WO2016148168A1 - Method of evaluating capacity of probiotics to be phagocytosed and screening method using same - Google Patents

Abstract

Provided is a more accurate method of evaluating the capacity of probiotics to be phagocytosed at the cellular level, which is also a method of uniformly evaluating the capacity of probiotics to be phagocytosed, which can easily be performed in nearly in-vivo conditions without depending on burdensome surgical operations. The method of evaluating the capacity of probiotics to be phagocytosed comprises a step of contacting a probiotic with a phagocytic cell that has never contacted a bacterium.

Description

Evaluation method for phagocytic ability of probiotics and screening method using the same

The present invention relates to a probiotic evaluation method, and more particularly, to a quantitative evaluation method for probiotic phagocytosis at a cellular level and a screening method using the same.

In recent years, instead of treatment with drugs, treatment is performed using useful microorganisms that can improve the flora in the digestive tract and have beneficial effects on the host, and their growth promoting substances, so-called probiotics. Attention is focused on the method. For example, it is known that a specific Lactobacillus gasseri-derived oligonucleotide is recognized through toll-like receptor 9 and thus exhibits activity in lymphocyte blastogenesis (Patent Document 1). In addition, administration of Lactobacillus jensenii strain TL2937 (accession number: FERM BP-11272) is known to have antiviral inflammatory effects and the like, and its mechanism is increased expression of IFN-β and expression of MCP-I. It is suggested that this is due to suppression (Patent Document 2).

By the way, in order to elucidate the mechanism when such probiotics are administered, analysis at the gene level and protein level of cytokines and nuclear transcription factors is proceeding. Analysis of the behavior of biotics, that is, phagocytosis (uptake) into cells in a living body, is not sufficient (Non-patent Documents 1 and 2).

In this regard, when cells were actually collected from in vivo organs and tested for the phagocytic ability of probiotics, the cells in in vivo organs had already taken up endogenous bacteria, etc. and were used for the test. It is difficult to distinguish whether the cell itself has a low phagocytic ability or whether the administered probiotic itself is difficult to phagocytose, and even in tests using fluorescent labels, the fluorescence intensity varies from strain to strain. It was difficult to accurately evaluate whether the administered probiotics themselves were phagocytosed (Non-patent Document 3). Furthermore, no method has been reported so far for evaluating probiotics by quantifying how much the probiotics are phagocytosed by cells in the living body.

In addition, when cells such as the large intestine and small intestine are used in vivo, it is possible to evaluate the phagocytic ability of probiotics in a state closer to that in the living body. On the other hand, in collecting these cells, physical and mental Surgical operation with a heavy burden is necessary, and in experimental animals such as pigs, cells of living organs are collected by slaughtering. Therefore, there is a demand for construction of an evaluation system for probiotic phagocytic ability in a state close to the living body of the individual by collecting cells by a method that does not depend on a heavy surgical operation or slaughter of experimental animals. In addition, in order to adjust target cells from a living organ, complicated cell purification steps are required, and time, reagents for purification, equipment, and purification training are also required. Furthermore, in order to evaluate the phagocytic ability of probiotics themselves, it is necessary to accurately measure the growth environment of the individual used and the variation due to the position where the same organ was collected. There is also a cost problem in that it is necessary to increase the number of individuals and tests.

JP 2006-232790 A International Publication No. 2011/1186060

Therefore, in view of the above circumstances, the subject of the present invention is a more accurate probiotic evaluation method of the phagocytic ability at the cellular level, and at the same time, a simple and easy method that does not depend on a heavy surgical operation and the like. To provide a method for evaluating the phagocytic ability of uniform probiotics, which can be used as an alternative for biological evaluation.

In order to solve the above-mentioned problems, the present inventors have intensively studied to bring a cell that has never been in contact with a bacterium into contact with the probiotic, thereby more accurately determining the phagocytosis ability of the probiotic. As a result of finding out that it can be evaluated and further researching it, the present invention has been reached.

That is, the present invention includes the following.
[1] A method for evaluating the phagocytic ability of probiotics, comprising the step of bringing probiotics into contact with cells having phagocytosis that have never been in contact with bacteria.
[2] The method according to [1], wherein the cell is a cell obtained by induction of differentiation from an undifferentiated cell.
[3] The method according to [1] or [2], wherein the cell expresses at least one of BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, and CD11R1.
[4] The method according to any one of [1] to [3], further comprising the step of observing cells and measuring the phagocytosed probiotic.
[5] The method according to any one of [1] to [4], wherein the probiotic contains bacteria.
[6] A kit for evaluating the phagocytic ability of probiotics, comprising cells having a phagocytic action that has never contacted bacteria.
[7] A step of bringing a control probiotic or a test probiotic into contact with a cell having a separate phagocytosis that has never been brought into contact with bacteria, observing each of the cells, and contacting the control probiotic Comparing the phenomenon that occurs when the test probiotic is brought into contact with the phenomenon that occurs when the test probiotic is contacted, and obtaining a comparison result, and selecting the test probiotic based on the comparison result A method for screening probiotics.
[8] A probiotic screening kit comprising cells that have not been contacted with bacteria.
[9] A cell having a phagocytic action that has never been in contact with bacteria expresses at least one of the markers of BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, and CD11R1 Probiotics to be devoured.
[10] An animal feed comprising the probiotic according to [9].
[11] An IL-1β elevation-regulating agent, wherein the probiotic according to [9] is a TL2937 strain (Accession Number: FERM BP-11272), and the TL2937 strain is administered.

According to the method for evaluating the phagocytosis of probiotics of the present invention, the phagocytosis of probiotics can be accurately evaluated at the cellular level. In addition, it is possible to easily evaluate probiotics in a state close to the living body of the individual, regardless of the surgical method or slaughter. Can be provided at low cost.

It is a dot plot by double staining using the flow cytometry data of the cells in Example 1 and Example 2 and the cells in Comparative Example 1 and Comparative Example 2. It is a histogram by the data of the flow cytometry after making the cell in Example 1 and Example 2, and the cell in Comparative Example 1 and Comparative Example 2 contact the TL2766 strain and TL2937 strain. It is the graph which showed the average fluorescence intensity in the contact time with the TL2766 strain | stump | stock and TL2937 strain | stump | stock, and each strain | stump | stock in the cell of the comparative example 1.

It is the photograph of the scanning electron microscope of the cell in Example 3 and Comparative Example 3 after making TL2766 strain and TL2937 strain contact, and the cell which is not made to contact any said strain | stump | stock. It is the photograph of the laser microscope after contacting the cell in Example 3 and the comparative example 3 with TL2937 strain | stump | stock, and dye | staining the nucleus by blue, endosome by red, and bacteria by green.

It is a photograph of the transmission electron microscope after making TL2766 strain and TL2937 strain contact the cell in Example 3 and Comparative Example 3. It is the graph which measured the number of bacteria taken in each cell after making TL2766 strain and TL2937 strain contact the cell in Example 3. FIG.

It is a graph of the analysis of the cytokine by the flow cytometry 16 hours after making the cell in Example 1 and Example 2 contact and irritate | stimulate with TL2766 strain | stump | stock or TL2937 strain | stump | stock, LPS, and Pam3CSK4. It is a graph of the analysis of the cytokine by the flow cytometry 24 hours after making the cell in Example 1 and Example 2 contact and irritate | stimulate with TL2766 strain | stump | stock or TL2937 strain | stump | stock, LPS, and Pam3CSK4.

In this specification, “probiotics” refers to useful microorganisms that improve the flora (bacteria flora) in the digestive tract and can have beneficial effects on the host, and act on the flora (bacteria flora) in the digestive tract to the host. Useful microorganisms that can bring about beneficial effects, growth-promoting substances derived from early microorganisms, and substances that act on useful microorganisms that can act on the flora in the digestive tract to bring about beneficial effects on the host . Therefore, the probiotics of the present invention have a beneficial effect on not only bacteria such as lactic acid bacteria, bifidobacteria, propionic acid bacteria, and E. coli that form bacterial flora, but also substances that promote the growth of such bacteria and the host. And useful microorganisms such as yeast, spore-producing fungi and fungi and substances produced by these microorganisms (culture of microorganisms).

In addition, “phagocytic ability” in this specification refers to the ability to be phagocytosed (incorporated) by cells, and phagocytosis includes both phagocytosis and drunk action.

The cells used in the present invention are phagocytic cells that have never been in contact with bacteria. If they have not been in contact with bacteria, cells that have phagocytosis can be collected directly from the living body, or Differentiating from undifferentiated cells, even if they have been contacted, are cells that have phagocytosis obtained by once collecting them from the living body and inducing differentiation, or from already established undifferentiated cells. You may use the cell which has a phagocytosis obtained by making it induce. From the viewpoint of reducing the physical and mental burden of the individual to be collected and improving the reliability of the test results, it is preferable to use cells having a phagocytosis effect obtained by induction of differentiation from undifferentiated cells.

In addition to undifferentiated cells, depending on the cells to be evaluated, not only stem cell cells such as iPS cells, embryonic stem cells (ES cells), hematopoietic stem cells, etc., but differentiation has already progressed to some extent. Cells having the ability to differentiate into other cells may be used. For example, when the phagocytosis of probiotics related to intestinal immunity is to be evaluated, cells having the ability to differentiate into iPS cells, hematopoietic stem cells, and blood cells are preferred, and in particular, monocytes, granulocytes More preferred are cells having the ability to differentiate into lymphocytes, especially preferred are cells having the ability to differentiate into monocytes. In the case of direct collection from an individual, peripheral blood-derived cells can also be used from the viewpoint that the physical and mental burden of the individual is small.

Furthermore, examples of the individual from which the stem cells and blood cells are derived or collected include vertebrates such as mammals and birds to be tested. Examples of mammals include humans. Further, it includes, but is not limited to, domestic animals such as pigs, cows, monkeys, cats, dogs, goats, sheep, mice, rats, breeding, or experimental animals. Examples of birds include, but are not limited to, domestic animals such as chickens, ducks and ducks, breeding, or experimental animals. On the other hand, humans can also be excluded from subjects. Note that the treatment methods described later are also intended for the vertebrates listed above.

Furthermore, cells having phagocytosis may be cells that have even phagocytosis, and may be completely differentiated into specific cells, even if they are not completely differentiated into specific cells, or are undergoing differentiation. It may be converted.

Further, the differentiation-inducing factor used for inducing differentiation may be appropriately used and changed depending on the degree of cell differentiation (degree) and induced differentiation. For example, from the viewpoint of inducing differentiation into dendritic cells, interleukin-4 (IL-4), granulocyte monocyte colony-stimulating factor (GM-CSF), and lipopolysaccharide (LPS) can be mentioned. It is not something.

Furthermore, the number of days for inducing differentiation may be appropriately changed depending on the degree (degree) of cell differentiation and induced differentiation. For example, when differentiation is induced into intestinal tract-derived dendritic cells using blood cells, it is preferably 2 to 7 days from the start of differentiation induction. From the viewpoint of having the same expression level as that of the intestinal tract-derived dendritic cell in the gene expression level of the cytokine, the 3rd to 5th days from the start of differentiation induction are more preferable.

Further, as an index for determining whether or not to have phagocytosis, cell expression markers can be mentioned, and BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, CD11R1, TLR2 or TLR4 is expressed. From the viewpoint of phagocytosis, cells expressing MHC class II, CD172a, CD11R1, TLR2 or TLR4 markers are more preferable. Needless to say, a plurality of the markers may be expressed.

Also, when using already differentiated cells, dendritic cells, macrophages and neutrophils are preferred. Dendritic cells are more preferable from the viewpoint of evaluating a strong antigen presenting action from a phagocytic action.

Bacteria that have not been brought into contact with bacteria are bacteria that are present in the cells of the individual to be evaluated. For example, oral bacteria, intestinal bacteria, stomach bacteria, skin resident bacteria ( Skin bacteria), vaginal bacteria, microorganisms and the like. Furthermore, the term “never contacted” means not only physical contact with the above-mentioned conventional bacteria, but also, for example, that the above-mentioned conventional bacteria are not phagocytosed (taken in). Of course.

The step of contacting the probiotic includes an act of bringing the cell and the probiotic into contact, taking in, phagocytosing, stimulating, and reacting. In addition, the ratio of cells and probiotics used in the same step may be appropriately changed depending on the phagocytic ability and state of probiotics, cells having phagocytosis, the index or method to be evaluated, and 1:10 ² : ˜1: 10 ⁴ is preferred, 1:10 ² ˜1: 10 ³ is more preferred, and 1:10 ² is particularly preferred.

Furthermore, probiotics labeled with fluorescence and radioisotopes may be used, and the probiotics labeled with fluorescence are not limited to measurement facilities, and are easy to evaluate qualitatively and / or quantitatively. Is preferred. The probiotics to be evaluated may be live bacteria themselves, or may be dead bacteria that have been subjected to ultrasonic crushing and heat sterilization. From the viewpoint that they are not affected by fluctuations in the number of bacteria being measured, they are heat sterilized. It is preferable to use probiotics. Of course, not only one type of probiotic but also a plurality of types of probiotics can be mixed and used.

Next, the contact time may be appropriately changed depending on the phagocytic ability and state of the probiotic, the cell having phagocytosis, the index or method to be evaluated, and preferably 30 minutes to 4 hours, and 1 hour to 3 hours. Time is more preferable, and 1 to 2 hours is particularly preferable. The contacting temperature may be appropriately changed depending on the phagocytic ability and state of probiotics, cells having phagocytosis, the index or method to be evaluated, and is preferably 4 ° C. to 45 ° C., preferably 22 ° C. to 40 ° C. Is more preferable, and 36 ° C to 38 ° C is particularly preferable. Moreover, pH adjustment and a buffer can be used suitably in the guideline or method to evaluate, contact conditions, etc.

Further, in addition to the above steps, a step of observing the contacted cell may be included, and the observation is preferably measuring phagocytosed probiotics, and more preferably the inside of the contacted cell. From the viewpoint of observing the state of phagocytosis, it is particularly preferable to observe a cross section of the contacted cell. More preferably, it is observed.

In addition, examples of the observation apparatus include a microplate reader, flow cytometry, fluorescence microscope, laser microscope, scanning electron microscope, and transmission electron microscope. Is not to be done.

Next, the evaluation kit for the phagocytic ability of probiotics, which includes cells having phagocytosis that have never been in contact with the bacterium of the present invention, includes cells having phagocytosis that have not been in contact with the bacterium described above. In addition, instructions and protocols, experimental reagents and buffers for evaluation and observation, and the like may be included as appropriate.

The screening method for probiotics of the present invention includes a step of separately contacting a control probiotic or a test probiotic using cells having phagocytosis that have never been in contact with the bacteria described above. included. Control probiotics may be, for example, probiotics that are more easily phagocytosed than test probiotics or probiotics that are less susceptible to phagocytosis. It may be changed as appropriate depending on the screening conditions.

Furthermore, in the process of observing each contacted cell and comparing the phenomenon that occurs when the control probiotic is contacted with the phenomenon that occurs when the test probiotic is contacted, The observation method mentioned above is mentioned. In addition, the comparison result in the same step may be appropriately changed depending on the screening conditions. For example, in addition to the number of phagocytosed test probiotics, the comparison result is larger than the cells phagocytosed in the control probiotics. Alternatively, changes in the position of the nucleus of the phagocytized cell, that is, changes in the phagocytized cell itself may be used as an index, but not limited thereto. Needless to say, some of the above indices may be combined. The method further includes a step of selecting a test probiotic based on the comparison result.

The probiotic screening kit of the present invention includes cells having phagocytosis that have not been in contact with the above-mentioned bacteria, and further includes instructions and protocols, and experimental reagents and buffers for evaluation and observation as appropriate. May be.

Next, a phagocytic cell that has never contacted the bacterium of the present invention is at least one of BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, CD11R1, TLR2 or TLR4. Probiotics that express the marker and are phagocytosed by the cells include TL2937 strain, OLL2768, and the like.

Further, the composition containing the probiotics includes feed, food, food composition, beverage composition, medicine, pharmaceutical composition (pharmaceutical), oral cosmetic (cosmetic) and the like. Since these probiotics can be administered orally, the form of feed is animal feed, and the form of food such as beverage composition or food composition is yogurt (fermented milk) or cheese. Dairy products and the like are preferred respectively. The probiotic is a TL2937 strain, and a composition comprising administering the TL2937 strain includes an IL-1β elevation regulator, an IL-6 elevation regulator, an IL-8 elevation regulator, Chemokine (C—C motig) ligand2 (CCL2) elevation regulator, and IL-1β elevation regulator is preferred.

Further, one embodiment of the present invention includes a method for administering or treating the probiotic or the composition containing the probiotic, and further includes the probiotic or the probiotic. Use for the manufacture of a composition comprising is included.

Next, the present invention will be described in more detail with reference to examples, comparative examples, and test examples. However, the present invention is not limited to these examples.

(1) not suffering from such as peripheral blood-derived cell preparation infections, outside of one month prior to the mature pig shipments (to F _l of Landrace and Large Yorkshire were crossed the Duroc, LWD pigs (Hills)) Blood was collected from the jugular vein and placed in a vacuum blood collection tube (TERUMO). Then, using the specific gravity method of Lympholyte-Mammal (CEDARLANE), the cell suspension was gently layered on the tube into which Lympholyte was dispensed from this blood, and then centrifuged (1800 rpm, 60 min, 20 ° C.). Used to fractionate mononuclear cells. Here, the mononuclear cells were seeded at 1 × 10 ⁷ cells / well and held for 1 hour for attachment. After that, RPMI medium containing IL-4 (R & D system) and GM-CSF (R & D system) at 20 ng / mL each was added and cultured (under 37 ° C. and 5% CO ₂ ). After 72 hours from the start of the culture, the medium was replaced with a new medium. Furthermore, this culture (under conditions of 37 ° C. and 5% CO ₂ ) was continued for 5 days to obtain differentiated peripheral blood-derived dendritic cells.

Example 1 and Example 2
(2-1) Preparation of cells by flow cytometry For the cells derived from the peripheral blood, CD172a and CD11R1, which are porcine dendritic cell surface markers, were co-stained with fierythrin (PE) and periodinin (PerCP), respectively. The sample was subjected to flow cytometry. As a result, dot plot analysis by double staining is shown in FIG. The vertical axis represents the number of CD172a positive cells, and the horizontal axis represents the number of CD11R1 positive cells. And the upper square of FIG. 1A shows the CD172a ⁺ CD11R1 ⁺ subset of the cell population with high expression of both surface markers, and this was used as the cells of Example 1 (FIG. 1A). The lower square in FIG. 1A shows a CD172a ^low CD11R1 ⁺ subset of a cell population with ^low expression of CD172a and high expression of CD11R1, which was used as the cells of Example 2 (FIG. 1A).

Example 3
(2-2) Preparation of cells by magnetic bead separation (MACS) method In order to separate CD172a positive cells from the above-mentioned peripheral blood-derived cells using the magnetic bead separation method, 0.5% BSA, 2 mM The cells were washed with MACS buffer containing EDTA and PBS, and 1 × 10 ⁷ of the peripheral blood-derived cells were mouse anti-porcine monocyte / granulocyte-biotin (Southern Biotech, Catalog Number 4525-08) ( anti-CD172a) was added at 10 μL, followed by incubation (4 ° C., 10 min). Thereafter, the cells were washed with MACS buffer, and Streptavidin-labeled microbeads (MiltenyiBiotec) were added at 10 μL, followed by incubation (4 ° C., 15 min). Further, it was washed with a MACS buffer and suspended in the MACS buffer.

Next, the LS column (MiltenyiBiotec, Catalog Number 130-042-401) was washed with MACS buffer, and a suspension of peripheral blood-derived cells labeled with magnetic beads was applied to the column. Further, the cells were washed with a MACS buffer, and using a plunger, CD172a positive cells held on the column were pushed out to obtain CD172a positive cells derived from peripheral blood. This was used as the cell of Example 3.

(3) Preparation of cells derived from the intestinal tract Peyer's patches are collected from the intestinal tissue of healthy mature pigs (LWD) that are not affected by infections and washed with PBS containing 1% Streptomycin / Penicilin (Invitrogen). did. Next, the Peyer's board tissue was homogenized using scissors. Then, RPMI medium containing 1 mg / mL collagenase and 15 μg / mL DNase was added, followed by shaking culture (37 ° C., 60 min). Here, the supernatant of the tissue fluid was removed with an aspirator, and PBS containing 10% FCS and 0.02% EDTA was added, followed by incubation (37 ° C., 5 min). Further, two layers of gauze were laid on PETRI DISH (Corning), and the tissue of Peyer's plate cultured thereon was crushed to prepare a cell suspension.

Furthermore, the supernatant of the cell suspension was removed with an aspirator, 0.2% NaCl solution was added, and lightly mixed to rupture erythrocytes. Then, an equal amount of 1.5% NaCl solution was immediately added, and then returned to the same level as physiological saline. Next, after adding an appropriate amount of RPMI medium, the supernatant was removed with an aspirator and suspended again in RPMI medium. Then, after filtering the RPMI medium using Cell Strainer (BD), immunocompetent cells were fractionated using the specific gravity method of Lympholyte-Mammal to obtain intestinal-derived dendritic cells.

Comparative Example 1 and Comparative Example 2
The intestinal tract-derived cells were prepared in the same manner as in Example 1 and Example 2 using flow cytometry. Here, the upper square in FIG. 1B represents CD172a ⁺ CD11R1 ⁺ subset, which was used as cells of Comparative Example 1 (FIG. 1B). Moreover, the lower square of FIG. 1B showed CD172a ^low CD11R1 ⁺ subset, and this was used as the cell of the comparative example 2 (FIG. 1B).

Comparative Example 3
Using the magnetic bead separation method, CD172a positive cells were obtained from the intestinal tract-derived cells in the same manner as in Example 3. This was used as a cell of Comparative Example 3.

(4) Preparation of Bacteria (Strain) The strains to be brought into contact with each of the above cells include Lactobacillus (L.) plantarum TL2766 (hereinafter sometimes referred to as TL2766 strain) and Lactobacillus (L.) owned by Meiji Co., Ltd. jensenii TL2937 (Accession number: FERM BP-11272) (hereinafter sometimes referred to as TL2937 strain) was used. At this time, using the MRS medium, culturing the above two strains (37 ° C., 24 h) was regarded as one passage and three passages.

Then, the TL2937 strain, against 1.0 × ^{10 11} pieces, Carboxyfluorescein Diacetate (Sigma) (hereinafter, simply referred to as CFDA) so as to have a ratio of 15 mg, was mixed with PBS, and in TL2766 strain , 1.0 × 10 ¹¹ , CFDA was mixed with PBS to a ratio of 0.15 mg. Furthermore, using a water bath, these PBS mixtures were heat-treated (37 ° C., 60 min) while shielding light to incorporate CFDA into the strain. Thereafter, each strain was washed with PBS three times to obtain CFDA-labeled TL2937 and TL2766 strains. Next, these labeled strains TL2937 and TL2766 were sterilized (60 ° C., 45 min), and then these strains were used in order to evaluate the phagocytic ability of probiotics.

The TL2937 strain used in the present invention is an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (IPOD, Sakai AIST) (Japan, 〒305-8565, 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, 6th) Deposited as FERM BP-11272 on May 14, 2010. As stated in Budapest Notification No. 282 (http://www.wipo.int/treaties/en/notifications/budapest/treaty_budapest_282.html), the National Institute of Technology and Evaluation (IPOD, NITE) Independent administrative agency National Institute of Advanced Industrial Science and Technology (AIST) has succeeded in depositing patent microorganisms, and is now an independent administrative corporation Product Evaluation Technology Foundation (IPOD, NITE) (Kazusa Kamashika, Kisarazu City, Chiba Prefecture) 2-5-8 Room 120) (Accession number FERM BP-11272).

Test example 1
(5-1) Observation using flow cytometry Cells of Example 1 and Example 2, Comparative Example 1 and Comparative Example 2 obtained by gating above were seeded at 5 × 10 ⁶ cells / well, Incubated overnight. Next, TLR2 antibody (BioLegend., Catalog Number 309710) and TLR4 antibody (BioLegend., Catalog Number 312808) were added to these media at 200 ng / mL, followed by incubation (37 ° C., overnight). . Then, after sterilizing the TL2766 strain and the TL2937 strain, the cells were contacted at 5 × 10 ⁸ cells / well.

Next, the cells brought into contact with the fluorescently labeled strain were collected by pipetting, and these cells were seeded at 2 × 10 ⁶ cells / well, and 2% FCS, 0.01% NaN ₃ / PBS was added. Each cell was washed twice with the wash buffer it contained. Thereafter, each cell was reacted with an anti-CD172a antibody (SouthernBiotech., Catalog Number 4525-09) and an anti-CD11R1 antibody (AbD., Catalog Number MCA1220) (4 ° C., 40 min), and then using a washing buffer. Each cell was washed twice. Thereafter, a secondary antibody (Santa Cruz Biotechnology., Catalog Number sc-45103) was added to cause an antibody reaction (4 ° C., 40 min), and then each cell was washed twice using a washing buffer. . Thereafter, 50 μL of a washing buffer containing 1% paraformaldehyde was added to each well, followed by fixation (4 ° C., 15 min). Further, each cell was washed with a washing buffer and then suspended in 150 μL of the washing buffer.

Using FACSAccuri C6 (BD), measure the bacterial fluorescence intensity per cell, count the cells in contact with the above-mentioned fluorescently labeled cells, and then the cells present on the surface and inside of each cell The quantity was quantified. These values were analyzed using FlowJo software (Tree （star). At this time, gates were set by FSC and SSC, and these values were analyzed except for CD172a-positive granulocytes.

Using a histogram, FL1 (CFDA) fluorescence values were shown for cells that were not in contact with the strain (control cells), cells that were contacted with the TL2766 strain, and cells that were contacted with the TL2937 strain. Here, the horizontal axis shows the fluorescence intensity of bacteria, the vertical axis shows the number of cells, the dotted histogram shows the results of the control cells, the solid histogram shows the cells contacted with the TL2766 strain and TL2937. The results for cells contacted with the strain are shown. The bar above the histogram indicates the gate, the numerical value below the bar indicates the mean fluorescence intensity (MFI) of the cells present in the gate, and the numerical value above the bar indicates the total number of cells in the gate. Shows the percentage. Then, two gates including a peak including the peak obtained with the control cells and a gate not including the gate were prepared, and the ratio of the cells to the cell population and the average fluorescence intensity were calculated (FIG. 2). In addition, after sterilizing TL2766 strain and TL2937 strain, those fluorescence values were measured and the fluorescence values were corrected. In addition, a TLR2 inhibition test using a blocking antibody confirms that the difference in the amount of bacteria recognized is not due to a difference in TLR2 recognition (data not shown).

In Example 1, the percentage of cells that showed high fluorescence when contacted with the TL2766 strain was 8.62%, whereas the percentage of cells that showed contact with the TL2937 strain and showed high fluorescence. Was 68.0% (FIGS. 2A and 2B). In Example 2, the proportion of cells that showed high fluorescence when contacted with TL2766 strain was 4.31%, whereas cells that showed high fluorescence when contacted with TL2937 strain. The ratio was 9.46% (FIGS. 2C and 2D). That is, in any cell, the adhesion amount of the TL2937 strain was larger than that of the TL2766 strain.

In Comparative Example 1 and Comparative Example 2, the proportion of cells that showed high fluorescence when contacted with TL2937 was higher than the proportion of cells that showed contact with TL2766 and showed high fluorescence. Results were obtained (FIGS. 2E-2H).

Further, the cells of Comparative Example 1 were contacted with the TL2766 strain and the TL2937 strain, respectively. After contact with each strain for 5 minutes, 30 minutes, 60 minutes, and 120 minutes, the average fluorescence intensity of each cell was measured. Furthermore, the relationship between the measured contact time of each strain (cell) of cells and the average fluorescence intensity at the contact time was shown (FIG. 3). As a result, the cells brought into contact with the TL2937 strain showed a high fluorescence amount 2 hours after the start of the measurement, and maintained a high level thereafter. This suggested that the amount of TL2937 strain attached was larger than the amount of TL2766 strain attached to the cells. Further, when the TL2937 strain was brought into contact with the TL2937 strain for about 5 minutes, it was shown that the TL2937 strain was rapidly phagocytosed because it was brought into contact with the TL2937 strain for 120 minutes.

From these results, it was suggested that in the cells of the examples as well as the cells of the comparative example, the bacterial cells were attached to the surface, and the bacterial cells were phagocytosed inside the cytoplasm. In other words, it is suggested that the cells of the example also exhibit the same properties as the cells of the comparative example, and in the experimental system using probiotics, the cells derived from the peripheral blood of the example are used to produce the comparative example. It was suggested that the properties of cells derived from the intestinal tract can be examined, that is, the effects on the intestinal tract immune system can be evaluated with cells derived from peripheral blood.

Test example 2
(5-2) Observation using a scanning electron microscope (SEM) Using collagen diluted with hydrochloric acid (Nitta Gelatin), the membrane surface of the Transwell insert (0.4 μm small pore polyester membrane (Corning)) After coating, the cells of Example 3 and Comparative Example 3 obtained by the above magnetic bead separation method were seeded at 5 × 10 ⁶ cells / well to allow the cells to settle (overnight). Here, after sterilizing the TL2766 strain and the TL2937 strain, these cells were contacted at 5 × 10 ⁸ cells / well (37 ° C., 2 hours). Then, after washing with 0.2 M HEPES buffer, fixation treatment (25 ° C., 2 h) with 2.5% glutaraldehyde (Nisshin EM) was performed. Further, after washing with HEPES buffer, washing was performed 5 times with PBS. Then, after fixing with 0.25% tannic acid (25 ° C., 15 min), fixing with 0.5% tannic acid (25 ° C., 15 min), and then fixing with 1% tannic acid ( 25 ° C., 1 h). Here, after dehydrating with 50%, 70%, 90% and 100% ethanol (10 min), it was immersed in t-butyl alcohol twice (30 min) and replaced with t-butyl alcohol. Furthermore, after freeze-drying (overnight), the cell surface was treated with platinum, and these cells were observed with a scanning electron microscope.

Cells observed at a magnification of 3000 using a scanning electron microscope are shown in FIGS. 4A to 4E. At this time, in Example 3 and Comparative Example 3, compared to the cells contacted with the TL2766 strain (FIGS. 4A and 4C), the cells contacted with the TL2937 strain (FIGS. 4B and 4D) moved to the cell surface. It was suggested that there was much adhesion amount of the microbial cell.

Also, it was confirmed that the amount of bacterial cells attached to the cell surface was larger in the cells contacted with the TL2937 strain compared to the non-contacted cells not contacted with any of the above strains (FIG. 4E). A portion surrounded by a dotted line shows one non-contact cell.

From these results, it was observed that the cells of the example also adhered to the surface of the cells as in the comparative example, and the cells were phagocytosed inside the cytoplasm. It was suggested. In other words, it is suggested that the cells of the example also exhibit the same properties as the cells of the comparative example, and in the experimental system using probiotics, the cells derived from the peripheral blood of the example are used to produce the comparative example. It has been confirmed that the properties of cells derived from the intestinal tract can be examined, that is, the effect on the intestinal tract immune system can be evaluated with cells derived from peripheral blood.

Test example 3
(5-3) Observation using laser microscope observation Obtained by the above-mentioned magnetic bead separation method on a microscope observation disk (cover glass, φ12 mm, pepsin-soluble collagen type I coat (IWAKI) derived from porcine tendon) The cells of Example 2 and Comparative Example 2 were seeded. Then, CellLight Early Endosomes-RFP (Life technologies) was added to the wells at a ratio of 50 μL per 1 mL of the medium, and then the endosomes of the cells were stained (37 ° C., overnight). Here, after the TL2937 strain was sterilized, these cells were contacted in wells. Thereafter, the plate was washed with PBS, and fixed with PBS containing 4% paraformaldehyde (25 ° C., 20 min). Furthermore, after washing with PBS and encapsulating with Fluoroshield with DAPI (ImmunoBioScience), the phagocytic ability of the cells (TL2937 strain) inside the cells was examined using a confocal laser scanning microscope (Carl Zeiss Microscopy, LSM-700). Observed at.

The result of observation using a laser microscope is shown in FIG. Here, blue indicates a nucleus, red indicates an endosome, green indicates a cell (bacteria), and a white bar indicates 100 μm. At this time, in Comparative Example 3 and Example 3, it was observed that the fluorescently labeled bacterial cells were taken into endosomes in the cytoplasm (FIG. 5). 5B and 5D are the results of observation by the vertical cross section of FIGS. 5A and 5C. At this time, in Example 3 and Comparative Example 3, in the cells contacted with the TL2766 strain, almost no cells were observed in the cytoplasm (data not shown).

From these results, it was observed that in the cells of the example as well as the cells of the comparative example, the cells were attached to the surface and the cells were phagocytosed inside the cytoplasm. In other words, the cells of the examples are qualitatively visually confirmed to exhibit the same properties as the cells of the comparative examples, and the peripheral blood cells of the examples are used in an experimental system using probiotics. Thus, it was confirmed that the properties of the cells derived from the intestinal tract of the sputum comparative example can be examined, that is, the effect on the intestinal tract immune system can be confirmed by cells derived from peripheral blood. Furthermore, it was confirmed that the cells can be observed quantitatively by preparing a calibration curve for each bacteria actually counted.

Test example 4
(5-4) Observation using a transmission electron microscope (TEM) Using collagen (Nitta gelatin) diluted with hydrochloric acid, the membrane surface of the Transwell insert (0.4 μm small pore polyester membrane (Corning)) After coating, the cells of Example 3 and Comparative Example 3 obtained by the above magnetic bead separation method were seeded at 5 × 10 ⁶ cells / well to allow the cells to settle (1 h). Here, after sterilizing the TL2766 strain and the TL2937 strain, these cells were contacted at 5 × 10 ⁸ cells / well (37 ° C., 2 hours). Thereafter, the cells were washed with 0.2 M HEPES buffer and fixed with 2.5% glutaraldehyde (Nisshin EM) (4 ° C., overnight). Further, after washing with 0.2 M PBS, fixation with 4% osmium tetroxide (4 ° C., 4 h) was performed. Here, it was dehydrated (10 min) with 50%, 70%, 80%, 90%, 95% and 100% ethanol, and then replaced with QY1 (n-butyl glycidyl ether). Further, the membrane was replaced with epon resin, the membrane was excised from the chamber, and the membrane was embedded in epon resin. Then, after drying with hot air (60 ° C., 3d), trimming the specimen, a glass knife was attached to the ultramicrotome (Ultracut S, Leichert), and an ultrathin slice having a thickness of 0.2 μm was produced. did. Further, these sections were placed on a grid, electron-stained with platinum blue and lead staining solution, dried, and these cells were observed with a transmission electron microscope (ZeroA H-7650).

The result of observation at a predetermined magnification using a transmission electron microscope is shown in FIG. At this time, in Comparative Example 3, more cells were observed in the cytoplasm in the cells contacted with the TL2937 strain than in the cells contacted with the TL2766 strain. However, even in non-contact cells (control cells), many bacterial cells are observed in the cytoplasm, and it is difficult to distinguish bacterial cells that have been in contact with cells from bacteria that have already existed before being taken in from the intestinal tract. It was. On the other hand, in Example 3, bacterial cells were not observed inside the cytoplasm in the control cells, and cells that appeared to be TL2937 strains were observed inside the cytoplasm in the cells contacted with the TL2937 strain.
From these results, it was confirmed that the cells (bacteria) phagocytosed inside the cytoplasm could be counted quantitatively.

(6) Measurement (counting) of bacteria (bacteria) taken up inside the cell
From the photograph of the transmission electron microscope of the cell in Example 3, the number of cells phagocytosed inside the cell was quantitatively counted according to the following criteria. That is, (1) black bodies in endosomes (voids) in cells were used as microbial cells phagocytosed inside the cells. (2) Cells taken up by one or more cells were counted. (3) Cells with disrupted cell membranes were excluded from the subject of counting. (4) The cells to be counted were randomly selected.

The number of cells phagocytosed inside each cell was quantitatively counted (evaluated) for each of the 27 cells contacted with the TL2766 strain and the TL2937 strain in Example 3. Table 1 shows the arithmetic average calculated from the number of cells.

Here, the average number of cells in cells brought into contact with the TL2766 strain is 6.3, whereas the average number of cells in cells brought into contact with the TL2937 strain is 17.4. I was able to confirm that. That is, it was shown that the phagocytic ability of the TL2766 strain and the TL2937 strain can be quantitatively counted (evaluated). The TL2766 strain did not show cells that incorporated 20 or more cells, whereas the TL2937 strain accounted for 37% of cells that incorporated 20 or more cells. It was.

Further, after contacting TL2766 strain and TL2937 strain (bacteria) with cells, the number of cells phagocytosed per cell was counted, and the counted number was graphed (FIG. 7). The method for counting (counting) bacteria is based on the methods (1) to (4) described above. As a result, it was confirmed that the cells brought into contact with the TL2937 strain tend to increase the number of phagocytic cells. Vesicles were observed in the cells, and the fungus bodies (visible in black) were counted therein (FIG. 7). In addition, the bar (**) in FIG. 7 indicates that there is a significant difference (p <0.01) between the two groups.

Test Example 5
(7) Analysis of cytokines by flow cytometry Counting the number of bacterial cells incorporated inside cells showed that the TL2937 strain had higher phagocytosis ability to cells than the TL2766 strain. Here, the cells contacted with the TL2766 strain and the cells contacted with the TL2937 strain in Example 1 and Example 2 were analyzed by flow cytometry, and the expression status of IL-1β and IL-10 was examined.

Specifically, the cells in Examples 1 and 2, the the TL2937 strain 5 × 10 ⁸ cells / well or TL2766 strain 5 × 10 ⁸ cells / well, LPS and (Sigma-Aldrich) 1000ng / mL , Pam3CSK4 (InvivoGen) was contacted and stimulated (16 h or 24 h) at 200 ng / mL. These cells are collected and stained with CD172a and CD11R1 in the same manner as in cell preparation by flow cytometry, and then IL-1β and IL11 and IL11β according to the protocol of BD Cytofix / Cytoperm Plus Fixation / Permeabilization Kit (BD). -10 was stained. Furthermore, it was analyzed by flow cytometry and Flowjo software in the same way as observation by flow cytometry.

Note that the statistically significant difference between the two groups relative to the control was analyzed by Student's t test. IL-1β acts on the hypothalamus, muscles, and fats, and is said to suppress the growth of pathogenic bacteria by fever. It acts to mobilize neutrophils to promote phagocytosis and to act on the liver to produce acute proteins. The effect of activating opsonization is also known, and IL-10 is known as an anti-inflammatory cytokine. In the TLR2 inhibition test using a blocking antibody, it was confirmed that the difference in the amount of bacteria recognized was not due to the difference in TLR2 recognition (data not shown).

The results of flow cytometry are shown in FIGS. Here, the horizontal axis represents the mean fluorescence intensity (MFI), and “*”, “**”, and “***” in the figure are significant differences compared to the respective controls, p <0.05. , P <0.01, p <0.001.

First, the cells in Example 1 and Example 2 were contacted / stimulated (16 h) with TL2937 strain and TL2766 strain. Here, in the cells of Example 2, no significant difference was observed in IL-1β and IL-10. And in the cells of Example 1, a significant increase in expression was observed in IL-1β (FIGS. 8A and 8B). Further, in the cells of Example 1, no significant increase or decrease in expression was observed in IL-10 (FIGS. 8C and 8D).

Next, the cells in Example 1 and Example 2 were contacted / stimulated (24 h) with the TL2937 strain and the TL2766 strain. Here, in the cells of Example 2, no significant difference was observed in IL-1β and IL-10. And in the cells of Example 1, a significant increase in expression was observed in IL-1β (FIGS. 9A and 9B). Further, in the cells of Example 1, no significant increase or decrease in expression was observed in IL-10 (FIGS. 9C and 9D).

From these results, a significant increase in IL-1β was observed at the protein level in cells contacted with the TL2937 strain. Evaluating the phagocytic ability of bacterial cells, it was easy to select beneficial probiotics. It was confirmed that it was possible. Furthermore, it was confirmed that the effect on the intestinal tract immune system can be evaluated with cells derived from peripheral blood. Therefore, it was confirmed that probiotics (immunobiotics) that have an effect on the immune system of the intestinal mucosa of the host as well as the current healthy bowel effect can be easily selected.

Regardless of the burdensome surgical operation or slaughter, probiotics can be evaluated for phagocytosis in a state close to the living body. By using this, probiotics useful for the living body can be screened. be able to.

Claims (11)

1. Including contacting the probiotic with a phagocytic cell that has never contacted the bacterium,
   A method for evaluating the fertility of probiotics.
2. The method according to claim 1, wherein the cell is a cell obtained by induction of differentiation from an undifferentiated cell.
3. The method according to claim 1 or 2, wherein the cell expresses at least one of the markers of BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, and CD11R1.
4. The method according to any one of claims 1 to 3, further comprising the step of observing cells and measuring the phagocytosed probiotic.
5. The method according to any one of claims 1 to 4, wherein the probiotic comprises a bacterium.
6. A kit for evaluating the phagocytic ability of probiotics, including cells with phagocytosis that have never been in contact with bacteria.
7. Contacting a control probiotic or test probiotic, respectively, with separate phagocytic cells that have not been contacted with bacteria;
   A step of observing each cell and comparing a phenomenon that occurs when the control probiotic is brought into contact with a phenomenon that occurs when the test probiotic is brought into contact to obtain a comparison result; and a comparison result Screening the test probiotic based on
   Probiotic screening method.
8. A probiotic screening kit containing cells that have never been in contact with bacteria.
9. Cells having phagocytosis that have never been in contact with bacteria are phagocytosed by cells expressing at least one of BDCA1 (CD1c), BDCA3 (CD141), CD14, MHC class II, CD172a, and CD11R1 , Probiotics.
10. Animal feed comprising the probiotic according to claim 9.
11. An IL-1β elevation regulator comprising administering the TL2937 strain, wherein the probiotic according to claim 9 is a TL2937 strain (Accession Number: FERM BP-11272).

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