CN104651245B - Probiotic composition for treating pico+ribonucleic acid+virus infection and application thereof - Google Patents
Probiotic composition for treating pico+ribonucleic acid+virus infection and application thereof Download PDFInfo
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- CN104651245B CN104651245B CN201310580840.5A CN201310580840A CN104651245B CN 104651245 B CN104651245 B CN 104651245B CN 201310580840 A CN201310580840 A CN 201310580840A CN 104651245 B CN104651245 B CN 104651245B
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Abstract
The invention discloses a kind of composition for treating pico+ribonucleic acid+virus infection, its deposit number comprising lactobacillus paracasei (GMNL 33) is that CCTCC M206133, the deposit number of lactobacillus reuteri (GMNL 89) are CCTCC M207154 and Lactobacillus casei (GMNL 277) deposit number is at least one of CCTCC M2013197, and pharmaceutically acceptable supporting agent.Wherein Lactobacillus casei GMNL 277 is novel lactobacillus separation strains；Invention also discloses by said composition or the lactobacillus, to treat the novel use that pico+ribonucleic acid+virus infects, its mechanism of action is that probiotic strain is combined with virus, and reduces the ability of virus infected cell.
Description
Technical field
The present invention is the separation on novel lactobacillus separation strains, and its in the skill for the treatment of pico+ribonucleic acid+virus infection
Art field.
Background technology
Enteric virus71 type belongs to the enterovirus category of pico+ribonucleic acid+virus section (Family Picornaviridae)
(genus Enterovirus).Viral epitheca is in the structure of regular dodecahedron (icosahedral), no outer embrane parcel, environment
In acid, alkali and temperature change be all difficult the activity of influence virus, therefore can in the environment survive permanent and be broadcast by excrement oral instructions
Reach wide-scale distribution.The genosome of virus is made up of the sub-thread underlying stock RNA nucleic acid of 7440 sizes, can be translated out one and be contained 2194
Individual Amino acid polymeric protein (polyprotein), includes the P1 regions of structural protein, and non-structural protein P2 and
P3 regions.Polymeric protein cuts into P1, P2, P3 by 2A proteases (protease), is cut P1 regions by 3D proteases
Into tetra- structural proteins of VP1, VP2, VP3 and VP4, P2 regions are cut into 2A protease, 2B and 2C nucleotide
Triphosphatase, the non-structural proteins such as 3A and 3CD are cut into by P3 regions.Coat protein (capsid) is by VP1-
Tetra- structural proteins of VP4 are constituted, about 20-30 nanometers of size (nm).
According to the biological nature and molecular characterization of enterovirus, enterovirus was redistricted as five major classes in 2003, point
It is not that (1) polio is viral, (2) enterovirus A groups (CAV2 to CAV8, CAV10, CAV12, CAV14, CAV16 and EV71),
(3) enterovirus B groups (CAV9, CBV1 to CBV6, E1 to E7, E9, E11 to E21, E24 to E27, E29 to E33 and EV69 and
EV73), (4) enterovirus C crowds (CAV1, CAV11, CAV13, CAV15, CAV17 to CAV22 and CAV24), (5) enterovirus D crowds
(EV68 and 70).Enteric virus71 type initially in 1969 California, USA in the sufferer with central nervous diseases it is separated go out
Come, named by Melnick as enteric virus71 type within 1974.
After enteric virus71 type was separated from 1969, extensive prevalence is caused in different continents, such as 1969 to
In the U.S., in Japan, all once broken out in Hong Kong Australia Victoria city, Brazil, 1986 within 1986 big within 1978 within 1973
Prevalence simultaneously causes serious encephalitis and class polio disease group.In 1998, also outburst enteric virus71 type was very popular in Taiwan, always
Meter has 130,000 people infection, and 405 severe diseases are virgin because brothers mouthful disease merges aseptic meningitis, encephalitis or ACUte flaccid paralysis
And be in hospital, cause 78 diseases virgin dead, there is 75% to turn out to be enteric virus71 type in severe case and cause, and then have in Lethal cases
92% isolates enteric virus71 type.After from be very popular in 1998, annual spring and summer is alternately and child's sense can all occur in summer period
The case of enteric virus71 type is contaminated, is the healthy problem of the important public health in current one of Taiwan.
Enteric virus71 type has nerve infiltration, and the serious neurological disease of limb paralysis can be equally caused with polio virus
Shape, because three years old Infants Below is attacked in the infection of enteric virus71 type mostly and is easily caused serious central neuropathy, enteric virus71
Type can be considered as after another very important Neural invasion (neurotropic) virus after polio virus.It is generally acknowledged that intestines
Virus is that virus invades host by entrance of alimentary canal, and then other target organs are arrived in infection via excrement mouthful routes of infection infection,
Particularly nervous system.The infection of acute enteric virus71 type not yet has effective antiviral drugs so far, though treatment clinically
Using IVIG, but therapeutic effect is limited, at present still based on the supporting treatment of symptom treatment.Carry out infection prevention disease using vaccine
Disease has good history, but enteric virus71 type vaccine, still in research and development state, is extremely listed uncertain to Clinical practice time-histories at present；And epidemic disease
Seedling select good strains in the field for seed with whether have to other genotype or other kinds of enteroviruses cross-protection still need to research confirm.
In 1908, Russia Nobel Laureate Elie Metchnikoff had found newborn bar contained in the cow's milk after fermentation
Bacterium has benefit to the health of human body.To nineteen sixty-five, probiotics is then further defined as by Lilly and Stilweel：
It is any Intestinal flora to be promoted to balance, increase the microorganism of health benefit.More and more researchs are pointed out, for microorganism
Caused enteric infection, probiotics can promote the immune response of enteron aisle.The preceding case US7,666,407 of applicant in this case is pointed out
Lactobacillus paracasei GMNL-33, which have, suppresses the odontopathy purposes that bacterium is caused；US7,901,926 is pointed out
Lactobacillus reuteri GMNL-89 have anti-inflammatory purposes.But whether can effectively be treated for probiotics at present
The symptom of enterovirus, then not yet have whether reported in literature has its effect.
The content of the invention
The purpose of the present invention is to be to provide a kind of novel lactobacillus separation strains, Lactobacillus casei (Lactobacillus
Casei GMNL-277) deposit number be BCRC910585 and CCTCC M2013197.
Another object of the present invention is to be to provide a kind of medicinal combination for being used to treat pico+ribonucleic acid+virus infection
Thing, its comprising Lactobacillus casei (Lactobacillus casei GMNL-277) deposit number for BCRC910585 with
CCTCC M2013197。
To reach aforementioned invention purpose, wherein the medical composition can further include lactobacillus paracasei
The deposit number of (Lactobacillus paracasei GMNL-33) is BCRC910314 and CCTCC M206133 and sieve
The deposit number of Yi Shi lactobacillus (Lactobacillus reuteri GMNL-89) is BCRC910340 and CCTCC
M207154 is at least one.
To reach aforementioned invention purpose, wherein the pico+ribonucleic acid+virus is enterovirus.
To reach aforementioned invention purpose, wherein the enterovirus is enteric virus71 type.
To reach aforementioned invention purpose, the wherein medical composition is the formulation for being available for oral administration medicine supplying.Wherein the formulation is
Selected from following constituted group：Solution, suspension, emulsion, powder, lozenge, pill, syrup, mouth containing ingot, tablet, chewing
Glue, underflow and capsule.
Another object of the present invention is to be to provide a kind of food product, and it includes Lactobacillus casei (Lactobacillus
Casei GMNL-277) deposit numbering be BCRC910585 and CCTCC M2013197.
To reach aforementioned invention purpose, wherein the food product can further include lactobacillus paracasei
The deposit number of (Lactobacillus paracasei GMNL-33) is BCRC910314 and CCTCC M206133 and sieve
The deposit number of Yi Shi lactobacillus (Lactobacillus reuteri GMNL-89) is BCRC910340 and CCTCC
M207154 is at least one.
To reach aforementioned invention purpose, at least one probiotics in following group can further include：Newborn bar
Ella species (lactobacillus sp.), bacillus bifidus species (Bifidobacterium sp.), Streptococcus species
(Streptococcus sp.) and saccharomycete (yeasts).
To reach aforementioned invention purpose, edible material is can further include, the edible material includes water, fluid breast
Product, milk, Evamilk, Yoghourt, yogurt, freezing yogurt, lactobacillus ferment beverage, milk powder, ice cream, cheese, cheese, beans
Milk, fermented soybean milk, fruit and vegetable juice, fruit juice, sports drink, dessert, jelly, candy, baby food, healthy food, animal feed, in
Herbal medicine material or dietary supplement.
Another object of the present invention be to provide a kind of probiotics be used for prepare treatment pico+ribonucleic acid+virus infect
Composition method, wherein the probiotics include lactobacillus paracasei (Lactobacillus paracasei GMNL-33)
Deposit number be BCRC910314 and CCTCC M206133, lactobacillus reuteri (Lactobacillus reuteri
GMNL-89 deposit number) is BCRC910340 and CCTCC M207154 and Lactobacillus casei (Lactobacillus
Casei GMNL-277) deposit number it is at least one for BCRC910585 and CCTCC M2013197.
To reach aforementioned invention purpose, wherein the pico+ribonucleic acid+virus is enterovirus.
To reach aforementioned invention purpose, wherein the enterovirus is enteric virus71 type.
To reach aforementioned invention purpose, wherein said composition is medical composition, is the formulation for being available for oral administration medicine supplying.Wherein
The formulation is to be selected from following constituted group：Solution, suspension, emulsion, powder, lozenge, pill, syrup, mouth containing ingot, piece
Agent, chewing glue, underflow and capsule.
Another object of the present invention is to be to provide a kind of method for treating pico+ribonucleic acid+virus infection, includes combination
Thing；Wherein said composition is comprising the deposit number of lactobacillus paracasei (Lactobacillus paracasei GMNL-33)
CCTCC M206133, the deposit number of lactobacillus reuteri (Lactobacillus reuteri GMNL-89) are CCTCC
The deposit number of M207154 and Lactobacillus casei (Lactobacillus casei GMNL-277) is CCTCC M2013197
It is at least one.
To reach aforementioned invention purpose, wherein the pico+ribonucleic acid+virus is enterovirus.
To reach aforementioned invention purpose, wherein the enterovirus is enteric virus71 type.
To reach aforementioned invention purpose, wherein said composition is medical composition, is the formulation for being available for oral administration medicine supplying.Wherein
The formulation is to be selected from following constituted group：Solution, suspension, emulsion, powder, lozenge, pill, syrup, mouth containing ingot, piece
Agent, chewing glue, underflow and capsule.
Another object of the present invention is to be to provide a kind of composition for being used to treat pico+ribonucleic acid+virus infection, its
Deposit number of the middle said composition comprising lactobacillus paracasei (Lactobacillus paracasei GMNL-33) is CCTCC
M206133, the deposit number of lactobacillus reuteri (Lactobacillus reuteri GMNL-89) are CCTCC M207154
And the deposit number of Lactobacillus casei (Lactobacillus casei GMNL-277) is CCTCC M2013197 at least one
Kind.
To reach aforementioned invention purpose, wherein the pico+ribonucleic acid+virus is enterovirus.
To reach aforementioned invention purpose, wherein the enterovirus is enteric virus71 type.
To reach aforementioned invention purpose, wherein said composition is medical composition, is the formulation for being available for oral administration medicine supplying.Wherein
The formulation is to be selected from following constituted group：Solution, suspension, emulsion, powder, lozenge, pill, syrup, mouth containing ingot, piece
Agent, chewing glue, underflow and capsule.
Another embodiment of the present invention is enterovirus, and enterovirus is the general name of a group virus, comprising polio virus, gram
More than 60 types such as husky strange virus type A and Type B, her coe virus and enterovirus, find in recent years and successively a variety of types, according to gene sequence
Row analysis result is reclassified.
Another embodiment of the present invention is pico+ribonucleic acid+virus, pico+ribonucleic acid+virus section
(Picornaviridae) (Picornaviridae) Parvoviridae is no mantle, underlying stock RNA, positive 20 face body protein shell
The virus of body, its genosome has an albumen to be for the primer as rna replicon at 5' ends.Parvoviridae can be divided into
Nine category, respectively enterovirus genus (Enterovirus), representative species：Poliomyelitis virus (Poliovirus polio
Virus)；Rhinovirus (Rhinovirus), representative species：Human rhinovirus A (Human rhinovirus A)；Hepatovirus
(Hepatovirus), representative species：Hepatitis A virus (Hepatitis A virus, HAV)；Cardiovirus
(Cardiovirus), representative species：Encephalomyocarditis virus (Encephalomyocarditis virus)；Aphtho virus belongs to
(Aphthovirus), representative species：Foot and mouth disease virus (Foot-and-mouth disease virus)；Secondary intestinal cells lesion
Human orphan's Tobamovirus (the lonely Tobamovirus of Parechovirus pair intestines), representative species：Mankind's pair intestinal cells lesion human orphan's disease
Poison (the lonely virus of Human parechovirus people pair intestines)；Equine rhinoviruses belongs to (Erbovirus)；Ridge Tobamovirus (Kobuvirus)；
With prompt Shen Tobamovirus (Teschovirus).
It is of the present invention to be used to treat probiotic composition of pico+ribonucleic acid+virus infection and application thereof with following
Advantage and beneficial effect：
（1）Lactobacillus casei (Lactobacillus casei GMNL-277) of the present invention, it is in Wuhan, China
Preservation in China typical culture collection center (CCTCC), preservation date is on 05 09th, 2013, and deposit number is CCTCC
M2013197；
（2）GMNL-277, GMNL-33 and GMNL-89 of the present invention have suppresses enteric virus71 in different cell lines
The ability of type.
Brief description of the drawings
Fig. 1 is the microphoto of the probiotics of embodiment 2 (GMNL-277)；
Fig. 2 is the ability of the probiotics viral adsorption of embodiment 4.
Embodiment
All technical and scientific terminology described in this specification, is all the art unless defined in addition
The meaning that can be understood jointly with usual those skilled in the art.The present invention will be described further with regard to the following example, these right embodiments
Only illustrate, and be not necessarily to be construed as implementing the limitation of the present invention.
The present invention is a kind of composition for treating pico+ribonucleic acid+virus infection, and it includes lactobacillus paracasei (GMNL-
33) deposit number be CCTCC M206133, the deposit number of lactobacillus reuteri (GMNL-89) be CCTCC M207154 and
Lactobacillus casei (GMNL-277) its deposit number is at least one for CCTCC M2013197, and pharmaceutically acceptable supporting agent.
Wherein Lactobacillus casei GMNL-277 is novel lactobacillus separation strains.In addition, the present invention is also on utilizing said composition or the breast
Novel use of the bacillus to treat pico+ribonucleic acid+virus infection, its mechanism of action is probiotics GMNL-277, GMNL-33
And GMNL-89 bacterial strains are combined with virus, and reduce the ability of virus infected cell.
Wherein the lactobacillus separation strains also include the offspring of its squamous subculture, or mutant strain, but still have and institute of the present invention
Strain properties, genosome (genomic) or the purposes stated (are used to treat enterovirus) identical person.
Composition described herein can include, but are not limited to：Food, drink, healthy food, drinking water for animals additive, animal
Feed additive, animal are used and the mankind are applicable application shape of the invention with medical composition, food additives, beverage additives etc.
Formula.
Term " treatment ", " in treatment " and its class term refer to delay, improve, reduce or reverse just to torment patient at present
The illness or the related any symptom of the illness method and the method for preventing the illness or its any symptom just occurred.
Term " pharmaceutically acceptable " refers to that material or composition must be compatible with other compositions of composite, and to patient
It is harmless.
Term " pico+ribonucleic acid+virus section " is classified as the 4th guiding principle according to Baltimore classification, genosome size from
7.2kb to 9.0kb, the mRNA of Parvoviridae does not have CAP but a kind of protein referred to as VPg at 5' ends, and 3' ends equally have
There are poly A tail, there is UTR at genosome two ends（Un-translated region non-translation areas）.
The composition of the present invention be using being familiar with the technology that this those skilled in the art knows in detail, by above-mentioned lactobacillus separation strains,
With pharmaceutically acceptable supporting agent (pharmaceutically acceptable vehicle), it is prepared into and is applicable of the present invention group
The formulation of compound.Wherein the formulation including but not limited to：Solution (solution), emulsion (emulsion), suspension
(suspension), powder (powder), lozenge (tablet), pill (pill), mouth containing ingot (lozenge), tablet
(troche), chewing glue (chewing gum), capsule (slurry) and other similar or be applicable formulation of the invention.
Wherein the pharmaceutically acceptable supporting agent can be selected from following reagent comprising one or more：Solvent
(solvent), emulsifying agent (emulsifier), suspending agent (suspending agent), distintegrant (decomposer), cohere
Agent (binding agent), excipient (excipient), stabilization agent (stabilizing agent), chelating agent
(chelating agent), diluent (diluent), gelling agent (gelling agent), preservative (preservative),
Lubricant (lubricant), surfactant (surfactant), and it is other similar or be applicable supporting agent of the invention.
In above-mentioned composition, it also optionally can suitably add usually used molten in one or more above formulation art
Solve accessory agent, buffer, preservative agent, colouring agent, spices, flavouring agent etc..
In another preferred embodiment, foregoing provided by the present invention can further add edible material, to make
Standby is a kind of food product or health product.Wherein the edible material include, but are not limited to：Water (water), fluid dairy products
(fluid milk products), milk (milk), Evamilk (concentrated milk)；Fermented milk products
(fermented milk), such as Yoghourt (yogurt), yogurt (sour milk), freezing yogurt (frozen yogurt), breast
Bacillus fermentation beverage (lactic acid bacteria-fermented beverages)；Milk powder (milk powder)；Ice river in Henan Province
Drench (ice cream)；Cheese (cream cheeses)；Cheese (dry cheeses)；Soymilk (soybean milk)；Ferment beans
Milk (fermented soybean milk)；Fruit and vegetable juice (vegetable-fruit juices)；Fruit juice (juices)；Motion drink
Expect (sports drinks)；Dessert (confectionery)；Jelly (jellys)；Candy (candies)；Baby food
(infant formulas)；Healthy food (health foods)；Animal feed (animal feeds)；Chinese medicinal herbs
(Chinese herbals)；Dietary supplement (dietary supplements) etc..
In addition, the novel strain that is found of the present invention, also it can know strain with other and be contained in the lump in constituent.
Wherein said composition, can further include and at least one know probiotics in following group：Lactobacillus
Species (lactobacillus sp.), Streptococcus species (Streptococcus sp.), bacillus bifidus species
(Bifidobacterium sp.), saccharomycete (yeasts).
Wherein should know Lactobacillus species (lactobacillus sp.) including but not limited to：Lactobacillus lactis
(lactobacillus lactis), lactobacillus acidophilus (lactobacillus acidophilus), Lactobacillus helveticus
(lactobacillus helveticus), double qi lactobacillus (lactobacillus bifidus), milk Lactobacillus paracasei
(lactobacillus casei), secondary secondary cheese subspecies (the Lactobacillus paracasei of milk Lactobacillus paracasei
Subsp.paracasei), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus gasseri
(Lactobacillus gasseri), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus fermenti
(Lactobacillus fermentum) or its combination.
Wherein should know Streptococcus species (Streptococcus sp.) including but not limited to：Streptococcus lactis
(Streptococcus lactis), streptococcus thermophilus (Streptococcus thermophilus), cheese streptococcus
(Streptococcus cremoris) or its combination.
Wherein should know bacillus bifidus species (Bifidobacterium sp.) including but not limited to：Short double fork lever
Bacterium (Bifidobacterium breve), lactic acid bacillus bifidus (Bifidobacterium lactis), elongated bacillus bifidus
(Bifidobacterium longum), dual forked type bacillus bifidus (Bifidobacterium bifidum) or its combination.
Wherein should know saccharomycete (yeasts) including but not limited to：Saccharomyces cerevisiae (Saccharomyces
Cereviseae), Candida kefyr bacterium (Candida kefyr), not sieve rib saccharomycete (Saccharomyces
Florentinus) or its combination.
In addition, the present invention also provide foregoing lactobacillus be used for prepare treatment pico+ribonucleic acid+virus infect composition side
Method or purposes.
Composition provided by the present invention and its method for treating pico+ribonucleic acid+virus infection, its road of offeing medicine
Footpath, visual demand is suitably adjusted, and is not particularly limited, preferably be applicable formulation for oral administration.
The present invention is to give demonstration with the following examples to illustrate, but the present invention is not limited by following embodiments.This hair
Bright medicine used, biomaterial are all commercially available to be easy to obtain, following merely illustrative obtainable pipeline.
Probiotics, cell and virus
Cell culture, RD cells, human rhabdomyosarcoma (rhabdomyosarcoma) is incubated at containing 10% hyclone
The High-glucose-Dulbecco's Modified Eagle's Medium of (fetal bovine serum, FBS)
(DMEM) in.HT-29 cells, human colon cancer cells (colorectal adenocarcinoma), are incubated at containing 10%FBS's
In Roswell Park Memorial Institute-1640 (RPMI1640).Caco-2 cells, human colon cancer cells
(colorectal adenocarcinoma), then be incubated at the High- of the pyruvate containing 20%FBS and 1.0mM sodium
In glucose DMEM.Cell is in 37 DEG C, 5%CO2Incubator culture, when cell needs subculture, old culture medium is drawn and abandoned,
Cell is washed with sterile PBS, trypsin-EDTA is added and is acted on several minutes in 37 DEG C, appropriate cell culture medium is added and by cell
Break up.New cell culture fluid is added after cell is diluted according to proper proportion to continue to cultivate.
Virus culture, this research uses enteric virus71 type (EV71-N4643/TW98) Strain.By RD cell culture in
75cm2In flask, after cell is covered with, virus inoculation liquid treats cytopathy (cytopathic effect, CPE) about 90%
After collect virus liquid, and infected cell under scraping, after chilled defrosting and centrifugation, Aspirate supernatant is dispensed to virus preservation
Pipe, deposit in -80 DEG C it is standby.
Probiotics, this research is using three plants of different probiotics bacterium powders, and code name is respectively GMNL-33, GMNL-89 and GMNL-
277。
Experimental design and method
Cell toxicity test (Cytotoxicity assay)
RD cells are planted in 96 porose discs, 37 DEG C of culture overnight.Bacterium powder is configured with virus-culturing fluid, is separately added into dense
Degree 107And 108CFU/mL probiotics, processing cell 24 hours, adds WST-1 reagents, is inhaled with ELISA reader detectings per hole
Light value.
Antivirus test (Antiviral assay)
(a) pre-treatment cell (Pre-treatment of cell)：RD, HT-29 and Caco-2 cell are seeded to respectively
12 hole culture plates, 37 DEG C of culture overnight.Add probiotics and handle cell 6 hours in 37 DEG C, suck supernatant, it is clear with PBS
Wash after cell and add the infection of enteric virus71 type, after 37 DEG C are cultivated 24 hours, collect cell and supernatant, be stored in -80 DEG C and treat
Survey virus quantity.
(b) while handling cell (Co-incubation with virus) (competition assay)：Respectively will
RD, HT-29 and Caco-2 cell are seeded to 12 hole culture plates, 37 DEG C of culture overnight.Probiotics and enteric virus71 type is same
When add cell in 37 DEG C cultivate 24 hours, collect cell and supernatant, be stored in -80 DEG C of virus quantities to be measured.
(c) post processing cell (Post-treatment of cell)：RD, HT-29 and Caco-2 cell are inoculated with respectively
To 12 hole culture plates, 37 DEG C of culture overnight.Enteric virus71 type is added in after 37 DEG C of infection cells 6 hours, by probiotics plus
Enter infection cell to cultivate 24 hours in 37 DEG C, collect cell and supernatant, be stored in -80 DEG C of virus quantities to be measured.
Viral Quantification：Half tissue culture infection dose (50%tissue culture infective dose, TCID50)
RD cell culture is cultivated into overnight in 96 porose discs in 37 DEG C.Virus to be measured is taken with ten times of serial dilutions, will
The good virus liquid of serial dilution is sequentially added in RD cells, in 37 DEG C, 5%CO2Incubator culture 24 hours.After 24 hours, suck
Nutrient solution is diluted, 80% ice acetone is added and fixes cell.Enzyme immunoassay is carried out using the monoclonal antibody of anti-enteric virus71 type
(ELISA) virus quantity is determined.Light absorption value is determined under wavelength 450nm.After measure in acquired results, cell control group is calculated (only
Containing cell and dilution nutrient solution) average absorbance value, it is average after result be multiplied by 2, be determined as if light absorption value is more than this numerical value
The positive of virus infection, is then feminine gender less than this numerical value.After interpretation, the viral TCID is calculated in Reed-Muench methods50。
Probiotics and the reciprocation (EV71adsorption by the probiotics) of enteric virus71 type
By 100TCID50Virus liquid and probiotics (107CFU/mL) it is mixed in 1.5mL centrifuge tubes, is placed in 37 DEG C, 5%CO2
1.5 hours of incubator.Centrifugation takes supernatant to survey virus concentration (TCID after 10 minutes50)。
Statistical method
Whether experimental result has statistically difference using unpaired t test calibratings, is as a result represented with means ± SD, p
Value is considered as tool statistically difference less than 0.05.
Embodiment 1
Applicant in this case (Jing Yue biotechnologies company) is separation more than the 600 strains separation in health adult's intestines and stomach corpse or other object for laboratory examination and chemical testing
Strain, to set up separation strains strain library.Gather status, obtain time, picker, picker's contact information such as table 1 below.
Table 1, genetic resources information
The lactobacillus of enterovirus can be suppressed by being selected from strain library, select the front three lactobacillus of analysis result to be pressed down
Viral growth analysis processed.Deposit number, preservation date and the strain name of three plants of lactobacillus, as shown in table 2；Wherein secondary cheese
Lactobacillus (Lactobacillus paracasei) GMNL-33, lactobacillus reuteri (Lactobacillus reuteri)
GMNL-89 above-mentioned is all disclosed strain, documentary evidence original and the phase such as its strain characteristics, preservation are proved, survival test is reported
Information is closed, has been contained in multinational patent；Wherein, Lactobacillus casei (Lactobacillus casei) GMNL-277 is this hair
The bright novel lactobacillus separation strains newly isolated.Under with the strain characteristicses of embodiment this plant of lactobacillus of 2 division, Biolog microorganisms
Identification systems analysis, 16S rDNA analyses.
Table 2, the lactobacillus of the present invention are preserved and research center in Hsin-chu Foodstuff Industrial and Development Inst. living resources
(BCRC) or China typical culture collection center (CCTCC) preservation information
Embodiment 2
Lactobacillus casei (Lactobacillus casei) GMNL-277 scientific name identification, separation strains GMNL-277 is entrusted
Ask Hsin-chu Foodstuff Industrial and Development Inst. to carry out bacterium scientific name identification, be described below：Separation strains GMNL-277 background informations：
Separation source is human body intestines and stomach；Culture medium is MRS；Cultivation temperature is 37 DEG C；Pathogenicity：Nothing.
Analysis result shows that separation strains GMNL-277 is Gram-positive bacillus, does not have catalase, oxidizing ferment and motility, in
It can all be grown under aerobic environment and anaerobic environment, endospore (as shown in Figure 1) will not be produced.In addition, separation strains GMNL-277 16S
RDNA partial sequences such as SEQ ID No:Shown in 1, analyzed according to 16S rDNA, separation strains GMNL-277 is similar to Lactobacillus casei
Degree up to more than 99%.Analyzed again by Biolog microbial identification systems, separation strains GMNL-277 is closest to Lactobacillus casei.
Therefore, summary result is shown, separation strains GMNL-277 is Lactobacillus casei (Lactobacillus casei).
Embodiment 3
Probiotics suppresses the ability of enteric virus71 type：
This experiment tests three plants of cell lines using three kinds of different probiotics, make use of three kinds of different mode test probiotics
Suppress enteric virus71 type ability, respectively pre-treatment (pre-treatment), while processing (co-incubation) and rear place
In (post-treatment), followed by TCID50Assay determines the difference that probiotics handles restrovirus amount, probiotic concentration
107CFU/mL, for RD, HT-29 and Caco-2 cell line compared with no cytotoxicity, with 107CFU/mL probiotic concentration enters
OK；Suppress virus effectiveness with 100TCID50For optimal, experimental result such as table 3.
Table 3, three probiotics suppress suppression percentage (the unpaired t of enteric virus71 type on three plants of different cell lines
test,*:P<0.05)
As a result it is shown in pre-treatment experiment in RD cells, three probiotics of the invention can suppress the life of enteric virus71 type
It is long, and difference has statistically meaning；But imitated in the HT-29 cell culture suppression that then only GMNL-89 has statistically difference
Really, then also only GMNL-89 has the inhibition of statistically difference to Caco-2 cells.And handle result in experiment at the same time and send out
It is existing, more bacterial strain is tended to have it can be seen that inhibition in RD cells, including GMNL-277, GMNL-33 and GMNL-89, and in HT-29
There is no bacterial strain to have the inhibition of statistically difference in cell.But in being tested in rear place, its result is relatively differed, in HT-
Then there is more bacterial strain to have the rejection ability of statistically difference, including GMNL-277, GMNL-33, GMNL-89 in 29 cells；Caco-
2 cells then have GMNL-33 to have the inhibition of statistically meaning.
In summary experimental result is shown, confirms that GMNL-277, GMNL-33 and GMNL-89 bacterial strain have in different cells
Suppress the ability of enteric virus71 type in strain.So GMNL-277, GMNL-33 and GMNL-89 bacterial strain are considered as with anti-enterovirus
The main bacterial strain of potentiality carries out subsequent viral adsorption test.
Embodiment 4
Whether probiotics produces reciprocation and suppressing virus replication with enteric virus71 type：
Why this experiment further inquires into its possible mechanism of action by the potentiality bacterial strain of three plants of anti-enteric virus71 types, because
This first assess probiotics whether can viral adsorption so that suppress virus infected cell ability.We are respectively by this three probiotics
With enteric virus71 type co-incubation, then via centrifugation allow probiotics to precipitate, take supernatant Test Virus amount.As a result find
GMNL-277, GMNL-33 and GMNL-89 reduce enteric virus71 type virus quantity up to 66.04%, 56.09% and 63.15% (table 4 and figure
2), display probiotics GMNL-277, GMNL-33 and GMNL-89 can all be combined with enteric virus71 type, therefore probiotics may pass through
Combined with virus and reduce the ability of virus infected cell.
Virus quantity (the * of reduction after table 4, enteric virus71 type are adsorbed by probiotics:P<0.05)
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (14)
1. a kind of novel lactobacillus separation strains, it is characterised in that the lactobacillus separation strains are Lactobacillus casei, the cheese breast
Bacillus (Lactobacillus casei) GMNL-277 deposit number is CCTCC M 2013197.
2. a kind of medical composition for being used to treat pico+ribonucleic acid+virus infection, it is characterised in that described to be used to treat micro-
The medical composition of picornavirus infection includes Lactobacillus casei (Lactobacillus casei) GMNL-277, institute
The deposit number for stating Lactobacillus casei is CCTCC M 2013197, and the pico+ribonucleic acid+virus is enterovirus.
3. the medical composition as claimed in claim 2 for being used to treat pico+ribonucleic acid+virus infection, it is characterised in that institute
Medical composition is stated also comprising the newborn bar of lactobacillus paracasei (Lactobacillus paracasei) GMNL-33 and Luo Yishi
At least one of bacterium (Lactobacillus reuteri) GMNL-89；The deposit number of the lactobacillus paracasei is
CCTCC M 206133, the deposit number of the lactobacillus reuteri is CCTCC M 207154.
4. the medical composition as claimed in claim 2 for being used to treat pico+ribonucleic acid+virus infection, it is characterised in that institute
Enterovirus is stated for enteric virus71 type.
5. being used for the medical composition for treating pico+ribonucleic acid+virus infection as claimed in claim 2 or claim 3, its feature exists
In the medical composition is the formulation for oral administration medicine supplying.
6. the medical composition as claimed in claim 5 for being used to treat pico+ribonucleic acid+virus infection, it is characterised in that institute
It is to be selected from following constituted group to state formulation：Solution, suspension, emulsion, powder, lozenge, pill, syrup, mouth containing ingot, piece
Agent, chewing glue, underflow and capsule.
7. a kind of food product, it is characterised in that the food product includes Lactobacillus casei, the Lactobacillus casei
(Lactobacillus casei) GMNL-277 deposit number is CCTCC M 2013197.
8. food product as claimed in claim 7, it is characterised in that the food product also includes lactobacillus paracasei
(Lactobacillus paracasei) GMNL-33 and lactobacillus reuteri (Lactobacillus reuteri) GMNL-
At least one of 89, the deposit number of the lactobacillus paracasei is CCTCC M 206133, the lactobacillus reuteri
Deposit number is CCTCC M 207154.
9. food product as claimed in claim 7 or 8, it is characterised in that the food product is also selected from comprising at least one
Probiotics in following group：Lactobacillus species (lactobacillus sp.), bacillus bifidus species
(Bifidobacterium sp.), Streptococcus species (Streptococcus sp.) and saccharomycete (yeasts).
10. food product as claimed in claim 7 or 8, it is characterised in that the food product also includes edible material,
The edible material comprising water, fluid dairy products, milk, Evamilk, Yoghourt, yogurt, freezing yogurt, lactobacillus ferment beverage,
Milk powder, ice cream, cheese, cheese, soymilk, fermented soybean milk, fruit and vegetable juice, fruit juice, sports drink, dessert, jelly, candy, baby
Food, healthy food, animal feed, Chinese medicinal herbs or dietary supplement.
11. a kind of probiotics is used for the method for preparing the composition for the treatment of pico+ribonucleic acid+virus infection, it is characterised in that institute
State probiotics and include lactobacillus paracasei (Lactobacillus paracasei) GMNL-33, lactobacillus reuteri
(Lactobacillus reuteri) GMNL-89 and Lactobacillus casei (Lactobacillus casei) GMNL-277；Institute
The deposit number for stating lactobacillus paracasei is CCTCC M 206133, and the deposit number of lactobacillus reuteri is CCTCC M
207154, the deposit number of Lactobacillus casei is CCTCC M 2013197, and the pico+ribonucleic acid+virus is enterovirus.
12. probiotics as claimed in claim 11 is used for the side for preparing the composition for the treatment of pico+ribonucleic acid+virus infection
Method, it is characterised in that the pico+ribonucleic acid+virus is enterovirus, the enterovirus is enteric virus71 type.
13. probiotics as claimed in claim 11 is used for the side for preparing the composition for the treatment of pico+ribonucleic acid+virus infection
Method, it is characterised in that the composition is medical composition, is the formulation for oral administration medicine supplying.
14. probiotics as claimed in claim 13 is used for the side for preparing the composition for the treatment of pico+ribonucleic acid+virus infection
Method, it is characterised in that the formulation is to be selected from following constituted group：Solution, suspension, emulsion, powder, lozenge, ball
Agent, syrup, mouth containing ingot, tablet, chewing glue, underflow and capsule.
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Citations (3)
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|---|---|---|---|---|
| CN1556853A (en) * | 2001-07-26 | 2004-12-22 | Probiotics lactobacillus casei bacterial strain | |
| TW200808191A (en) * | 2006-08-09 | 2008-02-16 | Genmont Biotech Inc | Product containing Lactobacillus paracasei |
| TW200944215A (en) * | 2008-04-30 | 2009-11-01 | Genmont Biotech Inc | Lactobacillus isolates having anti-inflammatory activities and uses of the same |
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2013
- 2013-11-15 CN CN201310580840.5A patent/CN104651245B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556853A (en) * | 2001-07-26 | 2004-12-22 | Probiotics lactobacillus casei bacterial strain | |
| TW200808191A (en) * | 2006-08-09 | 2008-02-16 | Genmont Biotech Inc | Product containing Lactobacillus paracasei |
| TW200944215A (en) * | 2008-04-30 | 2009-11-01 | Genmont Biotech Inc | Lactobacillus isolates having anti-inflammatory activities and uses of the same |
Non-Patent Citations (1)
| Title |
|---|
| Prevention and treatment of enteric viral infections:possible benefits of probiotic bacteria;Florence Colbe`re-Garapin et al;《Microbes and Infection》;20071002;第1627页右栏最后一段，第1628页左栏第三段 * |
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