Composition Containing Living Microorganisms and Preparation Method Thereof

Abstract

[Problem] The present invention provides a composition containing viable microorganisms, which can achieve a rapid and reliable colonization effect regardless of the application method and application route. [Solution] A composition containing viable microorganisms, which comprises the following (I) to (III): (I) at least one viable microorganism, (II) a solvent, and (III) bubbles with a size below the nanoscale.

Description

Technical Field

[0001] The present invention relates to a composition containing viable microorganisms and containing minute bubbles, a method for producing the same, and the like. Background Art

[0002] In recent years, the functions of microorganisms existing in living organisms have attracted much attention, and the importance of the balance of the "intestinal flora" has been pointed out.

[0003] The term "intestinal flora" refers to the state in which intestinal microorganisms of the same species or nature are somewhat aggregated and live on the intestinal wall, which looks like a garden where the same kind of flowers are clustered and blooming, and thus is the term named after this.

[0004] The "species and the ratio between species" of the microorganisms in the "intestinal flora" is called the "intestinal flora balance", which varies depending on the species of the organism or each individual. In particular, as the saying goes, "all diseases start from the intestine", it is known that in the case of suffering from certain diseases or in poor physical condition, the "intestinal flora balance" is different from that of healthy organisms of the same species (in the case of humans, humans).

[0005] As a method for improving the "intestinal flora balance", for example, ingestion of representative lactic acid bacteria beverages, etc. can be cited, but it is difficult to significantly improve only by these.

[0006] For this reason, in 2013, a research group in the Netherlands used a groundbreaking method of "directly transplanting the intestinal flora itself derived from healthy individuals into the intestine of patients" (intestinal flora transplantation) (Non-Patent Document 1), and its effect has attracted much attention. As a result, there are now many projects for treating various diseases underway both in Japan and abroad (Non-Patent Document 2).

[0007] The "intestinal flora transplantation" specifically refers to "fecal (source) microbial transplantation", which is a technique for transplanting a composition containing intestinal microorganisms obtained by dissolving feces with a solvent such as physiological saline into other individuals.

[0008] However, it can be said that there are various aspects that should be improved in the existing "intestinal flora transplantation" technology.

[0009] First, in the existing method, there is a problem that it is considered that the transplanted viable microorganisms need several days to settle in the living body in a living form (hereinafter referred to as "engraftment").

[0010] Therefore, it is considered that the transplanted "intestinal flora" will be basically excreted during these several days, which is one of the reasons why it does not remain in the patient's body as a result.

[0011] Secondly, almost all of the currently used administration methods employ a colon endoscope method, thus causing a relatively large burden on patients.

[0012] A further problem is that, as the biggest issue, it has been reported that regardless of various attempts, the colonization rate of live microorganisms after transplantation (hereinafter referred to as "engraftment rate") remains only 20% to 30%, resulting in a major problem that the expected degree of effect has not been achieved in the treatment of the disease as the ultimate goal.

[0013] On the other hand, devices for generating minute bubbles with a diameter of several tens of μm or less, so-called microbubbles, or nanobubbles with a diameter of less than 1 μm, etc. have been developed (Patent Document 1, etc.), and solutions containing these bubbles have been used in various fields such as medicine, agriculture, and aquaculture.

[0014] However, a technical concept of using a solution containing minute bubbles (so-called nanobubble water) as in the present invention to enable bacteria to engraft in a living body in a viable state has not existed until now.

[0015] Prior Art Documents

[0016] Patent Documents

[0017] Patent Document 1: Japanese Patent Application Laid-Open No. 2012-582

[0018] Non-Patent Documents

[0019] Non-Patent Document 1: Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile (Els van Nood, et al, The new England journal of medicine, January 31, 2013, vol. 368, no. 5, P.407-415)

[0020] Non-Patent Document 2: The Past, Present, and Future of Fecal Microbiota Transplantation in Inflammatory Bowel Disease (Modern Media, Vol. 62, No. 3, 2016 [Intestinal Flora], P.69-74). Summary of the Invention

[0021] Problems to be Solved by the Invention

[0022] An object of the present invention is to provide a "composition containing live microorganisms" and a "composition for adjusting the balance of live microorganisms" using the same, which have an engraftment effect on a living body, particularly an excellent engraftment effect through the intestine or other mucous membranes, by co-containing live microorganisms and minute bubbles in a solvent.

[0023] Solutions for solving problems

[0024] The above object can be achieved by the following First Invention to Sixteenth Invention.

[0025] <First Invention>

[0026] A composition containing viable microorganisms, which comprises the following (I) to (III):

[0027] (I) At least one viable microorganism

[0028] (II) A solvent

[0029] (III) Bubbles with a size below the nanoscale

[0030] <Second Invention>

[0031] The composition described in the First Invention, wherein the gas component in the bubbles is one or more of the following:

[0032] (i) Air

[0033] (ii) Hydrogen

[0034] (iii) Nitrogen

[0035] (iv) Ozone

[0036] (v) Oxygen

[0037] (vi) Carbon dioxide

[0038] (vii) Argon

[0039] <Third Invention>

[0040] The composition described in the First Invention or the Second Invention, wherein (I) is intestinal bacteria

[0041] <Fourth Invention>

[0042] The composition according to any one of the First Invention to the Third Invention, wherein (I) is viable microorganisms from one or more individuals that are the same as or different from the administration subject

[0043] <Fifth Invention>

[0044] A composition for adjusting the balance of viable microorganisms, which comprises the composition according to any one of the First Invention to the Fourth Invention

[0045] <Sixth Invention>

[0046] The composition described in the Fifth Invention, wherein the balance of viable microorganisms is the balance of intestinal flora

[0047] <Seventh Invention>

[0048] The composition according to any one of the first to sixth inventions, wherein the administration route is an administration route from the oral cavity, eyes, ears, nose, vagina, urethra, skin or anus.

[0049] <Eighth Invention>

[0050] The composition according to any one of the first to seventh inventions, which is used for transmucosal engraftment.

[0051] <Ninth Invention>

[0052] A method for preparing the composition according to any one of the first to eighth inventions, which at least includes the steps of (1) and (2) below:

[0053] (1) A step of generating bubbles of (III) in the solvent of (II),

[0054] (2) A step of dispersing and / or dissolving (I) in (II),

[0055] wherein (I) is at least one or more live microorganisms,

[0056] (II) is a solvent,

[0057] (III) is bubbles with a size below the nanoscale.

[0058] <Tenth Invention>

[0059] The preparation method described in the ninth invention, which further includes the step of (3) below:

[0060] (3) According to the attributes and / or environment of the administration object, determining the "species and ratio between species" of the live microorganisms in (I), and / or a step of collecting one or more individuals of (I) therefrom.

[0061] <Eleventh Invention>

[0062] A method for determining the composition of the composition according to any one of the first to eighth inventions, including the following steps:

[0063] (A) A step of evaluating the type of "live microorganism balance" to be administered,

[0064] (B) A step of selecting "live microorganisms" from one or more individuals that are the same as or different from the administration object and that can achieve the "live microorganism balance type" evaluated in (A).

[0065] <Twelfth Invention>

[0066] An apparatus for administering the composition according to any one of the first to eighth inventions, the apparatus being an apparatus for administering the composition from the anus and having a tubular portion.

[0067] <The thirteenth invention>

[0068] An in vivo introduction assisting solvent, comprising the following (II) and (III):

[0069] (II) A solvent,

[0070] (III) Bubbles with a size below the nanoscale.

[0071] <The fourteenth invention>

[0072] An agent for improving physique and / or physical condition, comprising the composition according to any one of the first to eighth inventions.

[0073] <The fifteenth invention>

[0074] A method for introducing the composition according to any one of the first to eighth inventions into a living body for preventing and / or treating diseases, or improving physique and / or physical condition.

[0075] <The sixteenth invention>

[0076] A method for introducing a target substance into a living body using the in vivo introduction assisting solvent described in the thirteenth invention.

[0077] Effects of the invention

[0078] Compared with conventional methods, the composition of the present invention can be administered in a method that is less burdensome and simpler for patients, and can achieve a more rapid and reliable engraftment effect regardless of the administration method and route. Description of the drawings

[0079] Figure 1 is the result of measuring the size, number, etc. of bubbles in the "in vivo introduction assisting solvent (nanobubble water A)" of Example A used in the present invention (250-fold dilution for measurement).

[0080] Figure 2 is a graph showing the change in the intestinal flora of the "recipient" (atopic dermatitis patient) in the case of administering the composition of Example 2 (intestinal flora from the "donor").

[0081] Figure 3 is a graph showing the change in the intestinal flora of the "recipient" (lung adenocarcinoma patient) in the case of administering the composition of Example 3 (intestinal flora from the "donor").

[0082] Figure 4 ​​​​Graph showing the changes in the intestinal flora of the "recipient" (a patient with chronic pancreatitis) after administration of the composition of Example 4 (intestinal flora from the "donor").

[0083] Figure 5 Graph showing the changes in the intestinal flora of the "recipient" (a patient with type II diabetes) after administration of the composition of Example 5 (intestinal flora from the "donor").

[0084] Figure 6 Graph showing the changes in the intestinal flora of the "recipient" (a patient with diabetic dyslipidemia) after administration of the composition of Example 6 (intestinal flora from the "donor").

[0085] Figure 7 Graph showing the changes in the intestinal flora of the "recipient" (a patient with irritable bowel syndrome) after administration of the composition of Example 7 (intestinal flora from the "donor").

[0086] Figure 8 Graph showing the changes in the intestinal flora of the "recipient" (a patient with ulcerative colitis) after administration of the composition of Example 8 (intestinal flora from the "donor").

[0087] Figure 9 Graph showing the changes in the intestinal flora of the "recipient" (a patient with depression) after administration of the composition of Example 9 (intestinal flora from the "donor").

[0088] Figure 10-1 Graph showing the order of administration of the composition of the present invention (intestinal flora from the "donor") to the "recipient" (a patient with Giardia infection).

[0089] Figure 10-2 Graph showing the changes in the intestinal flora of the "recipient" (a patient with Giardia infection) after administration of the composition of the present invention (intestinal flora from the "donor").

[0090] Figure 11-1 Graph showing the order of administration of the composition of the present invention (intestinal flora from the "donor") to the "recipient" (a patient with chronic fatigue).

[0091] Figure 11-2 Graph showing the changes in the intestinal flora of the "recipient" (a patient with chronic fatigue) after administration of the composition of the present invention (intestinal flora from the "donor").

[0092] Figure 12 ​​​​​​​​​​Figure showing the results of comparing the storage stability when a living organism is contained in an auxiliary solvent for introducing a living organism (nano-bubble water B) in Example B with physiological saline. Detailed Description of the Invention

[0093] The present invention will be specifically described below.

[0094] [Composition Containing Living Microorganisms of the Present Invention]

[0095] The "composition containing living microorganisms" of the present invention includes the following (I) to (III):

[0096] (I) At least one or more living microorganisms,

[0097] (II) A solvent,

[0098] (III) Bubbles with a size below the nano-scale.

[0099] [Living Microorganisms of (I) Used in the Present Invention]

[0100] (General Classification of Living Microorganisms)

[0101] The living microorganisms of (I) used in the present invention refer to all microorganisms that inhabit all living parts of humans and other organisms, including inside and on the surface of the body, and are classified into, for example, bacteria, fungi, viruses, etc.

[0102] In addition, among these living microorganisms, in addition to microorganisms in the state directly collected from living organisms, those obtained by culturing them to artificially proliferate them, those that have mutated, or microorganisms artificially modified by transformation or other means are also included.

[0103] (Medium Classification in Living Microorganisms)

[0104] Among the above general classifications of living microorganisms, the following medium classifications are included respectively.

[0105] (Medium Classification of Bacteria)

[0106] As the medium classification of bacteria, specifically, bacteria that inhabit the intestine, eyes, ears, nose, mouth, vagina, respiratory tract, skin, etc. can be cited.

[0107] (Medium Classification of Fungi)

[0108] As the medium classification of fungi, yeast-like fungi, candida, yeasts, etc. can be cited.

[0109] (Medium Classification of Viruses)

[0110] As the middle classification of viruses, examples include: DNA and RNA viruses such as herpesvirus, adenovirus, smallpox virus, Epstein-Barr virus, and mumps virus.

[0111] In addition, when the composition of the present invention is a "composition for adjusting the balance of intestinal flora", as the living microorganism of (I), intestinal microorganisms are preferably listed, and intestinal bacteria are more preferably listed.

[0112] (Minor classification of intestinal bacteria)

[0113] As the minor classification of intestinal bacteria, specifically, the following can be listed for example:

[0114] Bifidobacterium,

[0115] Lactobacillales,

[0116] Bacteroides,

[0117] Prevotella,

[0118] Clostridium cluster IV,

[0119] Clostridium subcluster XIVa,

[0120] Clostridium cluster IX,

[0121] Clostridium cluster XI,

[0122] Clostridium cluster XVIII,

[0123] Other Clostridium.

[0124] In addition, as the intestinal bacteria, intestinal bacteria preferably from the same species (for example, when the subject is a human, human intestinal bacteria) are preferred.

[0125] (Number of middle classification and minor classification used)

[0126] As the "living microorganism (I)" used in the "composition containing a living microorganism" of the present invention, usually 1 type of the above "middle classification" is preferably used, but there are also cases where 2 or more types of "middle classification" are preferably used.

[0127] In addition, as "sub-classifications" in each middle classification, one or more than two of them can be appropriately selected and used according to the "attributes and / or environment" of the administration target (hereinafter sometimes referred to as "recipient") described later, or other circumstances.

[0128] For example, when the "composition containing viable microorganisms" of the present invention is used in the "composition for adjusting the balance of viable microorganisms" described later, (I) it is preferably a mixture of two or more viable microorganisms. In particular, when the "viable microorganism (I)" is an "intestinal microorganism", it is preferable to use two or more intestinal microorganisms from healthy humans or the entire "intestinal flora".

[0129] This is because if it is known which disease is caused by the deficiency of a specific intestinal microorganism, it may be sufficient to administer only that microorganism species. However, at present, the causal relationship between diseases and specific intestinal microorganisms has not been clarified to that extent in most cases. In such cases, it is considered that transplanting the intestinal microorganisms of healthy humans in multiple species or the entire "intestinal flora" is a more convenient way of treatment than administering the specific intestinal microorganism.

[0130] (Source of viable microorganisms)

[0131] The "viable microorganism (I)" used in the "composition containing viable microorganisms" of the present invention can be those from the same or different individuals as the administration target.

[0132] The term "the same (individual) as the administration target" refers to the administration target (recipient) itself.

[0133] In addition, when collecting the "viable microorganism (I)" having the "activity and in vivo balance desired as the composition of the present invention", those from different individuals than the administration target are preferred. Among them, the individuals different from the administration target are preferably healthy animals (preferably humans) (at least related to the diseases suffered by the administration target, etc.).

[0134] (Mixing of viable microorganisms)

[0135] Regarding the "viable microorganism (I)" used in the "composition containing viable microorganisms" of the present invention, there are also cases where those from two or more individuals are preferably mixed and used.

[0136] This is because even microorganisms belonging to the same classification have differences in the growth and development activity of the microorganisms themselves, the amount of enzymes produced, and other activities due to different source individuals. By using those from two or more individuals, it may be possible to more surely implant and survive in the administration target, which is useful in its treatment and the like.

[0137] (Types and ratios between types of living microorganisms)

[0138] Regarding the "types" and the "ratios between types" of "living microorganisms" when using multiple (I), it is preferably determined individually based on the following comparison: comparing the "types" and the "ratios between types" (living microorganism balance) of the "living microorganisms" originally present in the individual of the subject (including human animals) (recipient) who is expected to receive the "composition containing living microorganisms" of the present invention with the "living microorganism balance" of a healthy individual different from the "recipient".

[0139] In addition, regarding the "types" and the "ratios between types" of "living microorganisms", in addition to the above, it is preferably determined carefully by adding insights, experiences, etc. accumulated so far in the technical field of the "attributes and / or environment" of the "recipient" and "living microorganism transplantation" as described in step (3) etc. of the "method for manufacturing the composition of the present invention" described below.

[0140] As the (I) actually used, there is also a case where it is preferably used directly all at once the living microorganism population collected from one or more healthy individuals (preferably human, hereinafter referred to as "donor" in some cases) different from the "recipient" without performing screening of bacterial cells etc.

[0141] This is because it is possible to transplant the state maintaining the living microorganism balance that has been confirmed as a healthy individual to the "recipient", and in addition, it is also possible to administer various living microorganisms other than the main living microorganisms consciously selected for the expected therapeutic effect included in the living microorganisms of the healthy individual.

[0142] Such various other living microorganisms may also well constitute one reason for maintaining the health of the "donor".

[0143] (Content ratio of living microorganism (I) in the composition)

[0144] Regarding the concentration (content ratio) of the "living microorganism (I)" in the "composition containing living microorganisms" of the present invention, it varies depending on the "attributes and / or environment" of the "recipient" described below, the administration method, the number of administrations, the administration time, etc., and thus is appropriately determined.

[0145] (Method for obtaining living microorganisms)

[0146] There is no particular limitation on the method for obtaining the "living microorganism (I)" used in the "composition containing living microorganisms" of the present invention. However, considering aspects such as "reproducibility of the balance of living microorganisms for ensuring a healthy body" and "ease of acquisition", it is preferable to use the "living microorganism (I)" obtained by treating a "biological sample" from a healthy "donor" (in the case where the living microorganism is an intestinal bacterium, it is feces, etc.) using the following method, and those from healthy humans are particularly preferred.

[0147] (Method for treating biological sample)

[0148] For obtaining the "living microorganism (I)" used in the "composition containing living microorganisms" of the present invention from a biological sample, methods such as filtering and purifying the biological sample using the methods described below can be enumerated.

[0149] Immerse the biological sample in a biological sample treatment solution, and make it semi-naturally dissolve by gentle stirring. Then, filter out impurities such as food residues multiple times using a sterilized gauze, etc.

[0150] In addition, as the biological sample treatment solution, as long as it does not inactivate the "living microorganism (I)" used in the "composition containing living microorganisms" of the present invention, there is no particular limitation, and pure water (purified water), physiological saline, etc. can be used.

[0151] In addition, although it is also okay to replace it with the later-mentioned "(solvent (II))" after treating the biological sample with this biological sample treatment solution, in order to avoid risks such as inactivation of living microorganisms caused by performing an additional step, or in order to more quickly and surely wrap the "living microorganism (I)" with air bubbles, therefore, at this stage, it is preferable to use the "(solvent (II))" used in the "composition containing living microorganisms" of the present invention.

[0152] Furthermore, it is more preferable to generate "bubbles (III) with a size below the nanoscale" in this "(solvent (II))" in advance.

[0153] This is because although the method of generating "nanoscale bubbles (III)" from obtaining the "(solvent (II))" containing the living microorganism (I) can be used, it is considered that the inactivation of the "living microorganism (I)" can be minimized when preparing a solution containing (I) using the (II) in which (III) is generated in advance.

[0154] In addition, the amount of the solvent (II) used is not particularly limited, but methods such as using 1.5 - 4A ml (25 - 67 w / v%), for example, 2A ml (50 w / v%) of the solvent for about A g of the "biological sample" can be enumerated.

[0155] In addition, the amount of (II) can be appropriately determined according to the state of the "biological sample" (including the moisture content) and the like.

[0156] In addition, filtration is preferably repeated 1 to multiple times, preferably 3 times or more.

[0157] (Type of donor)

[0158] In addition, as the "donor" who is the provider of the "biological sample" for (obtaining I), especially when "feces" is used as the "biological sample", from a psychological aspect and the like, there is a tendency to preferably select a close relative within the second degree of kinship or closer of the "recipient".

[0159] However, it is considered that if the therapeutic effect is given top priority, it is also worth considering a method of deliberately selecting a third-party "donor" other than a close relative with a completely different "living microbial balance" instead of a close relative whose "living microbial balance" is considered to be close in terms of genetics or living environment.

[0160] (Number of donors)

[0161] In addition, the third-party "donor" is not limited to one person, and there are also cases where multiple donors are preferably used for long-term improvement of the "living microbial balance".

[0162] 《Solvent of (II) used in the present invention》

[0163] As the solvent of (II) used in the present invention, there is no particular limitation, and it can be: purified water, physiological saline, mineral water, soft drinks, etc.

[0164] Considering the viewpoints of improving the fusion with the living body and the colonization activity of the "composition containing living microorganisms" of the present invention and the antiseptic effect, etc., physiological saline or the like can be used as (II).

[0165] On the other hand, as the device for generating bubbles of (III) in the solvent of (II), in the case of using an instrument made of stainless steel or the like, from the viewpoint of preventing the instrument from rusting, there are cases where purified water and mineral water are preferably used.

[0166] Therefore, in the case of manufacturing the "composition containing living microorganisms" of the present invention, the same solvent (II) can be used from start to finish, or the types of the solvent of (II) can be separately used according to different manufacturing stages.

[0167] In addition, in the "composition containing living microorganisms" of the present invention, it includes compositions at various stages such as the freshly manufactured "stock solution" and the "diluent" to be immediately administered to the "recipient".

[0168] In the "composition containing live microorganisms" of the present invention, various additives can also be contained within the scope of the effects not hindered by the present invention, for example, within the range where the live microorganisms of (I) are not killed.

[0169] 《Bubbles below the nanoscale of (III) used in the present invention》

[0170] As the "gas component" in the bubbles below the nanoscale of (III) used in the present invention, for example, the following are preferred, but not necessarily limited to these.

[0171] (Type of gas in the bubble)

[0172] The "gas component" in the bubble is preferably one or more of the following:

[0173] (i) Air,

[0174] (ii) Hydrogen,

[0175] (iii) Nitrogen,

[0176] (iv) Ozone,

[0177] (v) Oxygen,

[0178] (vi) Carbon dioxide,

[0179] (vii) Argon.

[0180] From the aspect that special "gas components" such as (ii) to (vii) do not need to be prepared, the case of using only (i) air is more realistic and preferred.

[0181] In addition, it is not easy to accurately measure the "enclosure ratio of each gas component" in the "bubbles (III) generated in the solvent (II)" when "air" or "mixed gas of two or more kinds" is introduced into the solvent (II).

[0182] This is because during the process of generating bubbles (III) in the solvent (II), the "amount" and "speed" that can be dissolved in the solvent (II) vary depending on the "gas component", and moreover, it is not easy to accurately determine the "type" and "ratio" of the "gas component" enclosed in the generated bubbles (III) itself.

[0183] However, in the case of using, for example, a "gas component (assumed to be X)" composed of a single type, it is considered that the "enclosure ratio" of the "gas component (X)" in the "bubbles (III) generated in the solvent (II)" should be higher compared to the case of using a mixed gas such as air, and thus, the characteristics of the "gas component (X)" as described above can be utilized more effectively.

[0184] In addition, in the case of manufacturing a "composition containing viable microorganisms" using intestinal bacteria, if the ratio of hydrogen in the bubbles is higher than that in the atmosphere, the following advantages can be expected, and thus it is preferred:

[0185] 1. It is considered that the redox potential of the nano-bubble water of the present invention using hydrogen (target: -150 mV ± 15 mV) is as low as that of the intestinal tract (-50 mV to -250 mV). Therefore, the bacteria encapsulated by it are likely to colonize in the intestine.

[0186] 2. It is considered that in the case of inflammation in the intestine, there is a tendency for the redox potential to increase, and the immune response becomes hyperactive, so the transplanted bacteria are often repelled by the recipient's immunity. However, the anti-inflammatory effect brought about by the low redox potential of hydrogen can alleviate the said reaction.

[0187] 3. It is considered that by using the "nano-bubble water" of the present invention containing hydrogen molecules, either facultative anaerobes or obligate anaerobes, which are the main groups of intestinal bacteria, can be kept in an inactivated and non-dead state. During the period until the bacteria start to become active again in a suitable environment in the intestine after transplantation, the balance between the bacteria in the bacterial solution can be maintained without being disrupted, and the bacterial solution can be stored in a state of keeping the bacteria dormant for a long period.

[0188] For the ratio of hydrogen in the bubbles to be higher than that in the atmosphere, in addition to using hydrogen alone, methods such as combining air and hydrogen can be cited.

[0189] In this case, air and hydrogen alone can be sealed in simultaneously. Methods such as the concentration of hydrogen sealed in together with the gradually increasing air, or starting with using air alone and then sealing in hydrogen in the latter half stage can be cited. It is considered that by using these methods, the loss of hydrogen due to dissolution in the solvent or the like can be suppressed to the minimum, and thus more hydrogen can be sealed in the bubbles.

[0190] There is no particular limitation on the ratio in the case of using air and hydrogen simultaneously. However, it is considered that, for example, the amount of "(ii) hydrogen" used in the sealing operation is preferably 10 times or more, more preferably 100 times or more, the amount of "(i) air" used.

[0191] (Size of the bubbles)

[0192] The size of the bubbles used in the "composition containing viable microorganisms" of the present invention primarily needs to be below the nano size. For the specific size, it can be changed as appropriate according to the different "viable microorganisms (I)" used.

[0193] When the "living microorganism (I)" is a virus, in order to encapsulate the virus, especially to improve the engraftment rate through mucous membranes, etc., the size of the bubbles is preferably smaller than the size of the virus itself (generally 10 - 100 nm).

[0194] When the "living microorganism (I)" is a bacterium, in order to encapsulate the bacterium, especially to improve the engraftment rate through mucous membranes, etc., the size of the bubbles is preferably smaller than the size of the bacterium itself (generally about 1 μm (1000 nm)).

[0195] When the "living microorganism (I)" is a fungus, in order to encapsulate the fungus, especially to improve the engraftment rate through mucous membranes, etc., the size of the bubbles is preferably smaller than the size of the fungus itself (generally several μm).

[0196] (Ratio of bubbles with a size below the nanoscale in all bubbles)

[0197] In all the bubbles in the "composition containing living microorganisms", the "bubbles below the nanoscale (III)" do not have to be 100%.

[0198] However, compared with the conventional "composition for transplantation of living microorganisms using a solvent without bubbles", since it is considered that the reason why the "living microorganism (I)" contained in the "composition of the present invention" is more surely engrafted in the living body is that the "bubbles below the nanoscale (III)" smaller than the "living microorganism (I)" encapsulate the "living microorganism (I)" and thus contribute to the engraftment in the living body, it is preferably contained in a large amount so as to be able to encapsulate each "living microorganism (I)".

[0199] Furthermore, from the aspect that the denaturation of the composition caused by the collapse of bubbles larger than the nanoscale is less, and from the aspect that the engraftment rate of the "living microorganism (I)" is further improved, it is desirable that the ratio of the "bubbles below the nanoscale (III)" is as high as possible.

[0200] In addition, the size of the bubbles does not have to be homogeneous, and usually has a certain degree of bubble diameter distribution.

[0201] Therefore, as a specific index, it is considered that if the "average diameter" of the bubbles is smaller than the "living microorganism (I)" used, it is at a level capable of encapsulating the "living microorganism (I)" and also contains many small bubbles. For example, when the "living microorganism (I)" is a bacterium, it is preferable to use a solution with an average bubble diameter below 1000 nm, and more preferably an average bubble diameter of 900 nm or less.

[0202] (Number of bubbles in the composition)

[0203] The number of bubbles in the "composition containing living microorganisms" of the present invention is preferably in a larger case.

[0204] The specific quantity also varies depending on the concentration of (I) (the quantity of viable microorganism (I)), and it cannot be generally stated. However, it is confirmed that in the case of using a known manufacturing apparatus capable of producing "bubbles (III) with a size below the nanoscale", several thousand to several hundred million per ml, which can be normally produced, is sufficient.

[0205] (Coating of viable microorganism (I) with bubbles)

[0206] In addition, it is considered that "bubbles (III) with a size below the nanoscale" are combined by gently stirring or mixing a solvent (II) containing bubbles and "viable microorganism (I)", thereby coating the periphery of "viable microorganism (I)" naturally. It is considered that this coating is sufficient to prevent being blocked by mucous membranes such as the intestinal wall mucopolysaccharides, and can be more quickly and surely implanted in vivo.

[0207] (Method for manufacturing bubbles)

[0208] The "bubbles with a size below the nanoscale of (III)" used in the "composition containing viable microorganism" of the present invention can be generated by step (1) of the manufacturing method of the present invention described below.

[0209] (Method for manufacturing the composition)

[0210] The "composition containing viable microorganism" of the present invention can be manufactured by the manufacturing method of the present invention described below and the like.

[0211] (Dosage of the "composition containing viable microorganism" of the present invention)

[0212] The dosage of the "composition containing viable microorganism" of the present invention is also appropriately determined according to the concentration (content ratio) and quantity of "viable microorganism (I)" in the composition of the present invention, the "attributes and / or environment" of the "recipient" described below, etc. It is preferably about 50 ml to 300 ml per administration.

[0213] (Number of administrations of the "composition containing viable microorganism" of the present invention)

[0214] The number of administrations of the "composition containing viable microorganism" of the present invention is also appropriately determined according to the concentration (content ratio) and quantity of "viable microorganism (I)" in the composition of the present invention, the "attributes and / or environment" of the "recipient" described below, etc. There are cases where the effect can be observed after one administration, but usually there are also cases where, in order to ensure the implantation of microorganisms in the recipient's body, it is preferably administered in at least two or more divided doses. There is no particular limitation on the number of multiple administrations. Considering the burden on the patient, etc., there are cases where it is preferably set to within about 10 times.

[0215] In addition, when administering in multiple doses, it can be appropriately determined in consideration of the "attributes and / or environment" of the "recipient" described below, the length of the administration time, the number of administrations, the amount and concentration (content ratio) of the "living microorganism (I)" in the composition, etc. However, it is also possible to observe the process after each administration and appropriately and flexibly change the initial plan.

[0216] In addition, in the case of multiple administrations, since it is usually easy to reconcile with the living body and more surely implant, it is preferably the case where the concentration of the "living microorganism (I)" is changed and a concentration gradient is imparted for administration.

[0217] (Administration time of the "composition containing a living microorganism" of the present invention)

[0218] The administration time of the "composition containing a living microorganism" of the present invention is also appropriately determined according to the concentration (content ratio) and amount of the "living microorganism (I)" in the composition of the present invention, the "attributes and / or environment" of the "recipient" described below, etc. In order to improve the implantation effect, it is preferably separated by several hours to several days between one administration and the next administration. In addition, as the overall administration time, for example, in the case of administering in 10 doses, it is preferably within 1 to several months.

[0219] The case where the interval between administrations is not made too long is to easily exhibit the effect of continuous administration.

[0220] In addition, the administration interval does not need to be a constant interval. In addition to the case where the interval is gradually extended or gradually shortened for each case, various methods such as appropriately setting a random interval can be considered while observing the physical constitution, physical condition, degree of recovery of the disease, etc. of the "recipient".

[0221] (Type of the "composition containing a living microorganism" of the present invention for administration)

[0222] For the "composition containing a living microorganism", it can be manufactured in the form of a mixture from multiple "donors" from the beginning of manufacturing. There is also a case where it is preferable to pre-manufacture multiple compositions from a single "donor", and while observing the physical constitution, physical condition, degree of recovery of the disease, etc. of the "recipient", appropriately use the appropriate composition from the "donor" for each transplantation.

[0223] This is because the type of living microorganism to be introduced will naturally change depending on the physical constitution, physical condition, degree of recovery of the disease, or unexpected situations such as the presence or absence of poor physical condition of the "recipient".

[0224] (Administration route of the "composition containing a living microorganism" of the present invention)

[0225] The "composition containing live microorganisms" of the present invention can reach the administration target site by various administration methods in the form of compositions for administration to the oral cavity, eyes, ears, nose, vagina, urethra, skin, anus, etc.

[0226] In addition, among the "compositions containing live microorganisms" of the present invention, the compositions for engraftment via the "mucosa" are preferred because they make better use of the advantages of the present invention that can improve the engraftment effect of live microorganisms.

[0227] The "mucosa" refers to the soft tissue on the inner surface of the main hollow internal organs such as the digestive organs, respiratory organs, urinary organs, and genital organs. Specifically, for example, the intestinal mucosa containing mucopolysaccharides and the like on the inner side of the intestine can be cited.

[0228] (Administration device for the "composition containing live microorganisms" of the present invention)

[0229] As the device for administering the "composition containing live microorganisms" of the present invention, in addition to the "device for administering the composition" of the present invention described later, as well-known devices for administering the composition in vivo, for example, endoscopy devices such as colonoscopes and duodenoscopes, commercially available intestinal catheters, etc. can be cited.

[0230] From the aspect of being able to select the administration position using precise positioning, endoscopy devices etc. are preferred, but from the aspects of not requiring prior dietary restrictions, intestinal lavage, taking laxatives after administration, etc., and being superior in that the burden on the administration object is less when introducing a long tube into the body, a scheme of using the "device for administering the composition" of the present invention and commercially available intestinal catheters for administration is preferred.

[0231] In addition, when using an intestinal catheter for administration, it is not necessarily necessary to use a tube that encompasses the entire intestine. Using a tube of "ten-odd centimeters (for example, a commercially available rubber intestinal catheter)" can also sufficiently enable the "live microorganism (I)" to engraft in the living body.

[0232] This is because live microorganisms all carry their own inherent bio-action potential and have the property that the same kind of live microorganisms in the same charged state attract each other. Therefore, the live microorganisms administered from the anus will move towards the same kind of live microorganisms in the distant living body as the target.

[0233] In addition, since in the "composition containing live microorganisms" of the present invention, the "bubbles (III) below the nano size" coat the "live microorganism (I)", it is considered that there is a possibility that the bio-action potential is amplified by using these bubbles, making it easier for live microorganisms to approach each other.

[0234] (Form, etc. of the "composition containing viable microorganisms" of the present invention)

[0235] The "composition containing viable microorganisms" of the present invention can take the form of a known composition, such as preferably a liquid, a gel-like substance, etc., within the range that does not cause the collapse of "bubbles (III) below the nano size" in the composition, the inactivation of "viable microorganisms (I)", etc.

[0236] As representative products of the "oral composition" when administered orally, therapeutic compositions such as "drugs", "quasi-drugs", etc. for humans or non-human animals, "food and beverages", "nutritional supplements", etc. can be cited.

[0237] As the "ophthalmic composition", "eye drops", etc. can be cited.

[0238] As the "otolaryngological composition", "nasal drops", etc. can be cited.

[0239] As the "composition for transdermal administration", "patches" such as "pastes", "ointments", "liniments" such as "sprays" can be cited.

[0240] In addition, as a composition for anal administration, therapeutic compositions such as "drugs", "quasi-drugs", etc. can be cited.

[0241] [Composition of the present invention for adjusting the balance of viable microorganisms]

[0242] The "composition for adjusting the balance of viable microorganisms" of the present invention is characterized by containing the above-mentioned "composition containing viable microorganisms" of the present invention.

[0243] The "composition containing viable microorganisms" of the present invention is useful for adjusting the balance of microorganisms in the living body (the types of multiple microorganisms and the ratio between types, etc.) that has been disrupted due to diseases, etc.

[0244] Among them, it is particularly preferably used as the main component of the "composition for adjusting the balance of intestinal flora".

[0245] In addition, for the "balance of viable microorganisms", there are cases where it refers to the "overall balance of viable microorganisms" in the living body of an individual, and cases where it refers to the "balance of viable microorganisms in a specific living organ", but the "composition for adjusting the balance of viable microorganisms" of the present invention is particularly useful for adjusting the "balance of viable microorganisms" in a specific organ, among which for the "balance of viable microorganisms inhabiting the intestine", that is, the so-called "intestinal flora balance".

[0246] Its specific composition can be the above-mentioned "composition containing viable microorganisms" itself, or it can further contain other components useful for adjusting the balance of viable microorganisms.

[0247] The "application amount", "number of applications", "application time", "application route", "apparatus for application", "form", etc. of the "composition for adjusting the balance of living microorganisms" of the present invention are as described in the text of the "composition containing living microorganisms" of the present invention.

[0248] [Method for manufacturing the composition of the present invention]

[0249] The method for manufacturing the "composition containing living microorganisms" and the "composition for adjusting the balance of living microorganisms" (hereinafter, sometimes collectively referred to as the "composition of the present invention") of the present invention includes at least the following steps (1) and (2):

[0250] (1) A step of generating bubbles of (III) in the solvent of (II),

[0251] (2) A step of dispersing and / or dissolving (I) in (II),

[0252] where (I) is at least one or more living microorganisms,

[0253] (II) is a solvent,

[0254] (III) is bubbles having a size below the nanoscale (hereinafter, sometimes referred to as "nanobubbles").

[0255] 《Order of steps》

[0256] The above steps can be carried out in the order of (2), (1), but from the viewpoint of minimizing the risk of inactivation of living microorganisms in the manufacturing process, it is preferably carried out in the order of (1), (2).

[0257] In addition, other steps can be added before and after these respective steps.

[0258] 《Method of step (1)》

[0259] As the method of "generating bubbles of (III) in the solvent of (II)" in step (1), for example, the following methods can be cited, or methods in which they are used in combination, etc., but are not limited to these:

[0260] Gas-liquid mixing and shearing method:

[0261] A method of swirling a gas and a liquid at high speed together.

[0262] Ultrasonic method:

[0263] A method of applying a shock wave and cavitation to a liquid to further break the temporarily generated bubbles.

[0264] Pressurized dissolution method:

[0265] A method of applying pressure to a gas and a liquid and discharging them at once to generate bubbles.

[0266] Micro-porous method:

[0267] A method of supplying a gas while applying pressure using a throttle orifice or the like.

[0268] Electrolysis method:

[0269] A method of generating a gas from a thin wire immersed in an aqueous solution.

[0270] Among the above, the "gas-liquid mixing and shearing method" is preferred because it can generate stable bubbles with a size below the nano-scale by further shearing treatment of micro-bubbles.

[0271] In addition, specifically, for example, commercially available devices such as the following 1) and 2) can be used in combination to generate nano-bubbles:

[0272] 1) Generation of bubbles with a diameter of less than 1 micron using the micro-nano bubble generator "BUVITAS (registered trademark) HYK-25" manufactured by Kyowa Kikai Co., Ltd. (rotary shearing method),

[0273] 2) Nano-bubbling of micro-nano bubbles from "νG7 (registered trademark)" manufactured by Yaba Co., Ltd. (stainless steel filter).

[0274] 《Method of Step (2)》

[0275] As a method of "dispersing and / or dissolving (I) in (II)" in step (2), a method of treating "living microorganisms (I)" with the "solvent (II) containing bubbles (III)" prepared in the above step (1) can be cited.

[0276] "Treatment" means, as also described in the above (method for treating biological samples), for example, after gently stirring a "biological sample" containing the target "living microorganisms (I)" taken from the living body of a "donor" in the "solvent (II) containing bubbles (III)", using methods such as filtration to remove unwanted substances other than (I), and diluting the pre-purified "living microorganisms (I)" with a solvent such as the "solvent (II) containing bubbles (III)".

[0277] In addition, regarding the amount of the solvent (II) used, the number of filtration times, etc., it is also as described in the above (method for treating biological samples).

[0278] Alternatively, after changing "solvent (II) containing bubbles (III)" to using "(solvent (II))" for the above treatment, bubbles (III) can be generated in this solvent (II). However, in order to minimize the inactivation of "living microorganisms (I)", it is preferred to perform the treatment using "solvent (II) containing bubbles (III)".

[0279] 《Addition of Step (3)》

[0280] In the manufacturing method of "the composition of the present invention", the following step (3) can be further included together with (1) and (2):

[0281] (3) Determining the types and ratios between types of living microorganisms in (I), and / or collecting one or more individuals from (I) according to the "attributes and / or environment" of the "recipient".

[0282] The term "attributes" includes properties, characteristics, etc. possessed by the "recipient", such as: the physical condition of the "recipient" itself, the types and severity of diseases carried, age, gender, height, weight, body type, blood pressure, previous medical conditions, lifestyle habits including eating habits, smoking habits, etc.

[0283] In addition, the term "environment" includes living environments, etc., and the living environment includes the climate zone of the living area where the "recipient" lives or works, temperature, humidity, presence or absence of tobacco smoke, exhaust gas, etc., family composition, occupation, presence or absence of pets, season, climate, etc. at the time of administration.

[0284] When collecting "living microorganisms (I)" from a healthy "donor" in the form of a certain degree of "mass" and using it directly (for example, using the whole aggregate of multiple living microorganisms derived from a biological sample such as the entire "intestinal flora"), it is not necessarily required to have a step like the above (3), but there are also cases where: when mixing and using "masses" from multiple "donors", when it is necessary to deliberately change the ratio of specific living microorganisms significantly according to the type of disease and other conditions, etc., it is preferred to set a step like (3).

[0285] In addition, when implementing the step of (3), it can be carried out either before or after the step of (2).

[0286] [Method for Determining the Composition of the Composition of the Present Invention]

[0287] The method for determining the composition of "the composition of the present invention" includes the following steps:

[0288] (A) A step of evaluating the type of "living microorganism balance" to be administered,

[0289] (B) Step of selecting "living microorganisms" from one or more individuals, the same as or different from the administration subject, that can achieve the "type of living microorganism balance" evaluated in (A).

[0290] In addition, in the present invention, "composition" means that in the composition of the present invention,

[0291] except for the "types of living microorganisms and the ratio between types in living microorganism (I)" and the "concentration (content ratio) of living microorganism (I) itself",

[0292] it includes the "type and content ratio of (solvent (II))",

[0293] the "bubble diameter", "type of gas component", "number of bubbles" of "nano-sized bubbles (III)",

[0294] and other comprehensive compositions,

[0295] For the composition of the present invention, the "types of living microorganisms and the ratio between types in living microorganism (I)" and the "bubble diameter" and "number of bubbles" of "nano-sized bubbles (III)" are particularly important.

[0296] The "evaluation of the type of living microorganism balance" in the step of (A) means that in the case where "living microorganism (I)" is an aggregate of multiple types of living microorganisms, taking the ratio of specific types of microorganisms as an index, an ideal "living microorganism balance" considered effective for the treatment of the disease suffered is designed for each administration subject (patient).

[0297] As specific exploration items for design, for example, the following can be cited as examples:

[0298] (A)-i:

[0299] The "living microorganism balance" of the administration subject.

[0300] The "living microorganism balance" refers to the types and ratio (quantity ratio) of living microorganisms in "the whole individual" or "within a specific living organ", etc.

[0301] As a method for measuring the "living microorganism balance", various known methods are known, but there is no particular limitation. For example, methods such as using the "gene characteristic of the living microorganism" and DNA sequence possessed by each living microorganism for measurement can be cited.

[0302] Among them, since there is a certain correlation between "physical condition, disease", etc. and the characteristics of "intestinal flora balance", etc., as the attributes of the "recipient" in (A)-iii described later, for example, if the type and degree of physical condition and disease are explored, it is not necessary to have the exploration in (A)-i.

[0303] (A)-ii:

[0304] The tendency of "living microbial balance" of different individuals who are considered healthy at least with respect to the disease.

[0305] For example, in the result of the analysis in (A)-i, etc., when it is judged that compared with the tendency of the ratio of A in the living microbial balance of healthy individuals, a specific "intestinal bacterium A" in the "living microbial balance" of the administration subject is extremely insufficient, the "living microbial balance" that sufficiently contains "intestinal bacterium A" is designed as the ideal "living microbial balance" to be administered.

[0306] For the type of "living microbial balance" to be administered evaluated in the process of (A), in addition, it can be corrected by further confirming or exploring the following items, etc.

[0307] (A)-iii:

[0308] The "attributes and / or environment" of the administration subject ("recipient").

[0309] In addition, the "attributes and / or environment" described here refers to those described in the manufacturing method of the composition of the present invention above.

[0310] (A)-iv:

[0311] Insights based on the treatment results of "living microbial transplantation" accumulated so far.

[0312] In the process of (B), in a manner that can achieve the type evaluated in (A), select "living microorganisms" derived from one or more individuals who are the same as or different from the "recipient".

[0313] Specifically, for example, it can be implemented by identifying more (or fewer) living microorganisms compared with the "living microbial balance" of one or more healthy individuals, and selecting "living microorganisms" derived from one or more individuals with good balance among them.

[0314] [Apparatus for administering the composition of the present invention]

[0315] The "apparatus for administering the composition" of the present invention is an example of an apparatus for administering the above-mentioned "composition containing viable microorganisms", "composition for adjusting the balance of viable microorganisms", etc. (the "composition of the present invention") from the anus or the like, and it has a "tubular portion".

[0316] As the "tubular portion", a tubular object such as a commercially available intestinal catheter or the like can be used.

[0317] In addition, the length of the "tubular portion" also varies depending on the age, body type, etc. of the "recipient". For example, it is preferably 5 cm or more, more preferably 10 cm or more, further preferably 15 cm or more, and particularly preferably 18 cm or more.

[0318] Among them, it does not necessarily have to be so long as to cover the entire inside of the intestine. On the contrary, in order to avoid risks such as physical property changes of the "composition" caused by excessive length, there are also cases where it is preferably within 50 cm, for example.

[0319] The above-mentioned apparatus may also be provided with the following parts:

[0320] "Storage portion": A portion that can store the "composition" of the present invention, such as the "main body" of a syringe, the "container (Tank)" attached to a large intestine endoscope, the "liquid medicine container" in an irrigator, a drip infusion bag, etc.

[0321] "Extrusion mechanism": A mechanism such as a pump that can extrude the "composition" in the "storage portion".

[0322] In addition, it can also be formed into a mechanism that extrudes the stored material inside the storage portion with the "storage portion" itself, such as a disposable enema container like Ichijiku enema.

[0323] [Viable introduction auxiliary solvent of the present invention]

[0324] The "viable introduction auxiliary solvent" of the present invention contains the following (II) and (III), and is also referred to as "nano-bubble water" in the present invention:

[0325] (II) Solvent,

[0326] (III) Bubbles smaller than nano size.

[0327] In addition, "viable introduction" means, in addition to implantation and attachment, making it exist inside and outside blood vessels, in cavities inside the living body (inside the intestine), etc., and placing it in a state where the introduced substance can function inside the living body.

[0328] Specifically, as the administration route, examples include: oral, eye, ear, nose, vagina, urethra, skin or anus, among which administration via the mucosa is preferred, and administration via the intestinal wall is particularly preferred.

[0329] As the object for promoting in vivo introduction using this "in vivo introduction assisting solvent", mainly organic substances can be listed. Among them, living microorganisms are preferred, and intestinal bacteria are particularly preferred. However, inorganic substances such as minerals can also be used.

[0330] In addition, when the object for promoting in vivo introduction is a "living microorganism", the "living microorganism" can be introduced into the "mucus" on the mucosa using the "in vivo introduction assisting solvent" of the present invention, and thus organic acids and the like produced by the living microorganism can deeply penetrate into the living body "via the mucosa".

[0331] By using the "in vivo introduction assisting solvent" of the present invention, as can be known from the test examples described later, etc., the substance to be introduced as the target can be more effectively introduced into the living body.

[0332] [Agent for improving constitution and / or physical condition of the present invention]

[0333] The "agent for improving constitution and / or physical condition" of the present invention contains the composition described in any one of claims 1 to 8, and specifically contains the following:

[0334] (I) At least one or more living microorganisms,

[0335] (II) A solvent,

[0336] (III) Bubbles with a size below the nanoscale.

[0337] By introducing the "agent for improving constitution and / or physical condition" of the present invention, as can be known from the test examples described later, etc., it is possible to improve the constitution and / or physical condition of patients suffering from a large number of diseases and poor physical conditions, and further help in the prevention and / or treatment of diseases.

[0338] [Method for improving constitution and / or physical condition of the present invention]

[0339] The "method for improving constitution and / or physical condition" of the present invention is a method for introducing the composition described in any one of claims 1 to 8 into a living body for the prevention and / or treatment of diseases or the improvement of constitution and / or physical condition.

[0340] According to the "method for improving constitution and / or physical condition" of the present invention, as can be known from the test examples described later, etc., it is possible to improve the constitution and / or physical condition of patients suffering from a large number of diseases and poor physical conditions, and further help in the prevention and / or treatment of diseases.

[0341] [Method for introducing the object of the present invention into a living body]

[0342] The "method for introducing an object into a living body" of the present invention is characterized by using the "auxiliary solvent for in vivo introduction" of the present invention described above.

[0343] Through this "method for introducing an object into a living body", as can be seen from the test examples described later, etc., the object can be introduced into the living body of patients suffering from a large number of diseases and poor physical conditions with an efficiency superior to that of conventional methods.

[0344] Examples

[0345] Before the description of the examples of the "composition containing living microorganisms" of the present invention, the details of the "auxiliary solvent for in vivo introduction" of the present invention used in these examples, that is, the "(II) solvent" containing "(III) bubbles with a size below the nanoscale" (hereinafter referred to as "nano-bubble water") and the "intestinal flora balance" will be described.

[0346] [Example A: Auxiliary solvent for in vivo introduction]

[0347] 《Materials and Methods》

[0348] (Manufacture of "nano-bubble water A")

[0349] Using the above-mentioned device, etc., the "auxiliary solvent for in vivo introduction A (nano-bubble water A)" of the present invention was produced using mineral water and air.

[0350] Using "Multisizer 3" manufactured by Beckman Coulter, the "nano-bubble water A" produced above was diluted 250 times for easy measurement of the bubble diameter, and the size, quantity, etc. of the bubbles in the diluted solution were measured.

[0351] The results are shown in Figure 1 .

[0352] The analysis result of the "nano-bubble water A" used in the present invention is the broken line graph at the uppermost part in the figure indicated by "*".

[0353] Figure 1 Analysis result of:

[0354] Average bubble diameter: 830 nm

[0355] Number of bubbles: Approximately 435,000 bubbles / ml

[0356] Therefore, it is considered that the number of bubbles in the "nano-bubble water A" (before 250-fold dilution) used in the present invention is about 100 million bubbles / ml.

[0357] [Example B: In vivo introduction assisted solvent]

[0358] As the gas enclosed when generating bubbles, hydrogen gas was used together with the atmosphere, and the hydrogen gas enclosure time was set in the latter half of the enclosure. In addition to increasing the hydrogen concentration in the bubbles, the same procedure as in Example A was carried out to produce the "in vivo introduction assisted solvent B (nano bubble water B)" of the present invention.

[0359] In addition, about 250 times the amount of hydrogen gas in the atmosphere was used for the enclosure.

[0360] It is considered that the number of bubbles in the "nano bubble water B" (before 250-fold dilution) used in the present invention is also about 100 million / ml.

[0361] [Measurement of "intestinal flora balance"]

[0362] The "types and ratios (quantity ratios) between types of intestinal bacteria" in the solution obtained by filtering and purifying the feces collected from each "donor" or "recipient" were measured using known genes specific to each intestinal bacterium as an index, as the "intestinal flora balance" of each individual.

[0363] The genes specific to each intestinal bacterium were measured using the following device.

[0364] DNA sequencer: manufactured by Illumina, Inc., "MiSeq"

[0365] [Example 1: Composition (stock solution) containing live microorganisms (intestinal bacteria)]

[0366] "Establishment of intestinal flora library"

[0367] The "intestinal flora balance" in the feces from about 80 "donors" (healthy people) was measured, and the "composition containing live microorganisms (stock solution)" of the present invention was produced from this feces using the above "nano bubble water A" (before 250-fold dilution), and an "intestinal flora library" was established.

[0368] Specifically, the feces "50 - 150 g" collected from each "donor" were respectively immersed in 1.5 - 4 times, that is, "75 - 600 ml" of "nano bubble water A" (before 250-fold dilution), and gently stirred to make the feces semi-naturally dissolved.

[0369] In addition, the amount of "nano bubble water A" was appropriately determined according to the state of the feces of the "donor" (including the water content).

[0370] Next, the lysate was filtered multiple times using sterilized gauze until no unwanted substances such as food residues could be confirmed under an optical microscope (low magnification), and a "composition containing live microorganisms (stock solution)" was produced separately for each "donor".

[0371] In addition, sterilized gauze with voids of a size that allows "live microorganisms (I)" and "nanobubbles (III)" to pass through was used.

[0372] [Examples 2 to 9: Composition (dilute solution) containing live microorganisms (intestinal bacteria)]

[0373] (A) Evaluation of the type of "intestinal flora balance" to be administered:

[0374] (A)-i: Determination of the "balance of live microorganisms (intestinal flora)" of the administration subject

[0375] As the administration subjects, as Figures 2 to 9 shown, patients with various diseases were selected.

[0376] Feces of these patients (hereinafter referred to as "recipients") were collected, and their "intestinal flora balance" was determined by gene analysis of intestinal bacteria.

[0377] The results are shown in Figures 2 to 9 (the left bar graph).

[0378] (A)-ii: Confirmation of the tendency of the "intestinal flora balance" of the "donor"

[0379] For each "recipient", the tendency of the "intestinal flora balance" of a "donor" considered to be healthy was confirmed at least with respect to the disease each had.

[0380] Then, by comparing the "intestinal flora balance" of the "recipient" measured in (A)-i with the tendency confirmed in (A)-ii, and further comprehensively considering the accumulated insights of "intestinal flora transplantation" in (A)-iv other than the "attributes and / or environment" of each "recipient" in (A)-iii, the type of "intestinal flora balance" to be administered to each "recipient" was evaluated.

[0381] (C) Selection of "intestinal flora" from healthy "donors":

[0382] Depending on the "recipient", 4 to 6 "donors" with "intestinal flora" capable of achieving the "live microorganism balance type" determined in (B) were selected from the above "intestinal flora library" respectively.

[0383] (Manufacture of composition (dilute solution) containing live microorganisms)

[0384] For each "recipient" suffering from Figures 2 to 9 various diseases, 4 to 6 kinds of "compositions containing live microorganisms (stock solutions)" (stock solution mixtures) were mixed.

[0385] After that, the stock solution mixtures prepared for each "recipient" were respectively diluted to various concentrations with physiological saline to prepare a plurality of "compositions containing live microorganisms (intestinal bacteria) (diluted solutions)" having a concentration gradient of about 5 to 50 times.

[0386] The "intestinal flora balance" of the "compositions containing live microorganisms (intestinal bacteria) (diluted solutions)" selected from a plurality of "donors" for each of various diseases is shown in Figures 2 to 9 (the bar graph on the right).

[0387] [Example 10: Appliance for administering a composition containing live microorganisms (intestinal bacteria)]

[0388] An intestinal catheter of 12 fr (French: outer diameter) to 15 fr (about 50 cm in length) was used as the "tubular part", and an enema container or a douche or a drip infusion bag with the front end connected to the intestinal catheter was used as the "storage part" of the composition to manufacture the "appliance for administering" the composition of the present invention.

[0389] The outer diameter of the "tubular part" and the type of the "storage part" are determined according to complex factors such as the age, gender, and symptoms of the "recipient".

[0390] In the case where the "recipient" is a child, 12 fr was used, and in the case of an adult, those of 12 to 15 fr were used.

[0391] [Test Example 1: Confirmation test for engraftment of live microorganisms - a]

[0392] (Administration to the "recipient" (patient))

[0393] The "compositions containing live microorganisms (intestinal bacteria) (diluted solutions)" prepared in Examples 2 to 9 were administered to each "recipient".

[0394] Specifically, the intestinal catheter part of the appliance of Example 10 above was inserted into the anus of the "recipient" about 17 to 20 cm, and the "composition containing live microorganisms (diluted solution)" of the present invention was transplanted by the intestinal infusion method of 50 to 300 ml each.

[0395] The inserted length is appropriately determined according to the age, body type, and symptoms of the "recipient", and the transplanted amount is appropriately determined according to age, symptoms, or the number of administrations, etc.

[0396] In addition, for transplantation, while confirming the degree of improvement of the disease, physical condition, etc. of the "recipient", the above-mentioned dilution solution about 5 to 50 times is used to give a concentration gradient while changing the concentration of viable microorganisms, and it is administered in 1 to 10 divided doses over 1 day to several months.

[0397] (Result: Change in "intestinal flora balance")

[0398] Fecal samples were collected from each "recipient" after 1 to 10 times of transplantation (after the transplantation period ended), and the "intestinal flora balance" was measured by gene analysis of intestinal bacteria.

[0399] The results are shown in Figures 2 to 9 (the bar graph in the middle).

[0400] It was found that in Figures 2 to 9 any of the cases, the "intestinal flora balance (bar graph in the middle)" of the "recipient" was significantly improved and was very close to the "intestinal flora balance" of the "donor" (right bar graph).

[0401] In addition, there may also be the following view: The change in the "intestinal flora balance" after transplantation is due to the result that the transplanted composition is not engrafted but directly excreted.

[0402] However, since the absolute amount of bacteria contained in the transplanted composition is much less than the intestinal bacteria of the "recipient" itself, and the "intestinal flora balance" in the feces after the transplantation period ended changed significantly as shown in the figure, it is natural to think that the bacteria inhabiting under the intestinal mucosa proliferated logarithmically repeatedly and were reflected in the feces as the bacteria of the "recipient" itself.

[0403] Therefore, it is considered that Figures 2 to 9 (bar graph in the middle) the "intestinal flora balance" after transplantation is based on the result that the viable microorganisms after transplantation were actually engrafted in the body of the "recipient".

[0404] In addition, it has been reported that in conventional ("nanobubble water A" not used) "intestinal flora transplantation", the change in the "intestinal flora balance" after transplantation is small, and in addition, it does not meet the degree of improvement of the disease.

[0405] In contrast, according to the above test results, after administration, the "intestinal flora balance" in the feces was significantly improved and was greatly close to the "intestinal flora balance" of a healthy "donor".

[0406] That is, the above test results are very astonishing when compared with conventional "intestinal flora transplantation".

[0407] (Discussion)

[0408] The above results showed that by using "sub-nanoscale" bubbles, the intestinal bacteria, which are "living microorganisms as (I)", were more surely implanted and proliferated in the intestine, and an "intestinal flora" with a new balance of living microorganisms was formed. That is, the "composition containing living microorganisms" in Examples 2 to 9 functioned as a "composition for adjusting the balance of living microorganisms".

[0409] It is considered that this may be because the function of the intestinal mucosa composed of mucopolysaccharides, etc., which prevents the invasion of external substances into the living body, was alleviated by the tiny bubbles that enveloped the living microorganisms.

[0410] Therefore, among the "compositions of the present invention", as a composition for implanting in the living body of a "recipient" via the mucosa, the utilization value of the composition of the present invention is particularly high.

[0411] [Test Example 2: Confirmation test for implantation of living microorganisms - b]

[0412] (Administration of human intestinal flora to mice)

[0413] Using nano-bubble water B instead of nano-bubble water A, and otherwise performing the same as in Example 1, a "composition containing living microorganisms" (stock solution) was prepared from human feces. Then, the "composition containing living microorganisms" of the present invention (dilute solution) was prepared in the same manner as in Examples 2 to 9.

[0414] In addition, for comparison, a "composition containing living microorganisms" (dilute solution) for comparison was prepared in the same manner as the conventional method by using physiological saline instead of nano-bubble water.

[0415] 2 cc each of the "composition containing living microorganisms" (dilute solution) based on the present invention and the conventional method were respectively introduced into 3 mice.

[0416] The mice's tails were lifted and they were hung upside down, and the composition was introduced into the intestine by enema from above to minimize backflow.

[0417] (Collection of mouse feces)

[0418] After transplantation, the mice were placed in cages, and feces were collected before administration, immediately after administration, on the next day (around 9 o'clock and 17 o'clock), 2 days later (around 9 o'clock and 17 o'clock), 3 days later (around 9 o'clock and 17 o'clock), and 7 days later (around 9 o'clock and 17 o'clock). After refrigerated storage, the intestinal flora was analyzed using a next-generation sequencer (Miseq, Illumina).

[0419] (Result: Change in "intestinal flora balance")

[0420] On the 7th day, regardless of the intestinal flora balance of the transplanted "composition containing live microorganisms", the "Blautia genus" proliferated rapidly.

[0421] In addition, the increase rate of the "Blautia genus" was significantly higher in the case of the "composition containing live microorganisms" of the present invention even when compared with the "composition containing live microorganisms" of the conventional method.

[0422] (Discussion)

[0423] Regarding the "Blautia genus", it has been reported that when organ transplantation or the like is performed, it proliferates rapidly to protect the host and is involved in reducing the lethality caused by Graft-versus-Host Disease (Biology of Blood and Marrow Transplantation Volume 21, Issue 8, August 2015, Pages 1373-1383).

[0424] Therefore, it is considered that the above results confirm that at least a part of the transplanted "human-derived intestinal flora" is indeed "engrafted" in the intestines of mice, and the "engraftment amount" is significantly more than that of the conventional method.

[0425] In addition, such a rapid increase of the "Blautia genus" was not observed during human-to-human transplantation. The excellent engraftment effect of the "composition containing live microorganisms" of the present invention can be confirmed by using the changes unique to such "xenotransplantation".

[0426] [Test Example 3-1: Confirmation Test of the Improvement Effect on Constitution and / or Physical Condition - Case 1]

[0427] [Case 1]: A 34-year-old male

[0428] [Diagnosis]: Giardia infection

[0429] [Chief complaint]: Nausea

[0430] [Current medical history]: Stayed in India for 2 months in 2011. At that time, fruits were consumed (using tap water), and as a result, diarrhea symptoms persisted. After returning to the home country (the United States), there were also digestive system symptoms such as diarrhea and nausea. After a detailed examination, it was diagnosed as giardiasis based on Giardia lamblia, and antibiotics were administered intermittently for 6 months. After that, giardiasis was cured in the examination, but starting from several months to 1 year later, discomfort was felt when consuming sugar. In particular, the symptoms were significant when consuming foods using refined sugar such as desserts, alcohol, and milk (yogurt is okay), and there were no problems with fruits and other foods containing dietary fiber. In addition, symptoms also occurred when a large amount of rice was consumed, but whole wheat flour did not cause symptoms.

[0431] [Past medical history]: No special precautions

[0432] [Test method for confirming the improvement effect of constitution and / or physical condition]

[0433] The composition of the "composition containing living microorganisms" used in transplantation and the protocol of the intestinal flora transplantation implemented are shown below and Figure 10-1 .

[0434] [Composition of the "composition containing living microorganisms"]

[0435] As described below, the "composition containing living microorganisms" used for transplantation at each point of UB1 to UB3 in the protocol was prepared.

[0436] UB1: Used Figure 10-2 5 ml of the stock solution of the nano-bubble water B-treated feces from donor B described above diluted with 250 ml of physiological saline

[0437] UB2: Used Figure 10-2 8 ml of the stock solution of the nano-bubble water B-treated feces from donor B described above diluted with 250 ml of physiological saline

[0438] UB3: Used Figure 10-2 10 ml of the stock solution of the nano-bubble water B-treated feces from donor A described above diluted with 250 ml of physiological saline

[0439] [Protocol]

[0440] 1. Perform an intestinal flora test (feces) before transplantation (1)

[0441] 2. Adjust the transplanted bacterial solution with physiological saline and perform fecal transplantation from the flora library by enema (S1)

[0442] 3. Two weeks later, test the intestinal flora (feces) (2)

[0443] 4. Thereafter, the bacterial solution adjusted with ultrafine bubble water (nano-bubble water B) was transplanted into the feces by enema (UB1).

[0444] 5. Fecal transplantation based on ultrafine bubble water (nano-bubble water B) (UB2, UB3) was performed once a week for a total of 3 times.

[0445] 6. Two weeks later, the intestinal flora (feces) was examined (3).

[0446] 7. The changes in each symptom were grasped through interviews.

[0447] [After]

[0448] 《S1~(2)》

[0449] After the S1 transplantation, there was a strong feeling of abdominal distension and it was difficult to eat. There was diarrhea for 3 days 3 days after the transplantation.

[0450] 《UB1~UB2》

[0451] After UB1, there was no feeling of abdominal distension. There was no diarrhea either and the physical condition was good.

[0452] 《UB2~UB3》

[0453] The physical condition improved. Defecation was normal (one formed stool per day).

[0454] 《UB3~(3)》

[0455] The physical condition was good. Foods that had caused symptoms so far were eaten little by little, but no symptoms occurred.

[0456] The results of investigating the intestinal flora balance of the feces collected in the above (1), (2), and (3) are shown in Figure 10-2 .

[0457] [Results]

[0458] As Figure 10-2 shown, after the transplantation (UB1 - UB3) of the composition containing live microorganisms of the present invention using nano-bubble water B, Clostridium cluster XI, which could not be confirmed after the implementation of S1, appeared. In addition, the balance of Clostridium in the whole increased.

[0459] It is suggested that the immune regulation and mental symptoms were improved. In addition, for the overall balance, the overexpression of Bacteroides was also inhibited, and it is considered that there is an improvement tendency through the transplantation (UB1 - UB3) of the composition containing live microorganisms of the present invention.

[0460] [Discussion]

[0461] Compared with the situation where the physical condition deteriorated after implementing S1, which was the initial conventional method, the physical condition recovered smoothly after the implementation of UB1. Thus, it is clear that the "composition containing viable microorganisms" using the nano-bubble water B of the present invention is more useful than the conventional "composition containing viable microorganisms" in which viable microorganisms are only dissolved in physiological saline and can be used as a "physical constitution and / or physical condition improver".

[0462] [Test Example 3-2: Confirmation Test of Improvement Effect on Physical Constitution and / or Physical Condition - Case 2]

[0463] [Case 2]: 52-year-old male

[0464] [Chief Complaint]: Chronic fatigue symptoms

[0465] [Past Medical History]: Appendectomy (in his 20s), lumbar disc herniation (in his 30s)

[0466] [Current Medical History]: There is no obstacle in daily life, and he feels fatigue symptoms starting from the afternoon. He participated in this clinical trial aimed at evaluating the engraftment rate of fecal transplantation as a subject.

[0467] [Test Method for Confirming the Improvement Effect on Physical Constitution and / or Physical Condition]

[0468] The composition of the "composition containing viable microorganisms" used in the transplantation and the protocol of the intestinal flora transplantation implemented are shown below and Figure 11-1 .

[0469] [Composition of the "Composition Containing Viable Microorganisms"]

[0470] As described below, the "composition containing viable microorganisms" used for transplantation at each point of UB1 to UB3 in the protocol was prepared.

[0471] UB1: Used Figure 11-2 5 ml of the stock solution treated with nano-bubble water B of the feces from donor D diluted with 250 ml of physiological saline

[0472] UB2: Used Figure 11-2 8 ml of the stock solution treated with nano-bubble water B of the feces from donor C diluted with 250 ml of physiological saline

[0473] UB3: Used Figure 11-2 10 ml of the stock solution treated with nano-bubble water B of the feces from donor D diluted with 250 ml of physiological saline

[0474] [Protocol]

[0475] 1. Conduct an intestinal flora test (feces) before transplantation (1)

[0476] 2. Adjust the transplanted bacterial solution with normal saline and perform fecal transplantation from the fecal microbiota library using an endoscope (S1).

[0477] 3. After 2 weeks, examine the intestinal microbiota (feces) (2).

[0478] 4. Thereafter, perform fecal transplantation of the bacterial solution adjusted with ultrafine bubble water (nano bubble water B) by enema (UB1).

[0479] 5. Perform fecal transplantation based on ultrafine bubble water (nano bubble water B) once a week for a total of 3 times (UB2, UB3), and examine the intestinal microbiota (feces) after each transplantation ((3)(4)(5)).

[0480] 6. Grasp the changes in each symptom through interviews.

[0481] [After]

[0482] 《S1 to UB1》

[0483] On the day after implementing S1, the fatigue increased further, accompanied by borborygmus with hyperactive abdominal peristalsis, and intermittent abdominal discomfort persisted.

[0484] 《UB1 to UB3》

[0485] Immediately after implementing UB-1, the abdominal discomfort disappeared, and the self-perceived symptoms such as fatigue also improved.

[0486] 《After UB-3》

[0487] Although there is a bit more flatulence perceived by oneself, there is no accompanying abdominal discomfort or fatigue.

[0488] The results of investigating the intestinal microbiota balance of the feces collected in the above (1)(2)(3)(4)(5) are shown in Figure 11-2 .

[0489] [Results]

[0490] As Figure 11-2 shown, after implementing S1, Clostridium cluster IX increased significantly, and the balance was greatly disrupted. It can be seen that in order to regain the disrupted balance, the bacteria transplanted in a way to approach the target balance in the subsequent fecal transplantation after UB1 fought with the intestinal bacteria of the subjects. Regarding the mental state, it can also be seen that there is a possibility of a temporary deterioration after implementing S1, and it converges to a generally good balance and a stable mental state after the end of UB3.

[0491] [Discussion]

[0492] Compared with the situation where the physical condition deteriorated after implementing S1, which was the initial conventional method, the physical condition recovered smoothly after the implementation of UB1. Thus, it is clear that the "composition containing viable microorganisms" using the nanobubble water of the present invention is significantly more useful than the conventional "composition containing viable microorganisms" in which viable microorganisms are only dissolved in physiological saline, and can be used as an "agent for improving physique and / or physical condition".

[0493] [Test Example 4: Confirmation Test of Improvement Effect on Various Diseases]

[0494] In addition, using the "composition of the present invention", the above-mentioned engraftment confirmation test was conducted on more than about 300 cases of patients with diseases other than those shown in Test Example 1 ( Figures 2 to 9 ).

[0495] As a result, an improvement in the balance of viable microorganisms to the same extent (or higher) was observed in a considerable number, and further improvement was also observed in various diseases. Figures 2 to 9

[0496] Furthermore, more than 800 cases were subjected to "intestinal flora transplantation" (including cases where the above-mentioned engraftment confirmation test was not conducted), and effects such as improvement of disease symptoms were also observed in a considerable number of these cases.

[0497] The results show that especially for "ulcerative colitis", "irritable bowel syndrome", "Crohn's disease", "bipolar disorder", "depression", "atopic dermatitis", "constipation", etc., the degree of improvement is relatively large, and it can be greatly expected that the transplantation of the "composition of the present invention" into the living body will be an effective treatment method for these diseases.

[0498] [Test Example 5: Confirmation Test of Engraftment Rate]

[0499] In the engraftment confirmation test of more than about 300 cases described in Test Example 4, although it varies depending on the "attributes and / or environment" of the "recipient", etc., when using the "composition of the present invention", in addition to obtaining a real feeling of the transplantation effect brought about by temperature sensation, etc. to some extent within a few minutes after transplantation, changes in the "intestinal flora balance" were confirmed in the initial stool samples collected within several hours to the next day after the end of the transplantation period in many cases.

[0500] Based on the above results, it can be considered that in the "intestinal flora transplantation" based on the composition of the present invention, the intestinal bacteria engraft at an astonishing speed and more surely compared with the conventional "intestinal flora transplantation".

[0501] [Test Example 6: Usefulness Test of Auxiliary Solvent for Living Body Introduction (Microorganism Preservation Stability Test)]

[0502] From the above test examples, it was clarified that the "composition containing viable microorganisms" of the present invention using the "viable organism introduction assisting solvent" (nano-bubble water) of the present invention is suitable for introducing organic substances such as microorganisms into viable organisms. Hereinafter, the suitability of the "viable organism introduction assisting solvent" of the present invention as a preservation solution for microorganisms was tested.

[0503] (Test method: protocol)

[0504] After culturing the "composition containing intestinal bacteria" of the following 1) and 2) for one week using a semi-fluid high-layer live medium manufactured by Kyokuto Pharmaceutical Industry Co., Ltd., a comparative observation was made on the change in the medium caused by the proliferation of intestinal bacteria and the motility of the bacteria.

[0505] 1) Using the "viable organism introduction assisting solvent B" (nano-bubble water B) of Example B of the present invention, the "composition containing intestinal bacteria" of the present invention (bacterial solution: stock solution) was prepared from human feces in the same manner as in Example 1.

[0506] 2) A conventional "composition containing intestinal bacteria" (bacterial solution: stock solution) was prepared using physiological saline.

[0507] Using 50 μl of each of the above bacterial solutions, they were punctured and injected into the above medium using a Pasteur pipette in the same manner as in the bacterial culture test, and the situation after one week was compared and observed.

[0508] The results are shown in Figure 12 .

[0509] (Results)

[0510] As shown in the right tube of Figure 12 , in the conventional "composition containing intestinal bacteria" of 2), soon after puncture, due to the growth of bacteria, turbidity changes due to proliferation were observed in the "aerobic part (upper part of the medium)" and the "anaerobic part of the punctured high layer (middle to bottom of the medium)", but after one week, in the test tubes other than these, the color tone of the culture medium itself was maintained and the transparency was high.

[0511] That is, there was no tendency for all the intestinal flora to continue culturing, multiplying, and maintaining biological activity.

[0512] On the other hand, similarly as shown in the left tube of Figure 12 , in the "composition containing intestinal bacteria" of the present invention of 1), in addition to the turbidity changes due to proliferation in the "aerobic part (upper part of the medium)" and the "anaerobic part of the punctured high layer (middle to bottom of the medium)" caused by the growth of bacteria soon after puncture, the entire medium became turbid and the transparency of the entire medium significantly disappeared.

[0513] This shows that the overall biological activity of the intestinal flora is basically maintained, that is, the storage stability of the "composition containing intestinal bacteria" (bacterial solution: stock solution) prepared using the "living body introduction assisting solvent" (nano-bubble water B) of the present invention lasts for more than one week.

[0514] (Examination)

[0515] From the above results, the following two aspects can be known.

[0516] First, it can be simply considered that the "living body introduction assisting solvent" of the present invention is more suitable for the preservation of living microorganisms.

[0517] Second, it is considered that it shows that, as one of the reasons why the "living body introduction assisting solvent" of the present invention and the "composition containing living microorganisms" using the same can far more effectively introduce organic substances such as microorganisms into a living body compared with the prior art in other test examples, etc., it may be that they have high storage stability of living microorganisms.

[0518] Industrial Applicability

[0519] Compared with the conventional method, the "composition of the present invention" can be administered in a method that is less burdensome for patients and is simpler, and can achieve more rapid and reliable engraftment regardless of the method and route of administration. Therefore, great expectations can be placed on its application to the treatment of various diseases, etc.

Claims

1. A composition containing intestinal bacteria, which is used for reconstructing the balance of the intestinal flora, wherein the composition comprises the following (I) to (III): (I) One or more intestinal bacteria obtained by removing insoluble substances, (II) A solvent selected from one of purified water, physiological saline or mineral water, (III) Bubbles with a nano-size or less with an average bubble diameter of less than 1000 nm, wherein the gas component in the bubbles consists of hydrogen.

2. The composition according to claim 1, wherein, (I) is intestinal bacteria from one or more healthy individuals.

3. The composition according to claim 1 or 2, wherein the composition comprises bacteria resident in the intestine, and the composition is a preparation suitable for administration via the administration route of the anus or mouth.

4. The composition according to claim 1 or 2, wherein the composition is for transmucosal colonization.

5. A method for preparing the composition defined in any one of claims 1-4, which at least comprises the following steps (1) to (3): (1) A step of generating the bubbles of (III) in the solvent of (II), (2) A step of dispersing (I) in the solvent of (II) containing the bubbles of (III) obtained in the step of (1), wherein, (I) is one or more intestinal bacteria obtained by removing insoluble substances, (II) is a solvent selected from one of purified water, physiological saline or mineral water, (III) is bubbles with a nano-size or less with an average bubble diameter of less than 1000 nm, wherein the gas component in the bubbles consists of hydrogen, (3) A step of determining one or more individuals for preparing (I) according to the attributes of the administration object and the changes in the environment.

6. A method for determining the composition of the composition defined in any one of claims 1-4, comprising the following steps: (A) A step of selecting the type of "intestinal flora balance" suitable for treatment to be administered, (B) A step of selecting "intestinal bacteria" from one or more individuals different from the administration object that can achieve the "intestinal flora balance type" selected in (A).

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