甚至出现局部出血现象；短双歧杆菌B1干预组(B1)支气管附近出现中度炎症浸润，而治疗组(Treatment)和短双歧杆菌干预组(CCFM1026)组小鼠肺部则炎症情况相对较轻。
以上动物实验表明，流感感染可使小鼠患流感性肺炎，肺部被病毒侵染后，其组织结构被破坏，出现大量炎症浸润；而本发明的短双歧杆菌可缓解小鼠肺部炎症状况，减轻其肺炎病症，同流感常用药利巴韦林药物治疗组的效果相当。
实施例2-6：短双歧杆菌对流感小鼠肺部病毒载量的影响
取体重20-24g的健康ICR雌鼠40只，随机分成5组，5组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃短双歧杆菌B1的短双歧干预组(B1组)，每组8只。
实验前2周，每天给短双歧杆菌CCFM1206干预组(CCFM1206)灌胃10 9CFU菌量的短双歧杆菌菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余4组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，短双歧杆菌干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，短双歧杆菌(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，短双歧杆菌B1干预组(B1)灌胃同样数量的短双歧杆菌B1菌悬稀释液，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。攻毒后的第5天取血后处死小鼠，处死小鼠后，取小鼠右肺组织置于1ml的TRIZOL中，冻存于-80℃冰箱备用，提取时将右肺组织样品置冰上融化后，采用经DEPC处理的无菌研磨器研磨，加入200ul的氯仿，充分混匀后，于4℃、12000rpm下离心10min，去上清500μL，加入等体积的异丙醇，混匀后，于4℃、12000rpm下离心10min，去上清，加入75％的乙醇洗涤RNA一次，于4℃、12000rpm下离心10min，去上清后，待酒精挥发干，加入40μL的DEPC处理水，溶解RNA，提取的RNA冻存于-80℃冰箱备用。
利用qPCR法测定MxA相对表达量(机体对流感病毒会做出防御反应，以清除入侵病毒，恢复健康，MxA是其分泌的一种抗病毒蛋白，可有效阻止病毒的复制)，以GAPDH为内参，采用经典的2 -ΔΔt计算方法，以模型组作为对比处理数据，结果如图10所示。
由图10可知，药物治疗组(Treatment)小鼠肺部MxA表达量为模型组(Model)的2.62倍，短双歧杆菌B1干预组小鼠肺部MxA表达量为模型组(Model)的2.67倍，无显著性差异，而短双歧杆菌干预组(CCFM1026)小鼠肺部MxA表达量则为模型组(Model)的3.46倍，可显著提高MxA表达量(p值为0.0292)。
以上实验表明，本发明的短双歧杆菌CCFM1026可以明显增强流感小鼠免疫能力，促使其MxA抗病毒蛋白表达量增加，从而对抗病毒的复制，帮助机体恢复健康，呼吸道炎症明显减轻，效果甚至优于药物治疗。
实施例3-1：混菌对流感小鼠体重的影响
取体重20-24g的健康ICR雌鼠48只，随机分成6组，6组分别命名为：空白对照组 (Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃益生菌混合制剂组的菌剂干预组(CCFM1025+1026)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余5组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验的5天中，每天灌胃前称重，连续记录五日体重变化，结果如图11所示。
由图11可知，攻毒后的第3天小鼠体重开始出现下降，第4天时体重下降最为明显(P<0.05)，与对照组(Control)相比，模型组(Model)体重下降超过10％，药物治疗组(Treatment)体重下降约5％，粘膜乳杆菌干预组(CCFM1025)体重下降约为3％，短双歧杆菌干预组(CCFM1026)体重下降约为5％，而菌剂干预组(CCFM1025+1026)小鼠体重下降最小，为不到3％，比药物治疗组(Treatment)、粘膜乳杆菌干预组(CCFM1025)、短双歧杆菌干预组(CCFM1026)干预效果更佳，足以说明摄入本发明的益生菌混合制剂可明显缓解流感小鼠因流感感冒导致的体重下降。
实施例3-2：混菌对流感小鼠血液指标的影响
取体重20-24g的健康ICR雌鼠48只，随机分成6组，6组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃益生菌混合制剂组的菌剂干预组(CCFM1025+1026)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余5组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组 (CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，将取出的小鼠血液放置在抗凝管中，轻轻晃动，使血液于抗凝剂充分接触，随后送至动物医院进行血常规分析检测(流感病毒感染初期，大量天然免疫细胞，如中性粒细胞和淋巴细胞会参与防御过程，因此，血常规分析检测重点检测中性粒细胞和淋巴细胞变化)，结果如图12-13所示。
由图12-13可知，在小鼠患流感感冒后，其体内天然免疫细胞的比例会发生不同变化，即模型组(Model)小鼠体内中性粒细胞和淋巴细胞等免疫细胞水平出现不同趋势的变化，显著异于空白对照组(Control)，而摄入益生菌的干预组小鼠的各项指标趋于正常值，与空白对照组(Control)无显著性差异，其中，同模型组相比，摄入益生菌组中性粒细胞百分比均下降(Control：22.60％、CCFM1025+1026：33.10％、CCFM1025：35.75％、CCFM1026：37.10％)，相应淋巴细胞百分比提高(Control：73.70％、CCFM1025+1026：63.95％、CCFM1025：60.56％、CCFM1026：62.10％)，可以看出益生菌混合制剂组(CCFM1025+1026)趋向空白对照组(Control)程度略微强于两个单菌(CCFM1025、CCFM1026)组别，说明小鼠摄入的益生菌可参与到机体的免疫调节中，协助机体共同抵抗流感病毒的入侵，维持机体健康，而混菌制剂的效果优于单菌。
实施例3-3：混菌对流感小鼠呼吸道感染炎症的影响
取体重20-24g的健康ICR雌鼠48只，随机分成6组，6组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃益生菌混合制剂组的菌剂干预组(CCFM1025+1026)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余5组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂干预组(CCFM1025+1026)灌胃 0.2mL的10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，立即取出小鼠左肺置于4％多聚甲醛中固定，固定后进行组织病理学切片，切片后对小鼠肺部组织病理学切片进行苏木精-伊红染色，通过职业技术人员对小鼠肺部组织病理学切片进行组织病理评分，结果如图14所示。
由图14可知，空白组(Control)小鼠肺部组织结构较完整，无炎症细胞浸润；模型组(Model)小鼠则出现大规模炎症表征，甚至充血情况；治疗组(Treatment)和益生菌混合制剂干预组(CCFM1025+1026)小鼠肺部也可观察到一定中轻度的炎症，但支气管等结构还算完整，病理程度得到一定程度缓解，其中，混合菌剂干预组(CCFM1025+1026)相比两组单菌组，其支气管的绒毛完整度更好，周围炎症更少，尽管各组的病理组织评分相近，但仍可以说明，混合菌剂干预组(CCFM1025+1026)可以使流感感染小鼠的呼吸道感染程度得到一定程度的降低，而混菌制剂的效果优于单菌。
实施例3-4：混菌对流感小鼠肺部病毒载量的影响
取体重20-24g的健康ICR雌鼠48只，随机分成6组，6组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃益生菌混合制剂组的菌剂干预组(CCFM1025+1026)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余5组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，处死小鼠后，取小鼠右肺组织置于1ml的TRIZOL中，冻存于-80℃冰箱备用，提取时将右肺组织样品置冰上融化后，采用经DEPC处理的无菌研磨器研磨，加入200ul的氯仿，充分混匀后，于4℃、12000rpm下离心10min，去上清500μL，加入等体积的异丙醇，混匀后，于4℃、12000rpm下离心10min，去上清，加入75％的乙醇洗涤RNA一次，于4℃、12000rpm下离心10min，去上清后，待酒精挥发干，加入40μL的DEPC处理水，溶解RNA，提取的RNA冻存于-80℃冰箱备用。
利用qPCR法测定病毒载量，以GAPDH为内参，采用经典的2 -ΔΔt计算方法，以模型组作为对比处理数据，结果如图15所示。
由图15可知，摄入益生菌的干预组小鼠肺部病毒载量均有所降低，均低于50％，其中，混合菌剂干预组(CCFM1025+1026)和粘膜乳杆菌干预组显著降低，p值分别为0.0097和0.0032，平均值分别为27.68％、16.3％，药物治疗组相对病毒载量均值为38.03％，以上数据说明，益生菌混合制剂可显著降低流感感染小鼠肺部病毒载量，且效果优于药物治疗的效果。
实施例3-5：混菌对流感小鼠肺部病毒载量的影响
取体重20-24g的健康ICR雌鼠48只，随机分成6组，6组分别命名为：空白对照组(Control)、流感模型组(Model)、给药利巴韦林药物治疗组(Treatment)、灌胃粘膜乳杆菌CCFM1025的粘膜乳杆菌干预组(CCFM1025)、灌胃短双歧杆菌CCFM1026干预组(CCFM1026)、灌胃益生菌混合制剂组的菌剂干预组(CCFM1025+1026)，每组8只。
实验前2周，每天给粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第1天，除空白对照组(Control)外，其余5组均在乙醚轻微麻醉后鼻滴法以流感病毒对小鼠进行攻毒，当天仍需灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
实验第2天，给药利巴韦林药物治疗组(Treatment)腹腔注射药物利巴韦林进行治疗。
攻毒之后的4天继续灌胃，其中，粘膜乳杆菌干预组(CCFM1025)灌胃10 9CFU菌量的粘膜乳杆菌CCFM1025菌悬稀释液，短双歧杆菌CCFM1026干预组(CCFM1026)灌胃10 9CFU菌量的短双歧杆菌CCFM1026菌悬稀释液，益生菌混合制剂组的菌剂干预组(CCFM1025+1026)灌胃10 9CFU菌量的益生菌混合制剂(益生菌混合制剂中含有5×10 8CFU菌量的粘膜乳杆菌CCFM1025、5×10 8CFU菌量的短双歧杆菌CCFM1026)，其余组(Control、Model、Treatment)每天灌胃0.2mL生理盐水。
攻毒后的第5天取血后处死小鼠，处死小鼠后，取小鼠右肺组织置于1ml的TRIZOL中，冻存于-80℃冰箱备用，提取时将右肺组织样品置冰上融化后，采用经DEPC处理的无菌研磨器研磨，加入200ul的氯仿，充分混匀后，于4℃、12000rpm下离心10min，去上清500μL，加入等体积的异丙醇，混匀后，于4℃、12000rpm下离心10min，去上清，加入75％的乙醇洗涤RNA一次，于4℃、12000rpm下离心10min，去上清后，待酒精挥发干，加入40μL的DEPC处理水，溶解RNA，提取的RNA冻存于-80℃冰箱备用。
利用qPCR法测定MxA相对表达量(机体对流感病毒会做出防御反应，以清除入侵病毒，恢复健康，MxA是其分泌的一种抗病毒蛋白，可有效阻止病毒的复制)，以GAPDH为内参，采用经典的2 -ΔΔt计算方法，以模型组作为对比处理数据，结果如图16所示。
由图16可知，菌剂干预组(CCFM1025+1026)小鼠肺部抗病毒蛋白MxA表达量最高，可达模型组的5.311倍，说明本发明的益生菌混合制剂可有效参与机体对抗流感病毒的过程，增强其抗病毒蛋白MxA的表达量，共同协助机体抑制病毒复制，而短双歧杆菌干预组(CCFM1026)和粘膜乳杆菌(CCFM1025)也可一定程度增加抗病毒蛋白MxA的表达量(分别为模型组的3.464、3.074倍)，表达量比药物治疗组(Treatment)要多，但都不如混合菌剂干预组(CCFM1025+1026)效果好。
实施例3-6：含有混菌的固体饮料的制备
具体步骤如下：
将(Bifidobacterium breve)CCFM1026按照占培养基总质量5～8％的接种量接种到培养基中，于37℃的厌氧环境下培养30h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液；将重悬液真空冷冻法冻干，得到(Bifidobacterium breve)CCFM1026菌粉；
将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025按照占培养基总质量5～8％的接种量接种到培养基中，于37℃下培养18h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬(冻干保护剂和菌体的质量比为2:1)，得到重悬液；将重悬液真空冷冻法冻干，得到粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉；
将含有10 10CFU的粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉和短双歧杆菌(Bifidobacterium breve)CCFM1026菌粉分别同麦芽糊精1g进行混合，得到富含粘膜乳杆菌(Lactobacillus mucosae)CCFM1025和短双歧杆菌(Bifidobacterium breve)CCFM1026的固体饮料。
取10克此含有粘膜乳杆菌CCFM1025和短双歧杆菌CCFM1026的固体饮料用20毫升的生理盐水复溶，每只小鼠灌胃200微升，连续两周，可有效缓解流感小鼠症状，在治疗和/或预防流感有极好的效果。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2019082656-appb-000001
Figure PCTCN2019082656-appb-000002

Claims (15)

  1. 一种益生菌混合制剂，其特征在于，所述益生菌混合制剂含有粘膜乳杆菌(Lactobacillus mucosae)以及短双歧杆菌(Bifidobacterium breve)；
    所述粘膜乳杆菌(Lactobacillus mucosae)已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60460，保藏地址为广州市先烈中路100号大院59号楼5楼；
    所述短双歧杆菌(Bifidobacterium breve)已于2018年10月11日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60459，保藏地址为广州市先烈中路100号大院59号楼5楼。
  2. 如权利要求1所述的一种益生菌混合制剂，其特征在于，所述益生菌混合制剂中粘膜乳杆菌(Lactobacillus mucosae)的活菌数不低于1×10 6CFU/mL；所述益生菌混合制剂中短双歧杆菌(Bifidobacterium breve)的活菌数不低于1×10 6CFU/mL。
  3. 权利要求1或2所述的益生菌混合制剂在制备预防和/或治疗流感的产品中的应用。
  4. 如权利要求3所述的益生菌混合制剂在制备预防和/或治疗流感的产品中的应用，其特征在于，所述产品中，粘膜乳杆菌(Lactobacillus mucosae)的活菌数不低于1×10 6CFU/mL、短双歧杆菌(Bifidobacterium breve)的活菌数不低于1×10 6CFU/mL。
  5. 如权利要求3或4所述的益生菌混合制剂在制备预防和/或治疗流感的产品中的应用，其特征在于，所述产品包含食品、药品或保健食品。
  6. 如权利要求5所述的益生菌混合制剂在制备预防和/或治疗流感的产品中的应用，其特征在于，所述药品含有益生菌混合制剂、药物载体和/或药用辅料。
  7. 如权利要求5所述的益生菌混合制剂在制备预防和/或治疗流感的产品中的应用，其特征在于，所述食品包含使用含有益生菌混合制剂的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品包含含有益生菌混合制剂的固体饮料。
  8. 一种用于预防和/或治疗流感的产品，其特征在于，所述产品含有权利要求1-3任一所述的一种具有抗流感能力的益生菌混合制剂。
  9. 如权利要求8所述的一种用于预防和/或治疗流感的产品，其特征在于，所述产品中，粘膜乳杆菌(Lactobacillus mucosae)CCFM1025的活菌数不低于1×10 6CFU/mL、短双歧杆菌(Bifidobacterium breve)CCFM1026的活菌数不低于1×10 6CFU/mL。
  10. 如权利要求8或9所述的一种用于预防和/或治疗流感的产品，其特征在于，所述产品包含食品、药品或保健食品。
  11. 如权利要求10所述的一种用于预防和/或治疗流感的产品，其特征在于，所述药品含有益生菌混合制剂、药物载体和/或药用辅料。
  12. 如权利要求10所述的一种用于预防和/或治疗流感的产品，其特征在于，所述食品包含使用含有益生菌混合制剂的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品包含含有益生菌混合制剂的固体饮料。
  13. 如权利要求12所述的一种用于预防和/或治疗流感的产品，其特征在于，所述发酵剂的制备方法为将(Bifidobacterium breve)CCFM1026按照占培养基总质量5～8％的接种量接种到培养基中，于37℃的厌氧环境下培养30h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬，得到重悬液；将重悬液真空冷冻法冻干，得到(Bifidobacterium breve)CCFM1026菌粉；
    将粘膜乳杆菌(Lactobacillus mucosae)CCFM1025按照占培养基总质量5～8％的接种量接种到培养基中，于37℃下培养18h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2的磷酸盐缓冲液清洗2～4次后用含100g/L的海藻糖冻干保护剂重悬，得到重悬液；将重悬液真空冷冻法冻干，得到粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉；
    将得到的粘膜乳杆菌(Lactobacillus mucosae)CCFM1025菌粉与短双歧杆菌(Bifidobacterium breve)CCFM1026菌粉混合，得到发酵剂；
    所述冻干保护剂和菌体的质量比为2:1。
  14. 如权利要求13所述的一种用于预防和/或治疗流感的产品，其特征在于，所述培养基的pH为6.8。
  15. 如权利要求13或14所述的一种用于预防和/或治疗流感的产品，其特征在于，所述保护剂包含100g/L的脱脂奶粉、150g/L的海藻糖以及10g/L的L-谷氨酸钠。
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