CN110055185B - Bifidobacterium breve with anti-influenza capacity and application thereof - Google Patents
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Abstract
The invention discloses a bifidobacterium breve with anti-influenza capacity and application thereof, belonging to the technical field of microorganisms and medicines. The Bifidobacterium breve (Bifidobacterium breve) has the function of resisting influenza, and is specifically characterized in that: (1) the weight loss degree of influenza mice is obviously improved; (2) the blood index of the influenza mice is obviously improved; (3) the respiratory tract infection inflammation condition of an influenza mouse is obviously improved; (4) the expression quantity of the influenza mouse pulmonary antiviral protein MxA is obviously enhanced, so the Bifidobacterium breve has great application prospect in preparing products for preventing and/or treating influenza.
Description
Technical Field
The invention relates to bifidobacterium breve with anti-influenza capacity and application thereof, belonging to the technical field of microorganisms and the technical field of medicines.
Background
Influenza is often pandemic in autumn and winter, and is mainly induced by influenza viruses. Influenza viruses can be divided into influenza a viruses, influenza b viruses and influenza c viruses, and the influenza suffered by human beings is mainly caused by the influenza a viruses and the influenza b viruses. Animal influenza viruses do not generally infect humans, nor do human influenza viruses infect animals, but swine is the exception. The pigs can be infected with avian influenza virus and human influenza virus, but are mainly infected with avian influenza virus, and once the pigs are infected with avian influenza virus, the pigs are extremely easy to infect people, so that the human influenza pandemics are caused.
For example, "Spanish flu" that occurred in 1819-1920. "Spanish influenza" is the most serious influenza pandemic in the world history, and has a wide range of involvement, a clinical morbidity as high as 40%, and is accompanied by various pneumonia complications, causing 2000-4000 million deaths, and the number of deaths is far beyond the first world war. Because of the limitations of scientific and technical conditions, the etiological agent of "Spanish influenza" was not isolated until 1997, and it was reported by American scientists in Science that the influenza virus in 1918 was very similar to swine influenza virus, a virus closely related to influenza A virus (H1N 1). In the next last hundred years, a plurality of influenza pandemics still occur worldwide, and people with different degrees are difficult and have economic losses.
It can be said that since the onset of influenza, there has been no complete control, presenting intermittent outbreaks. One of the main reasons for the incomplete control is that most influenza viruses are heat-labile and can be inactivated by heating at 56 ℃ for 30 minutes, and the infectivity is lost rapidly at room temperature, but the influenza viruses have extremely high variation degree, wherein the influenza a virus with the most frequent variation belongs to a large antigenic variation every ten years, and a new strain is generated, and the variation is called antigenic transformation/antigenic qualitative change; small variations in antigen, mainly point mutations in the amino acid sequence of the antigen, known as antigenic drift/amount of antigen, also occur within influenza virus subtypes, thus making it impossible for people to have vaccines that are effective in preventing influenza for a long period of time.
Research on the pathogenic mechanism of influenza virus in the field of medicine has been carried out for a long time. Animal experiment research shows that a plurality of pathogenic pathways can induce respiratory tract infection to further cause serious respiratory system diseases, inflammation caused by the respiratory system diseases is mainly concentrated in animal lungs and is shown in a pneumonia form, for example, histopathology examination of respiratory tract infection of the lungs of a mouse can find lesions such as destruction of a mouse pulmonary alveolar structure, lung interval fracture, necrosis and shedding of pulmonary alveolar epithelial cells and the like; lung interval broadening can be seen in lung tissues of a small part of mice; epithelial cell proliferation was observed in mouse local lesion lung tissue.
Currently, the world health organization considers that annual influenza pandemic pre-vaccination is the most effective prophylactic. At present, trivalent influenza vaccines are marketed, including both inactivated influenza vaccine (TIV) and attenuated live vaccine (LAIV), and are composed of 3 viruses, including 2 influenza a virus strains and 1 influenza b virus strain. The medicine for treating related influenza infection mainly comprises two western medicines, one is neuraminidase inhibitor such as oseltamivir, zanamivir and peramivir, and the mechanism is that the virus particles can not invade human cells through glycoprotein neuraminidase acting on the surface of the virus; another class are M2 ion channel blockers such as amantadine and rimantadine, which act on the proton channel M2 protein to inhibit influenza a virus replication by blocking its protein ion channel.
However, vaccine injections do not effectively protect the body from infection by viruses for long periods of time, and drug therapy has side effects on the central nervous system while killing the virus. Therefore, there is still a need for a medicament or treatment that is effective in protecting the body from influenza virus infection for a long period of time, and that can also alleviate some of the clinical symptoms of influenza, while not causing side effects to the central nervous system of the patient.
In recent years, a number of studies have shown that intestinal microorganisms play an important role in maintaining human health, while the effects of probiotics on health in human intervention studies include improving acute diarrhea in children, alleviating childhood milk allergy, atopic dermatitis and alleviating irritable bowel syndrome in humans, and probiotics may exert an effect through intestinal mucosa, balance local microbiota by inhibiting the growth of pathogenic microorganisms, thereby enhancing local and systemic immune responses, and in addition, probiotics may also affect the composition and activity of microbiota in intestinal contents; it has also been reported that influenza caused by viruses can affect gut flora structure and that specific probiotics can be effective in reducing the duration and severity of acute rotavirus gastroenteritis. Therefore, starting from intestinal microorganisms, attempts are made to find new drugs or new methods for preventing and treating influenza, so as to overcome the defects of obvious side effects and the like of the existing therapeutic drugs and therapeutic methods.
Disclosure of Invention
In order to solve the problems, the invention screens out a Bifidobacterium breve. The Bifidobacterium breve has anti-influenza effect, and is specifically characterized in that: (1) the weight loss degree of influenza mice is obviously improved; (2) the blood index of the influenza mice is obviously improved; (3) the respiratory tract infection inflammation condition of an influenza mouse is obviously improved; (4) the expression quantity of the influenza mouse pulmonary antiviral protein MxA is obviously enhanced, so the Bifidobacterium breve has great application prospect in preparing products for preventing and/or treating influenza.
The technical scheme of the invention is as follows:
the invention provides a Bifidobacterium breve (CCFM1026) strain, wherein the CCFM1026 has been preserved in Guangdong province microbial strain preservation center in 2018, 10 and 11 months, the preservation number is GDMCC No.60459, and the preservation address is No. 59, 5-th building of Michelia Torrensis No. 100, Guangzhou City.
The Bifidobacterium breve (Bifidobacterium breve) CCFM1026 is obtained by separating a human excrement sample, the sequence of 16S rRNA of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence is compared in GenBank, and the result shows that the coverage rate (Query) of the strain and the Bifidobacterium breve is 100 percent and the similarity (Ident) is 99 percent, so the strain is judged to be the Bifidobacterium breve and named as the Bifidobacterium breve (Bifidobacterium breve) CCFM 1026.
The taxonomic characteristics are as follows: the thallus is in a short rod shape, gram-positive, irregular in methylene blue staining, free of spores, flagella and capsules and motionless.
The bacterial colony is characterized in that: circular white.
The growth characteristics are as follows: culturing at 37 deg.C under anaerobic condition for 30 hr to reach stationary phase, and performing atypical lactic acid fermentation with glucose.
The invention provides application of the Bifidobacterium breve (CCFM1026) in preparing a product for preventing and/or treating influenza.
In one embodiment of the invention, the viable count of Bifidobacterium breve (CCFM1026) in the product is not less than 1 × 106CFU/mL。
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the medicament contains Bifidobacterium breve (Bifidobacterium breve) CCFM1026, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the food product comprises a dairy product, a soy product or a fruit and vegetable product produced by using a starter culture containing Bifidobacterium breve (CCFM 1026); or the food product comprises a solid beverage comprising Bifidobacterium breve (CCFM 1026).
The invention provides a product for preventing and/or treating influenza, which contains the Bifidobacterium breve (CCFM1026) for preventing and/or treating influenza.
In one embodiment of the invention, the viable count of Bifidobacterium breve (CCFM1026) in the product is not less than 1 × 106CFU/mL。
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the medicament contains Bifidobacterium breve (Bifidobacterium breve) CCFM1026, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the food product comprises a dairy product, a soy product or a fruit and vegetable product produced by using a starter culture containing Bifidobacterium breve (CCFM 1026); or the food product comprises a solid beverage comprising Bifidobacterium breve (CCFM 1026).
In one embodiment of the invention, the preparation method of the leaven comprises the steps of inoculating (Bifidobacterium breve) CCFM1026 into a culture medium according to an inoculation amount accounting for 5-8% of the total mass of the culture medium, and culturing for 30 hours at 37 ℃ in an anaerobic environment to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the bacteria with phosphate buffer solution with the pH of 7.2 for 2-4 times, and then resuspending the bacteria with trehalose freeze-drying protective agent containing 100g/L to obtain a resuspension solution; freeze-drying the heavy suspension by vacuum freezing method to obtain (Bifidobacterium breve) CCFM1026 bacterial powder; the mass ratio of the freeze-drying protective agent to the thallus is 2: 1.
In one embodiment of the invention, the medium comprises 87.7% water, 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract solubles by total mass of the medium.
In one embodiment of the invention, the pH of the medium is 6.8.
In one embodiment of the invention, the protective agent comprises 100g/L skimmed milk powder, 30mL/L glycerin, 100g/L maltodextrin, 150g/L trehalose and 10g/L sodium L-glutamate.
Has the advantages that:
the invention screens out a Bifidobacterium breve CCFM1026, and the Bifidobacterium breve CCFM1026 has the function of anti-influenza, which is specifically represented as follows:
(1) the weight loss degree of influenza mice is obviously improved;
(2) the blood index of the influenza mice is obviously improved;
(3) the respiratory tract infection inflammation condition of an influenza mouse is obviously improved;
(4) obviously enhances the expression quantity of the influenza mouse pulmonary antiviral protein MxA,
therefore, the Bifidobacterium breve (Bifidobacterium breve) CCFM1026 has a huge application prospect in preparing products (such as food, medicines or health products and the like) for preventing and/or treating influenza.
Biological material preservation
A Bifidobacterium breve (CCFM1026) is classified and named as Bifidobacterium breve, is preserved in Guangdong province microorganism strain preservation center in 2018, 10 and 11 days, and has the preservation number of GDMCC No.60459 and the preservation address of No. 59 building and No. 5 building of No. 100 institute of Xieli Zhonglu, Guangzhou city.
Drawings
FIG. 1: body weight changes of different groups of influenza mice were compared.
FIG. 2: and comparing blood detection indexes (neutrophils) of different groups of influenza mice.
FIG. 3: and comparing blood detection indexes (lymphocytes) of different groups of influenza mice.
FIG. 4: lung histopathological sections of different groups of influenza mice were compared.
FIG. 5: and comparing the expression levels of the pulmonary antiviral protein MxA of the influenza mice of different groups.
Detailed Description
The invention is further illustrated with reference to specific examples.
The media involved in the following examples are as follows:
MRS plate (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H 20 0.1g/L、MnSO40.05g/L, Tween 801 ml/L, agar 20g/L and cysteine hydrochloride 0.5 g/L.
Example 1: screening and identification of Bifidobacterium breve CCFM1026 and B1
1. Screening
Taking human feces as sample, storing the sample in about 20% glycerol at-80 deg.C, thawing, mixing, sucking 0.5mL sample, adding into 4.5mL 0.9% physiological saline containing 0.05% cysteine, diluting in gradient, selecting 10-3、10-4、10-5And (3) coating the diluted solution on an MRS plate added with 0.05% cysteine, culturing for 48h at 37 ℃, selecting a typical colony on the MRS plate, streaking and purifying, selecting a single colony, transferring to a liquid MRS culture medium (containing 0.05% cysteine) for enrichment, and preserving by 30% glycerol to obtain the strains CCFM1026 and B1.
2. Identification
The genome of CCFM1026 and B1 is extracted, 16S rDNA of CCFM1026 and B1 is amplified and sequenced (Shanghai biological engineering Co., Ltd.), and the sequences are compared in GenBank, so that the coverage rate (Query) of the strain and the coverage rate (Query) of the strain are both 100 percent, and the similarity (Ident) of the strain and the Bifidobacterium breve is both 99 percent, so that the strain is judged to be the Bifidobacterium breve and named as Bifidobacterium breve (Bifidobacterium breve) CCFM1026 and Bifidobacterium breve (Bifidobacterium breve) B1.
Example 2: culture of Bifidobacterium breve
Culturing Bifidobacterium breve (CCFM1026) in MRS culture medium (containing 0.05% cysteine) at 37 deg.C for 48 hr, transferring into fresh MRS culture medium (containing 0.05% cysteine), culturing under the same conditions for 30 hr, centrifuging thallus for 15min at 6000g, washing thallus with 0.9% physiological saline, centrifuging again at 6000g for 10min to obtain thallus, resuspending with 30% sucrose solution, and freezing at-80 deg.C.
The thalli is observed, and the taxonomic characteristics are as follows: the thallus is in a short rod shape, gram staining is positive, methylene blue staining is irregular, spores, flagella and capsules do not exist, and the thallus does not move; the bacterial colony is characterized in that: round white; the growth characteristics are as follows: culturing at 37 deg.C under anaerobic condition for 30 hr to reach stationary phase, and performing atypical lactic acid fermentation with glucose.
Example 3: effect of Bifidobacterium breve on body weight of influenza mice
32 healthy ICR female mice with the weight of 20-24g are taken and randomly divided into 4 groups, and the 4 groups are respectively named as: blank Control group (Control), influenza Model group (Model), drug Treatment group (Treatment) for administering ribavirin, dry pretreatment group for intragastric bifidobacterium breve CCFM1026 (CCFM1026), dry pretreatment group for intragastric bifidobacterium breve B1 (group B1), and 8 of each group.
2 weeks before the experiment, Bifidobacterium breve intervention group (CCFM1026) was gavaged daily for 10 weeks9The suspension diluent of bifidobacterium breve with CFU bacterial quantity, the suspension diluent of bifidobacterium breve B1 with the same quantity as that of the dry group (B1) of bifidobacterium breve B1, and the suspension diluent of the other groups (Control, Model and Treatment) are intragastrically infused with 0.2mL of normal saline every day.
On day 1 of the experiment, 4 groups were included, except for the blank Control group (Control)The method comprises treating mice with influenza virus by nasal drip after light anesthesia with diethyl ether, and intragastric administering on the same day, wherein Bifidobacterium breve intervention group (CCFM1026) is intragastric administered 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
On the 2 nd day of the experiment, the drug ribavirin was administered to the ribavirin drug Treatment group (Treatment) and the drug ribavirin was administered intraperitoneally for Treatment.
Continuing the gavage 4 days after the challenge, wherein Bifidobacterium breve (CCFM1026) is gavage 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
For 5 days of the experiment, the weight was weighed before gavage every day, and five-day weight changes were recorded continuously, and the results are shown in fig. 1.
As can be seen from fig. 1, the body weight of the mice began to decrease at day 3 after challenge, and the body weight decreased most significantly at day 4 (P <0.05), whereas the body weight decreased by more than 10% in the Model group (Model), about 5% in the drug Treatment group (Treatment), and only 5% in the bifidobacterium breve intervention group (CCFM1026) and the bifidobacterium breve B1, as compared to the Control group (Control).
The bifidobacterium breve CCFM1026 and B1 can obviously improve the weight loss symptom of influenza mice.
Example 4: effect of Bifidobacterium breve on blood index of influenza mice
40 healthy ICR female mice with the weight of 20-24g are taken and randomly divided into 5 groups, and the 5 groups are respectively named as: blank Control group (Control), influenza Model group (Model), ribavirin drug administration Treatment group (Treatment), gavage bifidobacterium breve CCFM1026 dry prediction group (CCFM1026), and gavage bifidobacterium breve B1 dry prediction group (B1), wherein each group comprises 8.
2 weeks before the experiment, the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was intragastrically administered 109Bifidobacterium breve with CFU bacterial countAnd (3) intragastrically irrigating 0.2mL of physiological saline solution for the rest groups (Control, Model and Treatment) every day by using the strain suspension diluent, wherein the Bifidobacterium breve B1 dried group (B1) is intragastrically irrigated with the same quantity of Bifidobacterium breve B1 strain suspension diluent.
On the 1 st day of experiment, except for the blank Control group (Control), the other 4 groups were subjected to challenge with influenza virus by nasal drip method after light anesthesia with diethyl ether, and were subjected to intragastric administration on the same day, wherein Bifidobacterium breve intervention group (CCFM1026) was intragastric administered 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
On the 2 nd day of the experiment, the drug ribavirin was administered to the ribavirin drug Treatment group (Treatment) and the drug ribavirin was administered intraperitoneally for Treatment.
Continuing the gavage 4 days after the challenge, wherein Bifidobacterium breve (CCFM1026) is gavage 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
The mice were sacrificed after blood was taken on day 5 after challenge, and the blood taken out of the mice was placed in an anticoagulation tube, gently shaken to make the blood sufficiently contact with the anticoagulation agent, and then sent to an animal hospital for routine blood analysis and detection (at the initial stage of influenza virus infection, a large number of natural immune cells such as neutrophils and lymphocytes participate in the defense process, and therefore, the routine blood analysis and detection mainly detects changes of neutrophils and lymphocytes), and the results are shown in fig. 2-3.
As can be seen from fig. 2 to 3, the percentage of neutrophils and lymphocytes in the Model group (Model) mice was significantly abnormal compared to the Control group (Control), and significantly different from the Control group (p values of 0.0015 and 0.0011, respectively), whereas the percentage of neutrophils and lymphocytes in the bifidobacterium breve dry pretreatment group (CCFM1026) and the drug Treatment group (Treatment) mice tended to the Control group (Control) without significant difference.
The bifidobacterium breve has the same effect as ribavirin medicine, and can actively participate in the relevant influenza immunoregulation of mice to relieve the influenza symptoms of the mice.
Example 5: effect of Bifidobacterium breve on inflammation of respiratory tract infection in influenza mice
40 healthy ICR female mice with the weight of 20-24g are taken and randomly divided into 5 groups, and the 5 groups are respectively named as: blank Control group (Control), influenza Model group (Model), ribavirin drug administration Treatment group (Treatment), gavage bifidobacterium breve CCFM1026 dry prediction group (CCFM1026), and gavage bifidobacterium breve B1 dry prediction group (B1), wherein each group comprises 8.
2 weeks before the experiment, the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was intragastrically administered 109The suspension diluent of bifidobacterium breve with CFU bacterial quantity, the suspension diluent of bifidobacterium breve B1 with the same quantity as that of the dry group (B1) of bifidobacterium breve B1, and the suspension diluent of the other groups (Control, Model and Treatment) are intragastrically infused with 0.2mL of normal saline every day.
On the 1 st day of experiment, except for the blank Control group (Control), the other 4 groups were subjected to challenge with influenza virus by nasal drip method after light anesthesia with diethyl ether, and were subjected to intragastric administration on the same day, wherein Bifidobacterium breve intervention group (CCFM1026) was intragastric administered 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
On the 2 nd day of the experiment, the drug ribavirin was administered to the ribavirin drug Treatment group (Treatment) and the drug ribavirin was administered intraperitoneally for Treatment.
Continuing the gavage 4 days after the challenge, wherein Bifidobacterium breve (CCFM1026) is gavage 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day.
The mice were sacrificed after blood was taken on day 5 after challenge, the left lungs of the mice were immediately taken out and fixed in 4% paraformaldehyde, histopathological sections were performed after fixation, histopathological sections of the lungs of the mice were stained with hematoxylin-eosin after sectioning, histopathological sections of the lungs of the mice were scored by professional technicians for histopathology, and the results are shown in fig. 4.
As can be seen from FIG. 4, the lung tissue structure of the mice in the blank group (Control) is relatively complete and has no inflammatory cell infiltration; the Model group (Model) mouse has severe inflammatory infiltration phenomenon and even has local bleeding phenomenon; moderate inflammatory infiltration near the bronchi occurred in the bifidobacterium breve B1 intervention group (B1), while the lung was relatively mild in the mice in the Treatment group (Treatment) and bifidobacterium breve intervention group (CCFM).
The animal experiments show that influenza infection can cause mice to suffer from influenza pneumonia, and after the lung is infected by virus, the tissue structure of the mice is damaged, and a large amount of inflammatory infiltration occurs; the bifidobacterium breve can relieve the pulmonary inflammation of mice and relieve the pneumonia symptoms of the mice, and has equivalent effect to that of a common influenza medicine ribavirin medicine treatment group.
Example 6: effect of Bifidobacterium breve on pulmonary viral load in influenza mice
40 healthy ICR female mice with the weight of 20-24g are taken and randomly divided into 5 groups, and the 5 groups are respectively named as: blank Control group (Control), influenza Model group (Model), ribavirin drug administration Treatment group (Treatment), gavage bifidobacterium breve CCFM1026 dry prediction group (CCFM1026), and gavage bifidobacterium breve B1 dry prediction group (B1), wherein each group comprises 8.
2 weeks before the experiment, the Bifidobacterium breve CCFM1206 intervention group (CCFM1206) was intragastrically administered 109The suspension diluent of bifidobacterium breve with CFU bacterial quantity, the suspension diluent of bifidobacterium breve B1 with the same quantity as that of the dry group (B1) of bifidobacterium breve B1, and the suspension diluent of the other groups (Control, Model and Treatment) are intragastrically infused with 0.2mL of normal saline every day.
On the 1 st day of experiment, except for the blank Control group (Control), the other 4 groups were subjected to challenge with influenza virus by nasal drip method after light anesthesia with diethyl ether, and were subjected to intragastric administration on the same day, wherein Bifidobacterium breve intervention group (CCFM1026) was intragastric administered 109CFU (carbon fiber Unit) bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) gastric lavage same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the rest groups (Control, Model, Treatmen)t) gavage 0.2mL of physiological saline daily.
On the 2 nd day of the experiment, the drug ribavirin was administered to the ribavirin drug Treatment group (Treatment) and the drug ribavirin was administered intraperitoneally for Treatment.
Continuing the gavage 4 days after the challenge, wherein Bifidobacterium breve (CCFM1026) is gavage 109CFU bacterial amount of Bifidobacterium breve CCFM1026 bacterial suspension diluent, Bifidobacterium breve B1 dried group (B1) is intragastrically perfused with the same amount of Bifidobacterium breve B1 bacterial suspension diluent, and the other groups (Control, Model, Treatment) are intragastrically perfused with 0.2mL of normal saline every day. Blood is taken on the 5 th day after the challenge, the mouse is killed, after the mouse is killed, the right lung tissue of the mouse is taken and placed in 1ml of TRIZOL to be frozen and stored in a refrigerator at minus 80 ℃ for standby, when in extraction, a right lung tissue sample is placed on ice to be melted, then is ground by adopting a DEPC-treated sterile grinder, 200ul of chloroform is added, after the mixture is fully mixed, the mixture is centrifuged at 4 ℃ and 12000rpm for 10min, 500 mu L of supernatant is removed, isopropanol with the same volume is added, after the mixture is mixed, the mixture is centrifuged at 4 ℃ and 12000rpm for 10min, the supernatant is removed, 75% of ethanol is added to wash RNA once, the mixture is centrifuged at 4 ℃ and 12000rpm for 10min, after the supernatant is removed, after the ethanol is volatilized, 40 mu L of DEPC-treated water is added to dissolve the RNA, and the extracted RNA is frozen and stored in a refrigerator at minus 80 ℃ for standby.
The relative expression quantity of MxA (organism can perform defense reaction to influenza virus to eliminate invaded virus and recover health, MxA is an antiviral protein secreted by MxA and can effectively prevent virus replication) is determined by utilizing a qPCR method, GAPDH is used as an internal reference, and classical 2 is adopted-ΔΔtThe calculation method, using the model group as the comparison processing data, results are shown in fig. 5.
As can be seen from fig. 5, the pulmonary MxA expression level of the mice in the drug Treatment group (Treatment) is 2.62 times that of the Model group (Model), the pulmonary MxA expression level of the mice in the bifidobacterium breve B1 intervention group is 2.67 times that of the Model group (Model), and there is no significant difference, while the pulmonary MxA expression level of the mice in the bifidobacterium breve intervention group (CCFM1026) is 3.46 times that of the Model group (Model), and the pulmonary MxA expression level of the mice in the bifidobacterium breve intervention group (CCFM1026) can be significantly increased (p value is 0.0292).
The experiments show that the bifidobacterium breve CCFM1026 can obviously enhance the immunity of influenza mice and promote the expression quantity of MxA antiviral protein to be increased, thereby being capable of resisting virus replication and helping the body to recover health, obviously reducing respiratory inflammation and having the effect even better than that of medicament treatment.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> bifidobacterium breve with anti-influenza capacity and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1395
<212> DNA
<213> Artificial sequence
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caagtcgaac gggatccatc gggctttgcc tggtggtgag agtggcgaac gggtgagtaa 60
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tgctccatct caccgcatgg tgttttggga aagcctttgc ggcatgggat ggggtcgcgt 180
cctatcagct tgatggcggg gtaacggccc accatggctt cgacgggtag ccggcctgag 240
agggcgaccg gccacattgg gactgagata cggcccagac tcctacggga ggcagcagtg 300
gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatggaggcc 360
ttcgggttgt aaacctcttt tgttagggag caaggcattt tgtgttgagt gtacctttcg 420
aataagcacc ggctaactac gtgccagcag ccgcggtaat acgtagggtg caagcgttat 480
ccggaattat tgggcgtaaa gggctcgtag gcggttcgtc gcgtccggtg tgaaagtcca 540
tcgcttaacg gtggatccgc gccgggtacg ggcgggcttg agtgcggtag gggagactgg 600
aattcccggt gtaacggtgg aatgtgtaga tatcgggaag aacaccaatg gcgaaggcag 660
gtctctgggc cgttactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 720
ccctggtagt ccacgccgta aacggtggat gctggatgtg gggcccgttc cacgggttcc 780
gtgtcggagc taacgcgtta agcatcccgc ctggggagta cggccgcaag gctaaaactc 840
aaagaaattg acgggggccc gcacaagcgg cggagcatgc ggattaattc gatgcaacgc 900
gaagaacctt acctgggctt gacatgttcc cgacgatccc agagatgggg tttcccttcg 960
gggcgggttc acaggtggtg catggtcgtc gtcagctcgt gtcgtgagat gttgggttaa 1020
gtcccgcaac gagcgcaacc ctcgccccgt gttgccagcg gattgtgccg ggaactcacg 1080
ggggaccgcc ggggttaact cggaggaagg tggggatgac gtcagatcat catgcccctt 1140
acgtccaggg cttcacgcat gctacaatgg ccggtacaac gggatgcgac agcgcgagct 1200
ggagcggatc cctgaaaacc ggtctcagtt cggatcgcag tctgcaactc gactgcgtga 1260
aggcggagtc gctagtaatc gcgaatcagc aacgtcgcgg tgaatgcgtt cccgggcctt 1320
gtacacaccg cccgtcaagt catgaaagtg ggcagcaccc gaagccggtg gcctaacccc 1380
ttgcgggagg gagcc 1395
Claims (10)
1. A strain of Bifidobacterium breve (Bifidobacterium breve) is characterized in that the Bifidobacterium breve (Bifidobacterium breve) is preserved in the microbial strain preservation center of Guangdong province in 2018, 10 and 11 days, the preservation number is GDMCC No.60459, and the preservation address is No. 59 building 5 of Ji Dazhou No. 100 of Jieli Zhou, Guangzhou city.
2. Use of a strain of Bifidobacterium breve (Bifidobacterium breve) according to claim 1 for the preparation of a product intended for the prophylaxis and/or treatment of influenza.
3. Use of a strain of Bifidobacterium breve (Bifidobacterium breve) according to claim 2 for the preparation of a product for the prophylaxis and/or treatment of influenza, wherein the Bifidobacterium breve (Bifidobacterium breve) has a viable count of not less than 1 x 106CFU/mL。
4. Use of a strain of Bifidobacterium breve (Bifidobacterium breve) according to claim 2 or 3 for the preparation of a product for the prevention and/or treatment of influenza, wherein the product comprises a food, pharmaceutical or nutraceutical product.
5. Use of a strain of Bifidobacterium breve (Bifidobacterium breve) according to claim 4 for the preparation of a product for the prophylaxis and/or treatment of influenza, wherein the product comprises Bifidobacterium breve (Bifidobacterium breve), a pharmaceutical carrier and/or a pharmaceutical excipient.
6. A product for the prevention and/or treatment of influenza, comprising Bifidobacterium breve (Bifidobacterium breve) as claimed in claim 1.
7. The product for preventing and/or treating influenza according to claim 6, wherein the viable count of Bifidobacterium breve is not less than 1 x 106CFU/mL。
8. The product for the prevention and/or treatment of influenza according to claim 6 or 7, wherein the product comprises a food, a pharmaceutical or a nutraceutical.
9. The product for the prevention and/or treatment of influenza according to claim 8, wherein the product comprises Bifidobacterium breve (Bifidobacterium breve), a pharmaceutical carrier and/or a pharmaceutical excipient.
10. The product for preventing and/or treating influenza according to claim 8, wherein the food comprises a dairy product, a soy product or a fruit and vegetable product produced using a starter culture containing Bifidobacterium breve; or the food product comprises a solid beverage containing Bifidobacterium breve.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080107634A1 (en) * | 2004-11-16 | 2008-05-08 | Anidral S.R.L. | Probiotic Bacteria Based Composition and Use Thereof in the Prevention and/or Treatment of Respiratory Pathologies and/or Infections and in the Improvement of the Intestinal Functionality |
| CN104168905A (en) * | 2011-06-10 | 2014-11-26 | 杜邦营养生物科学有限公司 | Treatment of respiratory tract illness with bifidobacterium lactis bl-04 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080107634A1 (en) * | 2004-11-16 | 2008-05-08 | Anidral S.R.L. | Probiotic Bacteria Based Composition and Use Thereof in the Prevention and/or Treatment of Respiratory Pathologies and/or Infections and in the Improvement of the Intestinal Functionality |
| CN104168905A (en) * | 2011-06-10 | 2014-11-26 | 杜邦营养生物科学有限公司 | Treatment of respiratory tract illness with bifidobacterium lactis bl-04 |
Non-Patent Citations (2)
| Title |
|---|
| HISAKO YASUI et al..Protection against Influenza Virus Infection of Mice Fed Bifidobacterium breve YIT4064.《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》.1999,第6卷(第2期),摘要. * |
| Protection against Influenza Virus Infection of Mice Fed Bifidobacterium breve YIT4064;HISAKO YASUI et al.;《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》;19990331;第6卷(第2期);摘要 * |
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