CN110055185A - One plant of bifidobacterium breve and its application with anti influenza ability - Google Patents
One plant of bifidobacterium breve and its application with anti influenza ability Download PDFInfo
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- CN110055185A CN110055185A CN201811589535.1A CN201811589535A CN110055185A CN 110055185 A CN110055185 A CN 110055185A CN 201811589535 A CN201811589535 A CN 201811589535A CN 110055185 A CN110055185 A CN 110055185A
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- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- 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
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- 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
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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
- C12N1/205—Bacterial isolates
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The bifidobacterium breve for having anti influenza ability the invention discloses one plant and its application, belong to microorganisms technical field and pharmaceutical technology field.Bifidobacterium breve (Bifidobacterium breve) of the invention has the function of anti influenza, is embodied in: (1) significantly improving influenza mouse weight decline degree；(2) influenza mouse blood index is significantly improved；(3) the respiratory tract infection inflammatory conditions of influenza mouse are significantly improved；(4) expression quantity of influenza mouse lung antiviral protein MxA is significantly increased, therefore, bifidobacterium breve of the invention (Bifidobacterium breve) has huge application prospect in the product of preparation prevention and/or treatment influenza.
Description
Technical field
The present invention relates to one plant with anti influenza ability bifidobacterium breve and its application, belong to microorganisms technical field with
And pharmaceutical technology field.
Background technique
Influenza is often very popular in autumn and winter, is mainly induced by influenza virus.Influenza virus can be divided into influenza A virus,
Three kinds of influenza B virus, influenza virus C, the influenza that people is suffered from is mainly by influenza A virus and influenza B virus
It is caused.General animal influenza virus will not infect people, human influenza virus will not infection animal, but outside pig comparative example.Pig
Both avian influenza virus can be infected, human influenza virus, but main or infection avian influenza virus can also be infected, but, they
Once infecting avian influenza virus, it is just easily transmitted to people, human influenza is caused to be very popular.
For example, " spanish influenza " that 1819-1920 occurs." spanish influenza " is one of most serious in world history
Secondary flu outbreak, coverage is wide, and clinical incidence is up to 40%, and with a plurality of types of complications of pneumonia, causes
The death of ten thousand people of 2000-4000, death toll is far beyond the World War I.Because being limited by scientific and technical condition, when
When people fail to isolate the pathogenic original of " spanish influenza ", until 1997, American scientist delivered title on " Science "
Influenza virus in 1918 is quite similar with swine influenza virus, is a kind of and influenza A virus (H1N1) closely related disease
Poison.And in subsequent nearly 100 year time, multiple flu outbreak has still been broken out in world wide, causes different degrees of personnel
It suffers a calamity or disaster and economic loss.
It can be said that just be unable to completely control always since occurring from influenza, discontinuous outburst is presented.And it can not be complete
One main cause of control is that, although most influenza viruses are thermo-labile, heating 30 minutes at 56 DEG C can inactivate,
Infectiousness can also be lost quickly at room temperature, and still, the degree of variation of influenza virus is high, wherein making a variation most frequent should belong to first
Every the more than ten years one antigen Big mutation rate will occur for type influenza virus, generate a new strain, and this variation is referred to as antigen
The qualitative change of transformation/antigen；The small variation of antigen, the mainly point of antigen amino acid sequence can also occur in influenza virus sub-strain
Mutation, referred to as antigenic drift/antigen quantitative change, so that people can not possess the vaccine of permanently effective flu-prevention.
The pathogenesis of field of medicaments infected by influenza is carried out already.Animal experiment study shows that there are many cause a disease
Access can induce the infection of respiratory tract, then cause serious respiratory disease, and the inflammation master that respiratory disease causes
Concentrate on animal lung, show as pneumonia form, for example, histopathological examination respiratory tract infection mouse lung can find it is small
Mouse alveolar structure is destroyed, the fracture of lung interval, the lesions such as alveolar epithelial cells is downright bad, falls off；Small part mouse lung tissue is visible
Lung interval is broadening；The visible epithelial hyperplasia of mouse local patholoic change lung tissue.
Currently, the World Health Organization thinks that vaccine inoculation early period is most effective prevention hand on influenza pandemic peak every year
Section.At this stage, the trivalent flu vaccine listed has two kinds of inactivated influenza vaccine (TIV) and attenuated live vaccine (LAIV), by 3
Kind virus composition, includes 2 kinds of influenza A virus strains and a kind of influenza B virus strain.And the drug therapy of related influenza infection
There are two main classes Western medicine, one kind is neuraminidase inhibitor such as Oseltamivir, zanamivir, Peramivir, and mechanism is logical
The glycoprotein neuraminidase for acting on virus surface is crossed, makes virion that can not encroach on human body cell；Another kind of is M2 ion
Channel blocker such as amantadine and Rimantadine, such drug is to act on proton channel M2 albumen, by obstructing its albumen
Ion channel is to inhibit the duplication of influenza A virus.
But vaccine injection cannot permanent effective protection body not by virus infection, and drug therapy is killing virus
Central nervous system has side effect simultaneously.Therefore, there is still a need for a kind of drug or therapeutic modality, it can either permanent effective protection
Body is not infected by influenza virus, can also alleviate some clinical diseases of influenza, meanwhile, patient's central nervous system will not be given
Bring side effect.
In recent years, a large amount of studies have shown that enteric microorganism is for maintaining human health to have important role, meanwhile, benefit
Influence of the raw bacterium in human intervention research to health includes improving acute diarrhea in children, alleviates milk for children allergy, idiocrasy
Dermatitis and alleviation people's irritable bowel syndrome, also, probiotics may be had an impact by intestinal mucosa, by inhibiting cause of disease micro-
The growth of biology balances topical microbial group, and then enhances and be locally and systemically immunoreacted, in addition, probiotics can also affect on
The composition and activity of micropopulation in intestinal contents；Also it has been reported and points out, influenza caused by virus can influence intestinal flora
Structure, and specific probiotics can effectively mitigate duration and the severity of acute rotavirus gastroenteritis.Therefore, perhaps may be used
Start with from enteric microorganism, attempt to find prevention and treat the novel drugs or new method of influenza, to overcome existing therapeutic agent
And the defect for the treatment of method side effect obviously etc..
Summary of the invention
To solve the above problems, the present invention has filtered out one plant of bifidobacterium breve (Bifidobacterium breve).This
Bifidobacterium breve (Bifidobacterium breve) has the function of anti influenza, is embodied in: (1) significantly improving influenza
Mouse weight declines degree；(2) influenza mouse blood index is significantly improved；(3) respiratory tract infection of influenza mouse is significantly improved
Inflammatory conditions；(4) expression quantity of influenza mouse lung antiviral protein MxA, therefore, this bifidobacterium breve are significantly increased
(Bifidobacterium breve) has huge application prospect in the product of preparation prevention and/or treatment influenza.
Technical scheme is as follows:
The present invention provides one plant of bifidobacterium breve (Bifidobacterium breve) CCFM1026, the short bifids
Bacillus (Bifidobacterium breve) CCFM1026 has been preserved in Guangdong Province microorganism fungus kind guarantor on October 11st, 2018
Hiding center, deposit number are GDMCC No.60459, and preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
Bifidobacterium breve (Bifidobacterium breve) CCFM1026 is isolated from human excrement and urine's sample
, through sequencing analysis, 16S rRNA sequence compares in GenBank the bacterial strain as shown in SEQ ID NO.1, by the sequence
Right, the coverage rate (Query cover) of bacterial strain and bifidobacterium breve is 100% as the result is shown, similarity (Ident) is 99%,
Therefore determine that bacterial strain is bifidobacterium breve, it is named as bifidobacterium breve (Bifidobacterium breve) CCFM1026.
Its taxology feature are as follows: thallus is in corynebacterium, and Gram-positive, methylene blue staining coloring is irregular, nothing
Gemma, flagellum and pod membrane, do not move.
Its colony characteristics are as follows: circular white.
Its growth characteristics are as follows: cultivate 30h under 37 DEG C of anaerobic conditions up to stationary phase, it is different to carry out atypia using glucose
Property lactic fermentation approach.
It is pre- in preparation that the present invention provides above-mentioned one plant of bifidobacterium breve (Bifidobacterium breve) CCFM1026
Application in anti-and/or treatment influenza product.
In one embodiment of the present invention, in the product, bifidobacterium breve (Bifidobacterium breve)
The viable count of CCFM1026 is not less than 1 × 106CFU/mL。
In one embodiment of the present invention, the product includes food, drug or health care product.
In one embodiment of the present invention, the drug contains bifidobacterium breve (Bifidobacterium breve)
CCFM1026, pharmaceutical carrier and/or pharmaceutic adjuvant.
In one embodiment of the present invention, the food includes to use to contain bifidobacterium breve (Bifidobacterium
Breve dairy products, bean product or the fruit and vegetable product that) leavening of CCFM1026 produces；Or the food includes containing short
The solid beverage of Bifidobacterium (Bifidobacterium breve) CCFM1026
The present invention provides a kind of for preventing and/or treating the product of influenza, and the product contains above-mentioned short bifid bar
Bacterium (Bifidobacterium breve) CCFM1026.
In one embodiment of the present invention, in the product, bifidobacterium breve (Bifidobacterium breve)
The viable count of CCFM1026 is not less than 1 × 106CFU/mL。
In one embodiment of the present invention, the product includes food, drug or health care product.
In one embodiment of the present invention, the drug contains bifidobacterium breve (Bifidobacterium breve)
CCFM1026, pharmaceutical carrier and/or pharmaceutic adjuvant.
In one embodiment of the present invention, the food includes to use to contain bifidobacterium breve (Bifidobacterium
Breve dairy products, bean product or the fruit and vegetable product that) leavening of CCFM1026 produces；Or the food includes containing short
The solid beverage of Bifidobacterium (Bifidobacterium breve) CCFM1026.
In one embodiment of the invention, the preparation method of the leavening is by (Bifidobacterium
Breve) CCFM1026 is inoculated into culture medium according to the inoculum concentration for accounting for culture medium gross mass 5~8%, in 37 DEG C of anaerobic environment
Lower culture 30h, obtains culture solution；By medium centrifugal, thallus is obtained；Thallus is cleaned 2 with the phosphate buffer that pH is 7.2
It is resuspended after~4 times with the trehalose freeze drying protectant containing 100g/L, obtains re-suspension liquid；Re-suspension liquid vacuum-freezing process is lyophilized, is obtained
To (Bifidobacterium breve) CCFM1026 bacterium powder；Mass ratio between the freeze drying protectant and thallus is 2:1.
In one embodiment of the invention, the culture medium includes the water for accounting for culture medium gross mass 87.7%, 10%
Enzyme hydrolysis skimmed milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract dissolution.
In one embodiment of the invention, the pH of the culture medium is 6.8.
In one embodiment of the invention, the protective agent include the skimmed milk power of 100g/L, 30mL/L glycerol,
The L-sodium of the maltodextrin of 100g/L, the trehalose of 150g/L and 10g/L.
The utility model has the advantages that
The present invention has filtered out one plant of bifidobacterium breve (Bifidobacterium breve) CCFM1026, this short bifid
Bacillus (Bifidobacterium breve) CCFM1026 has the function of anti influenza, is embodied in:
(1) influenza mouse weight decline degree is significantly improved；
(2) influenza mouse blood index is significantly improved；
(3) the respiratory tract infection inflammatory conditions of influenza mouse are significantly improved；
(4) expression quantity of influenza mouse lung antiviral protein MxA is significantly increased,
Therefore, bifidobacterium breve (Bifidobacterium breve) CCFM1026 is in preparation prevention and/or treatment influenza
Product (such as food, drug or health care product) in, have huge application prospect.
Biomaterial preservation
One plant of bifidobacterium breve (Bifidobacterium breve) CCFM1026, taxology are named as
Bifidobacterium breve, is preserved in Guangdong Province's Culture Collection on October 11st, 2018, and preservation is compiled
Number be GDMCC No.60459, preservation address be the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
Detailed description of the invention
Fig. 1: the changes of weight comparison of different group influenza mouse.
Fig. 2: blood testing index (neutrophil leucocyte) comparison of different group influenza mouse.
Fig. 3: blood testing index (lymphocyte) comparison of different group influenza mouse.
Fig. 4: the pulmonary histopathology of different group influenza mouse is sliced comparison.
Fig. 5: the lung antiviral protein MxA of different group influenza mouse expression quantity comparison.
Specific embodiment
The present invention will be further elaborated combined with specific embodiments below.
Culture medium involved in following embodiments is as follows:
MRS plate (g/L): peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/
L, diammonium hydrogen citrate 2g/L, K2PO4·3H2O 2.6g/L、MgSO4·7H20 0.1g/L、MnSO40.05g/L, Tween 80
1ml/L, agar 20g/L, cysteine propylhomoserin salt 0.5g/L.
Embodiment 1: the screening and identification of bifidobacterium breve CCFM1026 and B1
1, it screens
Using human excrement and urine as sample, sample is stored in -80 DEG C of refrigerators in 20% or so glycerol, is taken out after thawing, is mixed
Even sample draws 0.5mL sample and is added to the 0.9% physiological saline progress gradient dilution that 4.5mL contains 0.05% cysteine, choosing
Select 10-3、10-4、10-5The dilution of extension rate is coated on the MRS plate for having added 0.05% cysteine, is cultivated in 37 DEG C
48h, picking colonies typical to the flat lining out purifying of MRS, picking single colonie are forwarded to liquid MRS culture medium (containing 0.05% half
Cystine) increase bacterium, 30% glycerol stocks obtain bacterial strain CCFM1026 and bacterial strain B1.
2, it identifies
The 16S rDNA of CCFM1026, B1 are expanded and are sequenced (Shanghai life by the genome for extracting CCFM1026, B1
Work bioengineering limited liability company), which is compared in GenBank, as the result is shown bacterial strain and bifidobacterium breve
Coverage rate (Query cover) be 100%, similarity (Ident) be 99%, therefore determine that bacterial strain is bifidobacterium breve,
It is named as bifidobacterium breve (Bifidobacterium breve) CCFM1026 and bifidobacterium breve (Bifidobacterium
breve)B1。
Embodiment 2: the culture of bifidobacterium breve
Bifidobacterium breve (Bifidobacterium breve) CCFM1026 (is contained into 0.05% half Guang ammonia in MRS culture medium
Acid) in after 37 DEG C of culture 48h, be transferred in fresh MRS culture medium (contain 0.05% cysteine), similarity condition culture 30h,
6000g is centrifuged thallus 15min, and 6000g is centrifuged 10min again after 0.9% brine thallus, thallus is obtained, with 30%
Sucrose solution is resuspended, and freezes stand-by at -80 DEG C.
Thallus is observed, taxology feature are as follows: thallus is in corynebacterium, Gram-positive, methylene blue dye
Chromatic colorant is irregular, and no gemma, flagellum and pod membrane do not move；Its colony characteristics are as follows: circular white；Its growth characteristics are as follows: in 37
Culture 30h utilizes glucose to carry out atypia specificity lactate fermentation approach up to stationary phase under DEG C anaerobic condition.
Embodiment 3: influence of the bifidobacterium breve to influenza mouse weight
Healthy ICR female mice 32 of weight 20-24g are taken, are randomly divided into 4 groups, 4 groups are respectively designated as: blank control group
(Control), influenza model group (Model), the medication therapy groups (Treatment) that Ribavirin is administered, the short bifid bar of stomach-filling
Bacterium CCFM1026 intervention group (CCFM1026), the short bifid intervention group (B1 group) of stomach-filling bifidobacterium breve B1, every group 8.
Bifidobacterium breve intervention group (CCFM1026) stomach-filling 10 is given in first 2 weeks of experiment daily9The bifidobacterium breve of CFU bacterium amount
Bacterium hangs dilution, and the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling hangs dilution, remaining group
(Control, Model, Treatment) stomach-filling daily 0.2mL physiological saline.
Test the 1st day, in addition to blank control group (Control), remaining 4 groups after light anaesthesia with diethyl ether nose drip method to flow
Influenza Virus carries out mouse to attack poison, and the same day still needs to stomach-filling, wherein bifidobacterium breve intervention group (CCFM1026) stomach-filling 109CFU bacterium
The bifidobacterium breve CCFM1026 bacterium of amount hangs dilution, the same amount of short bifid bar of bifidobacterium breve B1 intervention group (B1) stomach-filling
Bacterium B1 bacterium hangs dilution, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
It tests the 2nd day, administration Ribavirin medication therapy groups (Treatment) intraperitoneal injection of drugs Ribavirin is controlled
It treats.
Attack the continuation stomach-fillings in 4 days after poison, wherein bifidobacterium breve (CCFM1026) stomach-filling 109The short bifid of CFU bacterium amount
Bacillus CCFM1026 bacterium hangs dilution, and the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling is hanged dilute
Liquid is released, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
In 5 days of experiment, weigh before daily stomach-filling, it is continuous to record the variation of five daily weights, as a result as shown in Figure 1.
As shown in Figure 1, the 3rd day mouse weight after attacking poison starts to decline, weight loss (P the most obvious at the 4th day
< 0.05), compared with control group (Control), model group (Model) weight loss is more than 10%, medication therapy groups
(Treatment) weight loss about 5%, and bifidobacterium breve intervention group (CCFM1026) and bifidobacterium breve B1 group weight loss
It is also only 5%.
Illustrate that bifidobacterium breve CCFM1026 and B1 of the invention can significantly improve influenza mouse weight and mitigate symptom.
Embodiment 4: influence of the bifidobacterium breve to influenza mouse blood index
Healthy ICR female mice 40 of weight 20-24g are taken, are randomly divided into 5 groups, 5 groups are respectively designated as: blank control group
(Control), influenza model group (Model), administration Ribavirin medication therapy groups (Treatment), stomach-filling bifidobacterium breve
CCFM1026 intervention group (CCFM1026), the short bifid intervention group (B1 group) of stomach-filling bifidobacterium breve B1, every group 8.
Bifidobacterium breve CCFM1206 intervention group (CCFM1206) stomach-filling 10 is given in first 2 weeks of experiment daily9CFU bacterium amount it is short
Bifidobacterium bacterium hangs dilution, the outstanding dilution of the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling
Liquid, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
Test the 1st day, in addition to blank control group (Control), remaining 4 groups after light anaesthesia with diethyl ether nose drip method to flow
Influenza Virus carries out mouse to attack poison, and the same day still needs to stomach-filling, wherein bifidobacterium breve intervention group (CCFM1026) stomach-filling 109CFU bacterium
The bifidobacterium breve CCFM1026 bacterium of amount hangs dilution, the same amount of short bifid bar of bifidobacterium breve B1 intervention group (B1) stomach-filling
Bacterium B1 bacterium hangs dilution, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
It tests the 2nd day, administration Ribavirin medication therapy groups (Treatment) intraperitoneal injection of drugs Ribavirin is controlled
It treats.
Attack the continuation stomach-fillings in 4 days after poison, wherein bifidobacterium breve (CCFM1026) stomach-filling 109The short bifid of CFU bacterium amount
Bacillus CCFM1026 bacterium hangs dilution, and the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling is hanged dilute
Liquid is released, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
Mouse is put to death after taking blood within the 5th day after attacking poison, the mouse blood of taking-up is placed in anticoagulant tube, is shaked gently,
Come into full contact with blood in anti-coagulants, be subsequently sent to pets hospital carry out routine analysis of blood detection (influenza infection initial stage, greatly
Innate immune cells are measured, if neutrophil leucocyte and lymphocyte can participate in defence process, therefore, routine analysis of blood detects emphasis inspection
Survey neutrophil leucocyte and lymphocyte variation), as a result as Figure 2-3.
By Fig. 2-3 it is found that compared with blank group (Control), model group (Model) Murine Neutrophil and lymph are thin
Obviously there is exception in born of the same parents' percentage, significantly different from blank group (Control) (p value is respectively 0.0015 and 0.0011), however it is short
The neutrophil leucocyte and lymphocyte percentage of Bifidobacterium intervention group (CCFM1026) and medication therapy groups (Treatment) mouse
Than tending to blank control group (Control), and there was no significant difference.
Illustrate that bifidobacterium breve of the invention is the same with Ribavirin effect of drugs, the influenza phase of mouse can be played an active part in
It closes in immunological regulation, alleviates mouse influenza illness.
Embodiment 5: influence of the bifidobacterium breve to influenza mouse respiratory infection inflammation
Healthy ICR female mice 40 of weight 20-24g are taken, are randomly divided into 5 groups, 5 groups are respectively designated as: blank control group
(Control), influenza model group (Model), administration Ribavirin medication therapy groups (Treatment), stomach-filling bifidobacterium breve
CCFM1026 intervention group (CCFM1026), the short bifid intervention group (B1 group) of stomach-filling bifidobacterium breve B1, every group 8.
Bifidobacterium breve CCFM1206 intervention group (CCFM1206) stomach-filling 10 is given in first 2 weeks of experiment daily9CFU bacterium amount it is short
Bifidobacterium bacterium hangs dilution, the outstanding dilution of the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling
Liquid, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
Test the 1st day, in addition to blank control group (Control), remaining 4 groups after light anaesthesia with diethyl ether nose drip method to flow
Influenza Virus carries out mouse to attack poison, and the same day still needs to stomach-filling, wherein bifidobacterium breve intervention group (CCFM1026) stomach-filling 109CFU bacterium
The bifidobacterium breve CCFM1026 bacterium of amount hangs dilution, the same amount of short bifid bar of bifidobacterium breve B1 intervention group (B1) stomach-filling
Bacterium B1 bacterium hangs dilution, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
It tests the 2nd day, administration Ribavirin medication therapy groups (Treatment) intraperitoneal injection of drugs Ribavirin is controlled
It treats.
Attack the continuation stomach-fillings in 4 days after poison, wherein bifidobacterium breve (CCFM1026) stomach-filling 109The short bifid of CFU bacterium amount
Bacillus CCFM1026 bacterium hangs dilution, and the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling is hanged dilute
Liquid is released, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
Mouse is put to death after taking blood within the 5th day after attacking poison, the taking-up left lung of mouse is placed in 4% paraformaldehyde fixed immediately, Gu
Histopathology slide is carried out after fixed, and hematoxylin eosin staining is carried out to mouse lung histopathology slide after slice, is passed through
Vocational technology personnel carry out histopathology scoring to mouse lung histopathology slide, as a result as shown in Figure 4.
As shown in Figure 4, blank group (Control) mouse lung institutional framework is more complete, no inflammation cellular infiltration；Model group
(Model) then there is extensive inflammation infiltration phenomenon in mouse, or even local hemorrhage phenomenon occurs；Bifidobacterium breve B1 intervention group (B1)
Nearby there is moderate inflammation infiltration in bronchus, and treatment group (Treatment) and bifidobacterium breve intervention group (CCFM) organize mouse
Then inflammatory conditions are relatively light for lung.
Animal experiments show that, influenza infection can make mouse suffer from influenzal pneumonia above, and lung is organized by after virus infection
It is destructurized, there are a large amount of inflammatory infiltrations；And bifidobacterium breve of the invention can be relieved mouse lung inflammatory conditions, mitigate it
Pneumonia illness is suitable with the effect of influenza common medicine Ribavirin medication therapy groups.
Embodiment 6: influence of the bifidobacterium breve to influenza mouse lung virus load
Healthy ICR female mice 40 of weight 20-24g are taken, are randomly divided into 5 groups, 5 groups are respectively designated as: blank control group
(Control), influenza model group (Model), administration Ribavirin medication therapy groups (Treatment), stomach-filling bifidobacterium breve
CCFM1026 intervention group (CCFM1026), the short bifid intervention group (B1 group) of stomach-filling bifidobacterium breve B1, every group 8.
Bifidobacterium breve CCFM1206 intervention group (CCFM1206) stomach-filling 10 is given in first 2 weeks of experiment daily9CFU bacterium amount it is short
Bifidobacterium bacterium hangs dilution, the outstanding dilution of the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling
Liquid, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
Test the 1st day, in addition to blank control group (Control), remaining 4 groups after light anaesthesia with diethyl ether nose drip method to flow
Influenza Virus carries out mouse to attack poison, and the same day still needs to stomach-filling, wherein bifidobacterium breve intervention group (CCFM1026) stomach-filling 109CFU bacterium
The bifidobacterium breve CCFM1026 bacterium of amount hangs dilution, the same amount of short bifid bar of bifidobacterium breve B1 intervention group (B1) stomach-filling
Bacterium B1 bacterium hangs dilution, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.
It tests the 2nd day, administration Ribavirin medication therapy groups (Treatment) intraperitoneal injection of drugs Ribavirin is controlled
It treats.
Attack the continuation stomach-fillings in 4 days after poison, wherein bifidobacterium breve (CCFM1026) stomach-filling 109The short bifid of CFU bacterium amount
Bacillus CCFM1026 bacterium hangs dilution, and the same amount of bifidobacterium breve B1 bacterium of bifidobacterium breve B1 intervention group (B1) stomach-filling is hanged dilute
Liquid is released, remaining group (Control, Model, Treatment) 0.2mL physiological saline of stomach-filling daily.After the 5th day after attacking poison takes blood
Mouse is put to death, after putting to death mouse, mouse right lung tissue is taken to be placed in the TRIZOL of 1ml, freezes, extraction spare in -80 DEG C of refrigerators
When right lung tissue sample set melt on ice after, ground using the sterile grinder that is handled through DEPC, the chloroform of 200ul be added,
After mixing well, it is centrifuged 10min under 4 DEG C, 12000rpm, 500 μ L of supernatant is removed, isometric isopropanol is added, after mixing, in
4 DEG C, be centrifuged 10min under 12000rpm, remove supernatant, be added 75% ethanol washing RNA it is primary, be centrifuged under 4 DEG C, 12000rpm
10min, dry to alcohol volatilization after removing supernatant, the DEPC that 40 μ L are added handles water, dissolves RNA, and the RNA of extraction freezes in -80 DEG C
Refrigerator is spare.
Using qPCR method measurement MxA relative expression quantity, (body infected by influenza can make defense reaction, to remove invasion disease
Poison is got well, and MxA is a kind of antiviral protein of its secretion, can effectively prevent the duplication of virus), using GAPDH as internal reference,
Using classical 2-ΔΔtCalculation method handles data with model group, as a result as shown in Figure 5 as a comparison.
As shown in Figure 5, medication therapy groups (Treatment) mouse lung MxA expression quantity is the 2.62 of model group (Model)
Times, bifidobacterium breve B1 intervention group mouse lung MxA expression quantity is 2.67 times of model group (Model), and there was no significant difference, and
Bifidobacterium breve intervention group (CCFM1026) mouse lung MxA expression quantity is then 3.46 times of model group (Model), can significantly be mentioned
High MxA expression quantity (p value 0.0292).
The above experiment shows that influenza mouse immune ability can be remarkably reinforced in bifidobacterium breve CCFM1026 of the invention,
Its MxA antiviral protein expression quantity is promoted to increase, to help body recovery health, respiratory inflammation to antiviral duplication
Substantially reduced, effect is even better than drug therapy.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>one plants of bifidobacterium breves and its application with anti influenza ability
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1395
<212> DNA
<213>artificial sequence
<400> 1
caagtcgaac gggatccatc gggctttgcc tggtggtgag agtggcgaac gggtgagtaa 60
tgcgtgaccg acctgcccca tgcaccggaa tagctcctgg aaacgggtgg taatgccgga 120
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. one plant of bifidobacterium breve (Bifidobacterium breve), which is characterized in that the bifidobacterium breve
(Bifidobacterium breve) is preserved in Guangdong Province's Culture Collection, preservation on October 11st, 2018
Number is GDMCC No.60459, and preservation address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building.
2. one plant of bifidobacterium breve (Bifidobacterium breve) as described in claim 1 is in preparation prevention and/or controls
Treat the application in the product of influenza.
3. one plant of bifidobacterium breve (Bifidobacterium breve) as claimed in claim 2 is in preparation prevention and/or controls
Treat the application in the product of influenza, which is characterized in that in the product, bifidobacterium breve (Bifidobacterium breve)
Viable count be not less than 1 × 106CFU/mL。
4. one plant of bifidobacterium breve (Bifidobacterium breve) as claimed in claim 2 or claim 3 preparation prevention and/
Or the application in the product for the treatment of influenza, which is characterized in that the product includes food, drug or health care product.
5. one plant of bifidobacterium breve (Bifidobacterium breve) as described in claim 2-4 is any prevents in preparation
And/or the application in the product for the treatment of influenza, which is characterized in that the drug contains bifidobacterium breve (Bifidobacterium
Breve), pharmaceutical carrier and/or pharmaceutic adjuvant.
6. a kind of for preventing and/or treating the product of influenza, which is characterized in that the product contains as described in claim 1
Bifidobacterium breve (Bifidobacterium breve).
7. as claimed in claim 6 a kind of for preventing and/or treating the product of influenza, which is characterized in that in the product,
The viable count of bifidobacterium breve (Bifidobacterium breve) is not less than 1 × 106CFU/mL。
8. as claimed in claims 6 or 7 a kind of for preventing and/or treating the product of influenza, which is characterized in that the product
Include food, drug or health care product.
9. as claimed in claim 6 to 8 a kind of for preventing and/or treating the product of influenza, which is characterized in that described
Drug contains bifidobacterium breve (Bifidobacterium breve), pharmaceutical carrier and/or pharmaceutic adjuvant.
10. a kind of for preventing and/or treating the product of influenza as described in claim 6-9 is any, which is characterized in that described
Food includes dairy products, the beans produced using the leavening containing bifidobacterium breve (Bifidobacterium breve)
Product or fruit and vegetable product；Or the food includes the solid beverage containing bifidobacterium breve (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 |
-
2018
- 2018-12-25 CN CN201811589535.1A patent/CN110055185B/en active Active
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 (1)
| Title |
|---|
| HISAKO YASUI ET AL.: "Protection against Influenza Virus Infection of Mice Fed Bifidobacterium breve YIT4064", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》 * |
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