CN106635868A - Preparation method of artificial micro-ecological preparation capable of enhancing infant immunity - Google Patents
Preparation method of artificial micro-ecological preparation capable of enhancing infant immunity Download PDFInfo
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- CN106635868A CN106635868A CN201610839502.2A CN201610839502A CN106635868A CN 106635868 A CN106635868 A CN 106635868A CN 201610839502 A CN201610839502 A CN 201610839502A CN 106635868 A CN106635868 A CN 106635868A
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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
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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
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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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
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention discloses a preparation method of an artificial micro-ecological preparation capable of enhancing infant immunity. Probiotic microorganisms in vaginal secretions of a prepartal woman are separated, and the probiotic microorganisms, based upon identification, are prepared into microorganism freeze-dried powder; and the freeze-dried powder, after being dissolved in physiological saline, is smeared to the whole body of an infant, so that the immunizing capacity and resisting capacity of the infant delivered by C-section to the outside world are established and recovered. The preparation method provided by the invention has the advantages that vaginal probiotics are separated from the vaginal secretions of the prepartal woman and possible pathological bacteria are removed, so that microbial community structures of various body parts of the infant delivered by C-section are reconstructed and recovered; and the preparation method has the advantages of being high in safety, good in effectiveness, strong in operability and the like.
Description
Technical field
The present invention relates to a kind of preparation method of the artificial microbial ecological agent for strengthening infant immunity, and in particular to point
From, screen the microorganism with prebiotic effect in maternal vaginal secretions to be produced, preparation becomes microbial ecological agent and cuts open for recovery
Abdomen produces neonate symbiotic microorganism, belongs to microbial biotechnology and biomedicine field.
Background technology
The mankind that develop into of modern medical techniques create caesarean operation, and this new childbirth options have saved countless
Puerpera and the life of baby, are to ensure that the health of the mankind and population regrowth are made that tremendous contribution.Cannot reach in vaginal delivery
Into, or vaginal delivery to puerpera or neonate when may threaten, and just need cesarean.But in recent years, China is cutd open
Anemia of pregnant woman's quantity that abdomen is produced increases year by year, and nowadays the cesarean delivery rate of China has been up to 40%-50%, certain areas even more high.
The selection of childbirth options directly affects the composition of neonate symbiosis flora.In the baby of natural labor, it was observed that micro- life
Thing composition is often similar to parent birth canal and intestinal, and in caesarean baby, microorganism group is into often closer to mother
Skin and hospital environment include medical personnel and other neonates.Mother's vagina is mainly received in the flora field planting of vaginal delivery youngster
With the impact of perineal region flora, the newborn baby that cuts open the belly then mainly affected by hospital environment.By taking intestinal microbial population as an example, the former is initial
Intestinal microbial population is mainly the antibacterial around mother's vagina and vulva, mostly lactobacilluss, melaninogenicus, bacillus bifiduss etc.;And
The latter is mainly based on streptococcus, staphylococcuses, anaerobic cocci, bacteroid, propionibacterium.Wherein staphylococcus aureuses are to lead
The main cause of septicemia of newborn is caused, bifidobacteria is then very low even without and bifidus bacilluss are to body health and exempt from
Epidemic disease function plays an important role.
Baby can be extruded when birth canal by birth canal during normal vaginal, in the progress of labor and birth after not
Long, the antibacterial in mother's body will earliest enter field planting in fetus body by fetal respiration road and digestive tract, and this antibacterial is determined
Plant is healthy extremely important to baby's all one's life, and it is to set up new microbial ecological in a substantially aseptic neonate intestinal
System.The result that the antibacterial of these field planting is mainly worked in birth process, birth canal environmental microorganism.And it is neonatal this
The constituent of microbial ecosystem directly affects the immunologic function of baby.Cesarean neonate during birth because not having Jing birth canals
Extrude, can not in time set up symbiosis flora.Ultimately cause the composition of the microbial ecosystem of the newborn baby that cuts open the belly has very with natural labor youngster
Big difference.
Substantial amounts of research finds that the microorganism group observed in cesarean neonate occurs into change and various diseases at a specified future date
Risk is raised close relationship, including allergy, eczema, asthma, obesity, type 1 diabetes, chronic related inflammation disease
Deng.Britain research alert personnel says that caesarean child is more susceptible to suffer from asthma than the child of natural labor, and cesarean child suffers from the general of asthma
Rate is higher than the child of normal natural labor by 80%.Cesarean has deprived child's right that at first sensory integration is taken exercise so that the newborn baby that cuts open the belly holds
It is susceptible to suffer from sense system imbalance.And cesarean makes " wet lung disease " to increase.Generally speaking, compared with spontaneous labor, cesarean is neonatal
Immunity and anti-infection ability are poorer than the child of spontaneous labor.
The content of the invention
Present invention aims to prior art is not enough to provide a kind of artificial Tiny ecosystem for strengthening infant immunity
The preparation method of preparation.
The method of the invention comprises the following steps:
1st, the collection of vaginal secretionies:1 hour before caesarean operation is carried out, the suitable sterile gauze of a block size is placed
The 10min in the vagina of mother to be produced;
2nd, the preservation of vaginal microbial flora:The gauze for being soaked with vaginal secretionies is immersed in aseptic PBS solution, with aseptic nipper not
Disconnected extruding gauze, makes the vaginal secretionies in gauze be distributed in PBS, is centrifuged, and takes precipitation, and 20% glycerol is added in precipitation, revolves
Whirlpool agitator is mixed, and is preserved at -8O DEG C;
3rd, the separation and identification of vaginal microbial flora:
(1)The preparation of culture medium:Prepare the solid and liquid of BSM, MRS, the GAM solid mediums of improvement and BHI+5% skimmed milks
Culture medium;The BSM culture medium is used to cultivate bifidus bacilluss, and MRS culture medium is used to cultivate lactic acid bacteria, the GAM culture medium of improvement
For cultivating bacteroid, the solid and fluid medium of BHI+5% skimmed milks are used to cultivate total bacterium;
(2)Training method:Vaginal secretionies are taken out from -8O DEG C of refrigerator-freezer, vaginal secretionies is taken and is added to aseptic PBS
In solution, by 101~108Dilution proportion;100 μ L10 are taken respectively2、104、10648h is cultivated under coated plate, anaerobism and aerobic condition;
(3)Choose bacterium inoculation:In aseptic superclean bench according to the form of bacterium colony, size, color, neat in edge in flat board with
It is no, whether whether swell with degraded Lactose, choose single bacterium colony and be inoculated in corresponding fluid medium;
(4)Gram's staining:10 μ L bacterium solutions are taken, coated plate is placed in the morphosiss of observed under electron microscope bacterium on microscope slide;
(5)Sequencing:Different antibacterials are selected for being sequenced according to the result of Gram's staining, various micro- lifes in vaginal secretionies are determined
The race relation of thing;
4th, with the screening of prebiotic effect microorganism:The mixing of the microorganism with prebiotic effect is filtered out according to sequencing result to go forward side by side
Row culture.
5th, the preparation of vaginal microbial flora lyophilized powder:The 20% of equivalent sterilized non-fat breast is added toward bacterium solution, uniform mixing is cold
After lyophilizing is dry, lyophilized powder is made.
6th, the application of vagina beneficial flora lyophilized powder:Before neonate is given birth to, with aseptic physiological saline solution lyophilized powder,
Wiring solution-forming, after cesarean neonate gives birth to 1-3 minutes, with sterile gauze dip solution wipe successively neonatal mouth,
Other positions of the bodies such as face, anus.
7th, the identification of neonate symbiotic microorganism:For verify the transplanting of this separation screening vagina beneficial flora method whether
Neonate symbiotic microorganism can be recovered.Receiving respectively in 1,3,7,14,21 and 30 days this six time points of neonate birth
Try the positions such as anus, oral cavity and the skin of mother and baby and acquire sample altogether, the method being sequenced using 16S rRNA identifies its group's group
Into.
Beneficial effects of the present invention:The method directly separates vagina probioticss from Prepartal women vaginal secretionies, can
Can pathogenic bacterium exclude, for set up and recover cesarean infant parts of body Bacterial community rebuild, with it is simple and convenient,
Safely and effectively, it is operable, can commercialization the advantages of, contribute to preferably strengthening and improving the neonatal anti-infection ability of cesarean
And immunologic function.
Specific embodiment
The method of the invention comprises the following steps:
1st, the collection of vaginal secretionies:1 hour before caesarean operation is carried out, the suitable sterile gauze of a block size is placed
The 10min in the vagina of mother to be produced;
2nd, the preservation of vaginal microbial flora:The gauze for being soaked with vaginal secretionies is immersed in aseptic PBS solution, with aseptic nipper not
Disconnected extruding gauze, makes the vaginal secretionies in gauze be distributed in PBS, is centrifuged, and takes precipitation, and 20% glycerol is added in precipitation, revolves
Whirlpool agitator is mixed, and is preserved at -8O DEG C;
3rd, the separation and identification of vaginal microbial flora:
(1)The preparation of culture medium:Prepare the solid and liquid of BSM, MRS, the GAM solid mediums of improvement and BHI+5% skimmed milks
Culture medium;The BSM culture medium is used to cultivate bifidus bacilluss, and MRS culture medium is used to cultivate lactic acid bacteria, the GAM culture medium of improvement
For cultivating bacteroid, the solid and fluid medium of BHI+5% skimmed milks are used to cultivate total bacterium;
(2)Training method:Vaginal secretionies are taken out from -8O DEG C of refrigerator-freezer, vaginal secretionies is taken and is added to aseptic PBS
In solution, by 101~108Dilution proportion;100 μ L10 are taken respectively2、104、10648h is cultivated under coated plate, anaerobism and aerobic condition;
(3)Choose bacterium inoculation:In aseptic superclean bench according to the form of bacterium colony, size, color, neat in edge in flat board with
It is no, whether whether swell with degraded Lactose, choose single bacterium colony and be inoculated in corresponding fluid medium;
(4)Gram's staining:10 μ L bacterium solutions are taken, coated plate is placed in the morphosiss of observed under electron microscope bacterium on microscope slide;
(5)Sequencing:Different antibacterials are selected for being sequenced according to the result of Gram's staining, various micro- lifes in vaginal secretionies are determined
The race relation of thing;
4th, with the screening of prebiotic effect microorganism:The mixing of the microorganism with prebiotic effect is filtered out according to sequencing result to go forward side by side
Row culture.
5th, the preparation of vaginal microbial flora lyophilized powder:The 20% of equivalent sterilized non-fat breast is added toward bacterium solution, uniform mixing is cold
After lyophilizing is dry, lyophilized powder is made.
6th, the application of vagina beneficial flora lyophilized powder:Before neonate is given birth to, with aseptic physiological saline solution lyophilized powder,
Wiring solution-forming, after cesarean neonate gives birth to 1-3 minutes, with sterile gauze dip solution wipe successively neonatal mouth,
Other positions of the bodies such as face, anus.
7th, the identification of neonate symbiotic microorganism:For verify the transplanting of this separation screening vagina beneficial flora method whether
Neonate symbiotic microorganism can be recovered.Receiving respectively in 1,3,7,14,21 and 30 days this six time points of neonate birth
Try the positions such as anus, oral cavity and the skin of mother and baby and acquire sample altogether, the method being sequenced using 16S rRNA identifies its group's group
Into.
Claims (1)
1. it is a kind of strengthen infant immunity artificial microbial ecological agent preparation method, it is characterised in that:Methods described includes
The following steps:
The collection of A, vaginal secretionies:1 hour before caesarean operation is carried out, the suitable sterile gauze of a block size is placed
The 10min in the vagina of mother to be produced;
The preservation of B, vaginal microbial flora:The gauze for being soaked with vaginal secretionies is immersed in aseptic PBS solution, with aseptic nipper not
Disconnected extruding gauze, makes the vaginal secretionies in gauze be distributed in PBS, is centrifuged, and takes precipitation, and 20% glycerol is added in precipitation, revolves
Whirlpool agitator is mixed, and is preserved at -8O DEG C;
C, the separation of vaginal microbial flora and identification:
(1)The preparation of culture medium:Prepare the solid and liquid of BSM, MRS, the GAM solid mediums of improvement and BHI+5% skimmed milks
Culture medium;The BSM culture medium is used to cultivate bifidus bacilluss, and MRS culture medium is used to cultivate lactic acid bacteria, the GAM culture medium of improvement
For cultivating bacteroid, the solid and fluid medium of BHI+5% skimmed milks are used to cultivate total bacterium;
(2)Training method:Vaginal secretionies are taken out from -8O DEG C of refrigerator-freezer, vaginal secretionies is taken and is added to aseptic PBS
In solution, by 101~108Dilution proportion;100 μ L10 are taken respectively2、104、10648h is cultivated under coated plate, anaerobism and aerobic condition;
(3)Choose bacterium inoculation:In aseptic superclean bench according to the form of bacterium colony, size, color, neat in edge in flat board with
It is no, whether whether swell with degraded Lactose, choose single bacterium colony and be inoculated in corresponding fluid medium;
(4)Gram's staining:10 μ L bacterium solutions are taken, coated plate is placed in the morphosiss of observed under electron microscope bacterium on microscope slide;
(5)Sequencing:Different antibacterials are selected for being sequenced according to the result of Gram's staining, various micro- lifes in vaginal secretionies are determined
The race relation of thing;
D, the screening with prebiotic effect microorganism:The mixing of the microorganism with prebiotic effect is filtered out according to sequencing result to go forward side by side
Row culture;
The preparation of E, vaginal microbial flora lyophilized powder:The 20% of equivalent sterilized non-fat breast is added toward bacterium solution, uniform mixing, freezing is dry
After dry, lyophilized powder is made;
The application of F, vagina beneficial flora lyophilized powder:Before neonate is given birth to, with aseptic physiological saline solution lyophilized powder, it is made into
Solution, after cesarean neonate gives birth to 1-3 minutes, with sterile gauze dip solution wipe successively neonatal mouth, face,
Other positions of anus body;
The identification of G, neonate symbiotic microorganism:To verify that the method for this separation screening vagina beneficial flora transplanting whether can be with
Recover neonate symbiotic microorganism;Neonate birth 1,3,7,14,21 and 30 days this six time points respectively in tested mother
The anus of baby, oral cavity and skin part acquire altogether sample, and the method being sequenced using 16S rRNA identifies its group's composition.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110327376A (en) * | 2019-05-28 | 2019-10-15 | 曹阳 | A kind of Tiny ecosystem transfer vector, preparation and the application in microecology in vaginas transplanting |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105288605A (en) * | 2015-11-13 | 2016-02-03 | 刘传新 | First bacterination method for caesarean delivery babies |
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2016
- 2016-09-22 CN CN201610839502.2A patent/CN106635868A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN105288605A (en) * | 2015-11-13 | 2016-02-03 | 刘传新 | First bacterination method for caesarean delivery babies |
Non-Patent Citations (2)
Title |
---|
DOMINGUEZ-BELLO等: "Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer", 《NATURE MEDICINE》 * |
李勇超等: "婴幼儿肠道茵群定殖及对健康的影响研究进展", 《食品科学》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110327376A (en) * | 2019-05-28 | 2019-10-15 | 曹阳 | A kind of Tiny ecosystem transfer vector, preparation and the application in microecology in vaginas transplanting |
CN110327376B (en) * | 2019-05-28 | 2023-01-13 | 曹阳 | Microecological transfer vector, preparation and application in vaginal microecological transplantation |
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Application publication date: 20170510 |