CN112786113A - Method for establishing individualized probiotic database and application of method in screening probiotics - Google Patents
Method for establishing individualized probiotic database and application of method in screening probiotics Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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Abstract
The invention provides a method for establishing an individualized probiotic database and application thereof in screening probiotics. The probiotic database completed according to the establishing method comprises the sequence of a plurality of probiotic strains or different strains corresponding to cytokine information numerical values IL-10 secretion, IL-4/IFN-gamma and IFN-gamma/IL-4, and the sequence is used as a probiotic screening index. In particular, the individualized probiotic database is very suitable for being applied to screening of individualized probiotics, and the purpose of accurate health care of individuals is achieved.
Description
Technical Field
The invention relates to a method for establishing an individualized probiotic database and application thereof in screening probiotics. In particular, the personalized probiotic database comprises at least two or more probiotic screening indexes, so that the screening of the probiotics according to personalized differences is performed.
Background
The existing health industry is a health product designed for the public, and the health effect is very limited because of neglecting individual differences. The concept of 'precise medicine' is to make prevention and auxiliary strategies for individual constitution differences, and the concept is applied to the development of health products, so that the product design can be made due to individual differences to increase the utility of the health products.
In summary, in the health industry related to probiotics, how to perform individual differentiation probiotic research and screening on a platform with scientific basis according to individual conditions is still a subject to be urgently needed to break through and develop.
Disclosure of Invention
In view of the above background of the invention, in order to meet the industrial requirements, the invention provides a method for establishing an individualized probiotic database and an application thereof in screening probiotics, thereby achieving the purpose of precisely pairing the probiotics to an individual.
The following explains some commonly used words in the summary of the invention.
The individual in the present invention includes all mammals such as human, pet, livestock, etc. or other organisms which can use probiotics to regulate their physiological functions.
The screening and the application of the probiotics strain or different strains of different species belong to the range of individual health care, and do not relate to the treatment and diagnosis of diseases.
IL-4 of the present disclosure is interleukin 4; IL-10 is interleukin 10; IFN-gamma is gamma interferon。
The Th1 helper cells described in the present disclosure are first-type helper T cells (T helper 1 cells); the Th2 helper cells are type II helper T cells (T helper 2 cells).
The sorting according to the invention is performed in such a way that the numerical values are prioritized from high to low, the highest numerical value representing the first order, the next highest numerical value representing the second order, and the next highest numerical value representing the third order.
The first purpose of the invention is to provide a method for establishing an individualized probiotic database. The setup method includes, but is not limited to, the five steps described below.
The method comprises the following steps: providing a plurality of probiotic bacterial species or different bacterial strains of the same species; the probiotic strains refer to species of different genera, different species of the same genus or different strains of the same genus, and include Bifidobacterium (Lactobacillus bifidum), Lactobacillus bifidus (Bacillus longum), Lactobacillus infantis (Bacillus infantis), Lactobacillus griffithii (Lactobacillus gasseri), Lactobacillus lactis (Lactobacillus lactis), Bacillus coagulans (Bacillus coagulans), Lactobacillus bulgaricus (Lactobacillus delbrueckii) subsp Streptococcus thermophilus (Streptococcus thermophilus), Lactobacillus helveticus (Lactobacillus helveticus), Clostridium butyricum (Clostridium butyricum), Bifidobacterium lactis (Bifidobacterium lactis), Lactobacillus rhamnosus (Lactobacillus rhamnoides), Lactobacillus reuteri (Lactobacillus reuteri), Streptococcus thermophilus (Streptococcus thermophilus), Bacillus putrescentiae (Faecalibacterium prausnitzii) or Ackermanella (Akkermansia polycephala).
Step two: the plurality of probiotic strains or different strains of the same species are co-cultured in vitro with biological samples derived from the same individual, respectively. The biological sample comprises immune cells.
Step three; performing an analysis step, thereby obtaining the cell hormone information secreted by the biological sample co-cultured with the plurality of probiotic strains or different strains respectively, wherein the cell hormone information comprises the ratio of the secretion amount of IL-4 to the secretion amount of IFN-gamma (IL-4/IFN-gamma), the ratio of the secretion amount of IFN-gamma to the secretion amount of IL-4 (IFN-gamma/IL-4) and the secretion amount of IL-10.
Step four: and performing a sequencing step, wherein the sequencing step is to establish sequencing information of the values from high to low according to the values of the cytokine information, and the sequencing information comprises sequencing of the probiotics respectively corresponding to IL-4/IFN-gamma, sequencing of the probiotics respectively corresponding to IFN-gamma/IL-4, sequencing of the probiotics respectively corresponding to IL-10 secretion or any combination of the sequencing.
Step five: performing a pairing step, thereby completing the establishment of the individualized probiotic database; the matching step comprises matching the IL-4/IFN-gamma and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains respectively corresponding to the IL-10 secretion amount and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the plurality of probiotic strains or different strains to an individual with over-expression of Th1 helper cells; and IFN-gamma/IL-4 and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the IFN-gamma/IL-4 and the IL-10 secretion amount and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the plurality of probiotic strains or different strains are paired to an individual with over-expression of Th2 helper cells.
Innovatively, the individualized probiotic database established according to the method comprises an index platform for selecting individualized probiotics established by the cytokine information (IL-10 secretion, IL-4/IFN-gamma and IFN-gamma/IL-4), and the index platform for selecting at least more than two probiotic strains or screening indexes of the same different strains, so that screening of the probiotics conforming to individuation or individual immune differentiation can be performed.
Specifically, the probiotic strains or the screening indexes and information of the same different strains provided by the selection index platform conforming to the individualized probiotics comprise the sequencing of the probiotics respectively corresponding to IL-4/IFN-gamma, the sequencing of the probiotics respectively corresponding to IFN-gamma/IL-4, the sequencing of the probiotics respectively corresponding to the secretion of IL-10 and the pairing information of the individualized probiotics.
Specifically, the matching information of the individualized probiotics comprises a probiotic adaptation table of an individual with over-expression of Th1 helper cells or a probiotic adaptation table of an individual with over-expression of Th2 helper cells.
The probiotic adaptation table of the individual with over-expression of Th1 helper cells takes the secretion of IL-4/IFN-gamma and IL-10 as an index, and the corresponding probiotics strains or the ordered first three cis-probiotics of different strains of the same species are matched to the individual with over-expression of Th1 helper cells.
The probiotic adaptation table of the individual with over-expression of Th2 helper cells takes IFN-gamma/IL-4 and the secretion of IL-10 as indexes, and the corresponding probiotics of the multiple probiotic strains or the ordered first three cis-probiotics of different strains are matched to the individual with over-expression of Th2 helper cells.
The second purpose of the invention is to provide a method for screening probiotics for regulating individualized immunity. The screening method is used for screening the probiotics for adjusting the individualized immunity according to the information of the individualized probiotic database established by the first purpose. The process is not artificially and intelligently judged, and the information processing system directly outputs the screening result. In other words, the screening method is used for innovatively applying big data analysis of probiotics and further screening and pairing probiotics meeting individualized immunity.
Specifically, the screening method comprises screening IL-4/IFN-gamma and a plurality of probiotic strains respectively corresponding to the IL-4/IFN-gamma or a first ordered probiotic strain of the same species and different strains to give a Th1 helper cell over-expression individual; or screening IFN-gamma/IL-4 and a plurality of probiotics respectively corresponding to the same species or probiotics of the same different strains with the first order of the sequence to give an individual with over-expression of Th2 helper cells.
Th1 helper cells primarily function as immune responses against intracellular bacteria and protozoa. When Th1 helper cells are over-expressed and IFN-gamma is relatively over-produced, autoimmune diseases such as multiple sclerosis, psoriasis, rheumatoid arthritis, type I diabetes, organ transplant rejection and the like can be caused.
Th2 helper cells primarily function as immune responses against extracellular multicellular parasites. When Th2 helper cells are over-expressed and IL-4 is produced in a relatively excessive amount, allergy-related diseases such as allergic rhinitis, asthma and atopic dermatitis may be caused.
Preferably, the screening method further comprises screening for IL-10 secretion and corresponding first-cis probiotic strains of the plurality or strains of the same species for administration to the subject having over-expression of Th1 helper cells, the subject having over-expression of Th2 helper cells, or a subject having over-expression of Th1 helper cells and over-expression of Th2 helper cells together.
Accordingly, the second objective of the present invention is to provide a method for screening individual probiotics for regulating personalized immunity, which is a method for performing precise probiotic screening pairing on individuals with different T-helper cell manifestations according to the selection index platform of individual probiotics in the individual probiotic database. The second objective of the present invention is to provide a method for screening probiotics, which is to screen out suitable probiotics according to individual differences, so as to achieve the purpose of selecting suitable probiotics to perform accurate individual health care or adjust individual immunity according to individual differences.
The third purpose of the invention is to provide a method for screening probiotics with the function of regulating over-expression of Th1 helper cells, which is to screen the probiotics with the function of regulating over-expression of Th1 helper cells according to the individualized probiotics database obtained by the establishing method of the individualized probiotics database; wherein the probiotics with the first three cis-positions of IL-4/IFN-gamma and a plurality of probiotic strains respectively corresponding to the probiotics or different strains in the same species are screened as the probiotics for regulating over expression of Th1 helper cells.
Preferably, the method for screening the probiotics capable of regulating the over-expression of the Th1 helper cells further comprises screening the individuals with over-expression of the Th1 helper cells by screening the secretion amount of IL-10 and the corresponding probiotics of the plurality of probiotic strains or the first ordered probiotics of the same strain, so as to regulate the number of Th1 helper cells and the number of other immune cells of the individuals to balance by increasing the secretion amount of IL-10 of the individuals, such as Th2 helper cells, and simultaneously inhibit the inflammation and reduce the allergic or autoimmune reaction of the individuals.
The fourth purpose of the invention is to provide a method for screening probiotics with regulatory Th2 helper cell over-expression, which is to screen the probiotics with regulatory Th2 helper cell over-expression according to the individualized probiotics database obtained by the establishing method of the individualized probiotics database; wherein IFN-gamma/IL-4 and a plurality of probiotic strains respectively corresponding to the same or the first three-cis probiotics of different strains in the sequence are screened as the probiotics for regulating over expression of Th2 helper cells.
Preferably, the method for screening the probiotics capable of regulating the over-expression of the Th2 helper cells further comprises screening the individuals with over-expression of the Th2 helper cells by screening the secretion amount of IL-10 and the corresponding probiotics of the plurality of probiotic strains or the first ordered probiotics of the same strain, so as to regulate the number of Th2 helper cells and the number of other immune cells of the individuals to balance by increasing the secretion amount of IL-10 of the individuals, such as Th1 helper cells, and simultaneously inhibit the inflammation and reduce the allergic or autoimmune reaction of the individuals.
In summary, the present invention includes (1) providing a method for establishing an individualized probiotic database, the method establishes an individualized probiotic database including the aforementioned cytokine information as an indicator platform for selecting individualized probiotic strains or different strains, the database includes the sequences of the probiotics corresponding to IL-4/IFN- γ, the sequences of the probiotics corresponding to IFN- γ/IL-4, the sequences of the probiotics corresponding to IL-10 secretion, and the pairing information of the individualized probiotics, thereby serving as the basis for screening and adapting of the individualized probiotics. (2) The method is characterized in that probiotics suitable for different individuals are screened out by using a dual-index system according to different individual conditions, so that the aim of accurately selecting the suitable probiotics for individual health care or adjusting the individual immunity is fulfilled according to individual differences. (3) A method for screening out the probiotics for regulating the over expression of Th1 helper cells is provided, which comprises screening out the first three cis-position probiotics of IL-4/IFN-gamma and a plurality of corresponding probiotics strains or different strains respectively as the probiotics for regulating the over expression of Th1 helper cells, and simultaneously or further screening out the secretion of IL-10 and the first cis-position probiotics of the plurality of corresponding probiotics strains or different strains respectively as the probiotics for regulating the over expression of Th1 helper cells. (4) A method for screening out the probiotics for regulating the over expression of Th2 helper cells is provided, which comprises screening IFN-gamma/IL-4 and the first three cis-positioned probiotics of a plurality of corresponding probiotics strains or different strains respectively as the probiotics for regulating the over expression of Th2 helper cells, and simultaneously or further screening out the secretion of IL-10 and the first cis-positioned probiotics of the plurality of corresponding probiotics strains or different strains as the probiotics for regulating the over expression of Th2 helper cells. The method for screening each probiotic takes the individualized probiotic database as a screening and measuring system, applies big data analysis, and directly screens and pairs the probiotic strains according with individualized differences by an information processing system without artificial mental judgment.
Drawings
Is free of
Detailed Description
The foregoing and other aspects, features and advantages of the invention will be apparent from the following more particular description of a preferred embodiment, as illustrated in the accompanying drawings. In order that the invention may be fully understood, specific steps and components thereof will be set forth in the following description. It will be apparent that the invention may be practiced without limitation to specific details that are within the skill of one of ordinary skill in the art. In other instances, well-known components or steps have not been described in detail so as not to unnecessarily obscure the present invention. While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
A first embodiment of the present invention is to provide a method for establishing a personalized probiotic database. The setup method includes, but is not limited to, the five steps described below.
The method comprises the following steps: providing a plurality of probiotic bacterial species or different bacterial strains of the same species; the probiotic strains refer to species of different genera, different species of the same genus or different strains of the same genus, and include Bifidobacterium (Lactobacillus bifidum), Lactobacillus bifidus (Bacillus longum), Lactobacillus infantis (Bacillus infantis), Lactobacillus griffithii (Lactobacillus gasseri), Lactobacillus lactis (Lactobacillus lactis), Bacillus coagulans (Bacillus coagulans), Lactobacillus bulgaricus (Lactobacillus delbrueckii) subsp Streptococcus thermophilus (Streptococcus thermophilus), Lactobacillus helveticus (Lactobacillus helveticus), Clostridium butyricum (Clostridium butyricum), Bifidobacterium lactis (Bifidobacterium lactis), Lactobacillus rhamnosus (Lactobacillus rhamnoides), Lactobacillus reuteri (Lactobacillus reuteri), Streptococcus thermophilus (Streptococcus thermophilus), Bacillus putrescentiae (Faecalibacterium prausnitzii) or Ackermanella (Akkermansia polycephala).
Step two: the plurality of probiotic strains or different strains of the same species are co-cultured in vitro with biological samples derived from the same individual, respectively. The biological sample comprises immune cells.
Step three; performing an analysis step, thereby obtaining the cell hormone information secreted by the biological sample co-cultured with the plurality of probiotic strains or different strains respectively, wherein the cell hormone information comprises the ratio of the secretion amount of IL-4 to the secretion amount of IFN-gamma (IL-4/IFN-gamma), the ratio of the secretion amount of IFN-gamma to the secretion amount of IL-4 (IFN-gamma/IL-4) and the secretion amount of IL-10.
Step four: and performing a sequencing step, wherein the sequencing step is to establish sequencing information of the values from high to low according to the values of the cytokine information, and the sequencing information comprises sequencing of the probiotics respectively corresponding to IL-4/IFN-gamma, sequencing of the probiotics respectively corresponding to IFN-gamma/IL-4, sequencing of the probiotics respectively corresponding to IL-10 secretion or any combination of the sequencing.
Step five: performing a pairing step, thereby completing the establishment of the individualized probiotic database, wherein the pairing step comprises pairing the IL-4/IFN-gamma and the respectively corresponding first three cis-positioned probiotics of the plurality of probiotic strains or different strains and the secretion of the IL-10 and the corresponding first three cis-positioned probiotics of the plurality of probiotic strains or different strains to an individual with over-expression of Th1 helper cells; and IFN-gamma/IL-4 and the corresponding first three cis-arranged probiotic strains of the plurality of probiotics and the secretion amount of IL-10 and the corresponding first three cis-arranged probiotics of the plurality of probiotic strains or different strains are paired to an individual with over-expression of Th2 helper cells.
In one embodiment, the biological sample comprises immune cells.
In one embodiment, the immune cells are isolated from a blood sample or an oral mucosal sample.
In one embodiment, the assay used in the assay step comprises enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA), flow cytometry, chemiluminescence immunoassay (CLIA), or flow bead array multiplex assay systems. Preferably, the assay is an enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA).
In one embodiment, the ratio of the IL-4 secretion to IFN- γ secretion (IL-4/IFN- γ) and the IL-10 secretion is used as an indicator of probiotic selection in individuals with over-expression of Th1 helper cells.
In one embodiment, the ratio of the amount of IFN- γ to the amount of IL-4 secreted (IFN- γ/IL-4) and the amount of IL-10 secreted are indicators of probiotic selection in an individual with over-expression of Th2 helper cells.
In one embodiment, the sorting and pairing steps are performed by an information processing system.
In a representative embodiment, the process comprises the steps of: (1) identifying individual differences, which can be detected by allergen testing or individual investigation; (2) taking a blood or oral mucosa sample of said individual; (3) separating immune cells from the blood or oral mucosa sample; (4) co-culturing the immune cell with a plurality of probiotic strains or different strains of the same species; (5) analyzing the content of cell hormones secreted by the immune cells after co-culture with the plurality of probiotic strains or different strains of the same species by an ELISA technology, wherein the cell hormones comprise IFN-gamma, IL-4 and IL-10; (6) outputting the sequencing of the probiotic strains or the same different strains and the information related to the cytokines by a computer provided with a biological information statistical operation system, wherein the sequencing order is that the numerical values of the information related to the cytokines are arranged in sequence from high to low, and the sequencing comprises the sequencing of the probiotics respectively corresponding to IL-4/IFN-gamma, the sequencing of the probiotics respectively corresponding to IFN-gamma/IL-4 and the sequencing of the probiotics respectively corresponding to the secretion of IL-10; and (7) producing a personalized and differentiated probiotic adaptation table, wherein the IL-4/IFN-gamma and the respectively corresponding sequenced first three cis-positioned probiotics of the plurality of probiotic strains or different strains and the secretion amount of the IL-10 and the corresponding sequenced first three cis-positioned probiotics of the plurality of probiotic strains or different strains are paired to an individual with over-expression of Th1 helper cells; and IFN-gamma/IL-4 and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains respectively corresponding to the same, the secretion amount of the IL-10 and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the same are paired to an individual with over-expression of Th2 helper cells; thereby completing the establishment of the individualized probiotic database.
The second embodiment of the invention provides a method for screening probiotics for regulating individualized immunity, which is to screen the probiotics for regulating individualized immunity according to the individualized probiotic database obtained by the method for establishing the individualized probiotic database according to the first embodiment of the invention; it comprises screening IL-4/IFN-gamma and the corresponding probiotic strains or the first ordered probiotic of the same different strains to give a Th1 helper cell over-expressed individual; or screening IFN-gamma/IL-4 and a plurality of probiotic strains respectively corresponding to the same or first ordered probiotics of different strains to give an individual with over-expression of Th2 helper cells.
Preferably, the screening method of probiotics for regulating individualized immunity further comprises screening the individuals with over-expression of Th1 helper cells, over-expression of Th2 helper cells or over-expression of Th1 helper cells and Th2 helper cells together by the secretion amount of IL-10 and the corresponding first-cis probiotics of the plurality of probiotic strains or different strains.
The third embodiment of the invention provides a method for screening probiotics with the function of regulating over-expression of Th1 helper cells, which is to screen the probiotics with the function of regulating over-expression of Th1 helper cells according to the individualized probiotics database obtained by the method for establishing the individualized probiotics database as described in the first embodiment; wherein the probiotics with the first three cis-positions of IL-4/IFN-gamma and a plurality of probiotic strains respectively corresponding to the probiotics or different strains in the same species are screened as the probiotics for regulating over expression of Th1 helper cells.
Preferably, the method for screening probiotics capable of regulating over-expression of Th1 helper cells further comprises screening the individuals with over-expression of Th1 helper cells by using the probiotics with the secretion amount of IL-10 and the corresponding first order of the plurality of probiotic strains or different strains.
The fourth embodiment of the invention provides a method for screening probiotics with the function of regulating over-expression of Th2 helper cells, which is to screen the probiotics with the function of regulating over-expression of Th2 helper cells according to the individualized probiotics database obtained by the method for establishing the individualized probiotics database; wherein IFN-gamma/IL-4 and a plurality of probiotic strains respectively corresponding to the same or the first three-cis probiotics of different strains in the sequence are screened as the probiotics for regulating over expression of Th2 helper cells.
Preferably, the method for screening the probiotics capable of regulating the over-expression of the Th2 helper cells further comprises screening the individuals with over-expression of the Th2 helper cells by the aid of the secretion amount of IL-10 and the corresponding first-cis probiotic strain of the plurality of probiotic strains.
The following examples are experiments conducted according to the contents of the above embodiments, and are therefore provided as a detailed description of the present invention.
The experimental pre-operation comprises: preparing basal salt solution (or sterile PBS), preparing washing solution (PBS + 0.05% Tween-20), and preparing stop solution (2N H)2SO4) And a prepared medium (medium).
And (3) re-dissolving and sterilizing probiotics: the powder of the capsule is dissolved back in 5ml of sterile PBS; 1ml of the bacterial solution was taken at 80 ℃ for 30min (sterilized) and then diluted to the desired concentration for use in the co-culture step with the biological sample.
Individual biological samples and co-culture: taking individual leucocyte cells as a biological sample co-cultured with the probiotic strain, and sequentially performing the steps of separation and extraction and co-culture as follows: mixing the blood sample of the individual and the balanced salt solution 1:1 into a diluted blood sample; shaking the Ficoll-Paque PLUS uniformly; adding Ficoll-Paque PLUS into a centrifugal tube; diluted blood was carefully added; centrifuging at 400 Xg for 30-40 min (18-20 deg.C); removing the uppermost plasma (plasma); obtaining LymphocystesM of the second layeronocytes plants; adding at least three times of volume of balanced salt solution into the obtained cells and gently scattering the cells; centrifuging at 60-100 Xg for 10 min (18-20 deg.C); removing the supernatant, adding the same volume of balanced salt solution as step 8 and gently breaking up the cells; centrifuging at 60-100 Xg for 10 min (18-20 deg.C); removing the supernatant, scattering the cells by using a cell culture solution, and counting the cells; cells were seeded into 24-well plates, 4X105A/well; co-culturing with probiotics with different bacteria numbers for 40 hours; after the supernatant was aspirated and centrifuged at 4 ℃ to remove suspended bacteria, the supernatant was analyzed for IFN-. gamma.IL-10 and IL-4 by ELISA.
General procedure for ELISA analysis of cytokines
BioLegend's ELISA MAX were used for this ELISA assayTMThe Deluxe Set kit was used to analyze the secretion of IFN-. gamma.IL-4 and IL-10, respectively.
The analytical procedure comprises the following steps: fixing 100ul of diluted capture antibody (for example, IFN-gamma capture antibody is to be analyzed) on a 96-hole plate, and standing at 2-8 ℃ overnight; washing with PBS buffer solution for 4 times, adding 200ul of Assay diluent, and standing at room temperature for 1 hr; washing with PBS buffer solution for 4 times, adding 100ul diluted standard substance and biological sample to be tested, standing at room temperature for 2 hours; washing with PBS buffer for 4 times, adding 100ul diluted detection antibody (IFN-gamma if to be analyzed), standing at room temperature for 1 hr; washing with PBS buffer for 4 times, adding 100ul diluted Avidin (Avidin-HRP), and standing at room temperature for 0.5 hr; washing with PBS buffer solution for 5 times, adding 100ul of color developing solution (TMB substrate solution) in the dark, and standing at room temperature for 20 min; the reaction was stopped by adding 100ul of stop solution (stop solution) and then reading the instrumental analysis values at 450nm and 570 nm. Thereby obtaining the secretion amounts of IFN-gamma, IL-4 and IL-10, respectively.
Inputting the cell hormone information obtained by the ELISA analysis into an information processing system to complete the establishment of a database of the individualized probiotics.
Individual a: the individual A has symptoms related to autoimmune disorder and type I diabetes through physiological condition investigation; defining the individual A as the over-expression of Th1 cells.
The contents of the ordering and adaptation table part of the personalized probiotic database of the individual a prepared according to the present invention are shown in table one, and the sample for detection and analysis is a blood sample of the individual a.
Watch 1
Blood specimen
Individual B: the individual B has symptoms related to autoimmune disorder and rheumatoid arthritis through physiological condition investigation; defining the individual B as the over-expression of Th1 cells.
The contents of the ranking and adaptation table part of the personalized probiotic database of the individual B prepared according to the present invention are shown in table two, and the sample for detection and analysis is the oral sample of the individual B.
Watch two
Oral cavity sample
According to the first or second table, the probiotics with the order of IL-4/IFN-gamma of 1 or 2 or the probiotics with the order of IL-10(pg/mL) of 1 or 2 are preferentially screened and administered to the individual A or the individual B.
Individual C: through the investigation of physiological conditions, the individual C has allergic symptoms such as nasal allergy and atopic dermatitis; defining the individual C as the over-expression of Th2 cells.
The contents of the ordering and adaptation table part of the individualized probiotic database of the individual C prepared according to the present invention are shown in table three, and the sample for detection and analysis is a blood sample of the individual C.
Watch III
Blood specimen
Individual D: the individual D has allergy symptom to pollen by physical condition investigation; defining the individuals classified as over-represented by Th2 cells.
The contents of the ranking and fitting table part of the personalized probiotic database of the individual D prepared according to the present invention are shown in table four, and the sample for detection and analysis is the oral sample of the individual D.
Watch four
Oral cavity sample
According to Table three or Table four, the subjects C or D were preferentially selected for probiotics with order of 1 or 2 of IFN-gamma/IL-4 or probiotics with order of 1 or 2 of IL-10 (pg/mL).
While the present invention has been described with reference to the specific examples, it should be understood that the scope of the present invention is not limited thereto, and various changes and modifications may be made without departing from the spirit and scope of the present invention. In addition, the abstract and the title of the invention are provided for assisting the search of patent documents and are not intended to limit the scope of the invention.
Claims (15)
1. A method for establishing a personalized probiotic database, which is characterized by comprising the following steps:
(1) providing a plurality of probiotic bacterial species or different bacterial strains of the same species;
(2) the plurality of probiotic strains or different strains of the same species are co-cultured in vitro with biological samples from the same individual respectively;
(3) performing an analysis step, thereby obtaining cytokine information secreted by the biological sample co-cultured with the plurality of probiotic strains or the different strains, respectively, the cytokine information including a ratio of the secretion amount of IL-4 to the secretion amount of IFN-gamma (IL-4/IFN-gamma), a ratio of the secretion amount of IFN-gamma to the secretion amount of IL-4 (IFN-gamma/IL-4), and a secretion amount of IL-10;
(4) performing a sequencing step, wherein the sequencing step establishes sequencing information of the values from high to low according to the values of the cytokine information, and the sequencing information comprises sequencing of the probiotic strains or different strains respectively corresponding to IL-4/IFN-gamma, sequencing of the probiotic strains or different strains respectively corresponding to IFN-gamma/IL-4, sequencing of the probiotic strains or different strains respectively corresponding to IL-10 secretion or sequencing of the probiotic strains or different strains respectively corresponding to IL-10 or combination of the sequencing; and
(5) performing a pairing step, thereby completing the establishment of the individualized probiotic database, wherein the pairing step is to pair the IL-4/IFN-gamma and the respectively corresponding sequenced first three cis-positioned probiotics of the plurality of probiotic strains or different strains and the secretion amount of the IL-10 and the corresponding sequenced first three cis-positioned probiotics of the plurality of probiotic strains or different strains to an individual with over-expression of Th1 helper cells; and IFN-gamma/IL-4 and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the IFN-gamma/IL-4 and the IL-10 secretion amount and the ordered first three cis-position probiotics of the plurality of probiotic strains or different strains corresponding to the plurality of probiotic strains or different strains are paired to an individual with over-expression of Th2 helper cells.
2. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the probiotic strains refer to species belonging to different genera, or to the same species but different strains, and comprise bifidus, bifidus infantis, brefelacillus infantis, lactobacillus gasseri, lactobacillus lactis, lactococcus lactis, bacillus coagulans, lactobacillus bulgaricus, lactobacillus johnsonii, lactobacillus kei, lactobacillus paracasei, bifidobacterium adolescentis, lactobacillus salivarius, lactobacillus plantarum, lactobacillus fermentum, lactobacillus brevis, bifidobacterium breve, lactobacillus acidophilus, streptococcus thermophilus, lactobacillus helveticus, clostridium butyricum, bifidobacterium rette, lactobacillus rhamnosus, lactobacillus reuteri, streptococcus thermophilus, coprinus shigella, or akmansiella.
3. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the biological sample comprises immune cells.
4. The method of establishing an individualized probiotic database according to claim 3, characterized in that: the immune cells are separated from a blood sample or an oral mucosa sample.
5. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the assay used in this assay step may include enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA), flow cytometry, chemiluminescence immunoassay (CLIA), or flow bead array multiplex assay systems.
6. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the ratio of the IL-4 secretion to IFN-gamma secretion (IL-4/IFN-gamma) and the IL-10 secretion are used as probiotic selection indicators for individuals with over-expression of Th1 helper cells.
7. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the ratio of IFN-gamma secretion to IL-4 secretion (IFN-gamma/IL-4) and the IL-10 secretion are used as probiotic selection indicators for individuals with over-expression of Th2 helper cells.
8. The method of establishing an individualized probiotic database according to claim 1, characterized in that: the sorting step and the matching step are performed by an information processing system.
9. A screening method of probiotics for adjusting individualized immunity is characterized in that: the personalized probiotic database obtained according to the establishing method of the personalized probiotic database of claim 1 is used for screening the probiotics for adjusting personalized immunity; it comprises screening IL-4/IFN-gamma and the corresponding probiotic strains or the first ordered probiotic of the same different strains to give a Th1 helper cell over-expressed individual; or screening IFN-gamma/IL-4 and a plurality of probiotic strains respectively corresponding to the same or first ordered probiotics of different strains to give an individual with over-expression of Th2 helper cells.
10. The method for screening of probiotics for modulating personalized immunity according to claim 9, characterized in that: the method also comprises screening the secretion amount of IL-10 and corresponding probiotics of a plurality of probiotic strains or first order of different strains of the same species to give the individual with over-expression of Th1 helper cells, the individual with over-expression of Th2 helper cells or the individual with over-expression of Th1 helper cells and over-expression of Th2 helper cells at the same time.
11. The method for screening of probiotics for modulating personalized immunity according to claim 9, characterized in that: providing a plurality of probiotic bacterial species or different bacterial strains of the same species; the probiotic strains refer to species belonging to different genera, different species belonging to the same genus or different strains belonging to the same genus and different strains belonging to the same genus, and comprise bifidus, bifidus infantis, lactobacillus gasseri, lactobacillus lactis, lactococcus lactis, bacillus coagulans, lactobacillus bulgaricus, lactobacillus johnsonii, lactobacillus kei, lactobacillus paracasei, bifidobacterium adolescentis, lactobacillus salivarius, lactobacillus plantarum, lactobacillus fermentum, lactobacillus brevis, bifidobacterium breve, lactobacillus acidophilus, streptococcus thermophilus, lactobacillus helveticus, clostridium butyricum, bifidobacterium rette, lactobacillus rhamnosus, lactobacillus reuteri, streptococcus thermophilus, coprinus shigella or akmanella.
12. A method of screening for probiotics having the ability to modulate over-expression of Th1 helper cells, comprising: the screening of the probiotics with the regulation Th1 helper cell over-expression is carried out according to the individualized probiotics database obtained by the establishing method of the individualized probiotics database of claim 1; wherein the probiotics with the first three cis-positions of IL-4/IFN-gamma and a plurality of probiotic strains respectively corresponding to the probiotics or different strains in the same species are screened as the probiotics for regulating over expression of Th1 helper cells.
13. The method for screening of probiotic bacteria with modulated over-expression of Th1 helper cells according to claim 12, wherein: also comprises screening the secretion of IL-10 and corresponding probiotics of the plurality of probiotic strains or the first order of different strains of the same species to give the individuals with over-expression of Th1 helper cells.
14. A method of screening for probiotics having the ability to modulate over-expression of Th2 helper cells, comprising: the screening of the probiotics with the regulation Th2 helper cell over-expression is carried out according to the individualized probiotics database obtained by the establishing method of the individualized probiotics database of claim 1; wherein IFN-gamma/IL-4 and a plurality of probiotic strains respectively corresponding to the same or the first three-cis probiotics of different strains in the sequence are screened as the probiotics for regulating over expression of Th2 helper cells.
15. The method for screening of probiotic bacteria with modulated over-expression of Th2 helper cells according to claim 14, wherein: also comprises screening the secretion of IL-10 and corresponding probiotics of the plurality of probiotic strains or the first order of different strains of the same species to give the individuals with over-expression of Th2 helper cells.
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