Method for screening probiotic or prebiotic and medium for screening used therefor

Abstract

As regards the beneficial intestinal bacteria present in the intestines of each individual, to provide a method for screening, by a simple, safe in vitro test, probiotics and prebiotics that promote the proliferation of beneficial intestinal bacteria.　A screening method according to the present invention screens probiotics or prebiotics that promote the proliferation of beneficial intestinal bacteria present in the intestine of a subject or a test animal and comprises a step for adding a fecal sample collected from a subject or a test animal and a candidate probiotic or a candidate prebiotic to a liquid culture medium and culturing anaerobically, and a step for evaluating the state of proliferation of the beneficial intestinal bacteria in the liquid culture medium after anaerobic culture. The liquid culture medium used for anaerobic culture is prepared so that the concentration of the constituent components of the liquid culture medium is 1 / 3 to 1 / 6 of the concentration normally used in the culture of beneficial intestinal bacteria.

Description

Screening method for probiotics or prebiotics and screening medium used therefor

[0001] The present invention relates to a method for screening probiotics or prebiotics, and more specifically to a method for screening probiotics or prebiotics that can promote the growth of useful intestinal bacteria such as bifidobacteria and butyric acid-producing bacteria among the intestinal bacteria of a subject, and a screening medium used therefor.

[0002] It is known that the human intestinal microbiota varies from person to person due to factors such as dietary habits, lifestyle, stress, and history of antibiotic use. It is also known that improving the intestinal environment can improve or enhance health. Therefore, useful intestinal bacteria that benefit the human body, so-called "good bacteria," such as lactic acid bacteria and bifidobacteria (Bifidobacterium genus bacteria), are used as probiotics in foods, supplements, and pharmaceuticals. When these microorganisms or their metabolites are orally ingested, they reach the intestine and produce lactic acid and acetic acid. This lowers the intestinal pH, suppresses the growth of harmful bacteria, and activates the activity of lactic acid bacteria and bifidobacteria present in the host's intestine.

[0003] In recent years, so-called "butyric acid-producing bacteria" that have the property of producing butyric acid have attracted attention as more useful intestinal bacteria. Clostridium butyricum is a well-known example of butyric acid-producing bacteria, but other butyric acid-producing bacteria belonging to various genera, such as Agathobacter, Anaerobutyricum, Anaerostipes, Coprococcus, Faecalibacterium, and Roseburia, are also known. Butyric acid produced by butyric acid-producing bacteria is not only utilized as a major energy source for colonic epithelial cells, but also plays a role in regulating the energy metabolism system and immune system, maintaining homeostasis, and the like, via short-chain fatty acid receptors. Recently, it has been discovered that butyric acid acts on the intestinal immune system to enhance the host's immunity by promoting IgA secretion, and also has the function of promptly reducing postprandial blood glucose levels by promoting the gene expression of the intestinal hormone GLP-1. Therefore, the butyric acid-producing bacterium Clostridium butyricum is used in supplements and the like.

[0004] However, the inventors of the present application believe that ingesting specific butyrate-producing bacteria themselves in the form of supplements or the like may slow down the activity of the butyrate-producing bacteria possessed by the host, depending on the composition of the host's intestinal bacterial population, because the excess butyrate produced by the bacteria creates a negative feedback loop.

[0005] Therefore, in order to enhance butyrate production by butyrate-producing bacteria present in the host's intestine, probiotics or prebiotics that can promote the growth of butyrate-producing bacteria are needed. However, since the intestinal flora differs from person to person, the butyrate-producing bacteria possessed by each person also differ, and the probiotics or prebiotics that can promote their growth also differ from person to person. In such cases, a possible screening method is an in vivo test in which subjects orally ingest candidate probiotic and prebiotic materials for a predetermined period of time and examine changes in the intestinal environment of the subject before and after ingestion. However, with this method, if tests are conducted for each type and dose of candidate material, in addition to the complicated ethical review procedures, the test takes time, places a heavy burden on the subject, and has safety issues, making it extremely difficult to conduct the test.

[0006] Therefore, the present invention has been made in consideration of the above points, and an object of the present invention is to provide a method for screening probiotics and prebiotics that promote the growth of useful intestinal bacteria present in the intestines of individuals through a simple and safe in vitro test.

[0007] Another object of the present invention is to provide a screening medium for use in carrying out the above-mentioned screening method.

[0008] The present inventors have discovered a technique for screening probiotics and prebiotics that promote the growth of beneficial intestinal bacteria in an individual by adding candidate probiotics or prebiotics to a fecal sample collected from the individual during in vitro bulk culture and examining the growth state of beneficial intestinal bacteria in the culture medium. Based on the findings obtained as a result, the present invention has been completed.

[0009] In order to solve the above-mentioned problems, the screening method of the present invention is a method for screening probiotics or prebiotics that promote the growth of useful intestinal bacteria present in the intestine of a subject or test animal, and includes the steps of adding a fecal sample collected from the subject or test animal and a candidate probiotic or candidate prebiotic to a liquid medium and anaerobic culturing, and evaluating the growth state of the useful intestinal bacteria in the liquid medium after anaerobic culturing, wherein the liquid medium used for anaerobic culturing is a medium prepared so that the concentrations of the constituent components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in culturing useful intestinal bacteria. This prevents the liquid medium from immediately becoming saturated with fecal-derived viable bacteria during anaerobic culturing, allowing the useful intestinal bacteria to grow in the liquid medium due to the added probiotic or prebiotic, while allowing room for the fecal-derived viable bacteria to grow slowly, thereby making it possible to evaluate the growth-promoting effect of the added candidate material on useful intestinal bacteria. In the present invention, the "concentration normally used in culturing useful enterobacteria" refers to the concentration of each component normally used when culturing the useful enterobacteria using the liquid medium, and more specifically, the concentration of each component indicated in the recipe of a commercially available medium or a conventionally known medium, for example.

[0010] Furthermore, the liquid medium used in the screening method of the present invention is preferably a medium selected from the group consisting of reinforced clostridia (RCM) medium, GAM bouillon medium, modified GAM (mGAM) bouillon medium, Bryant-Berkey (BB) medium, TOS propionic acid medium, and a mixture thereof, and is also preferably a medium prepared by diluting this selected medium 3 to 6 times so that the concentrations of the components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in the cultivation of useful enterobacteria. This allows the selection of a medium that can be suitably and easily prepared as a liquid medium to be used in anaerobic cultivation and that can also be used for screening.

[0011] In addition, in the screening method of the present invention, the useful intestinal bacteria are preferably butyric acid-producing bacteria or Bifidobacterium bacteria, thereby selecting useful intestinal bacteria that improve or enhance the health condition of a subject and whose growth-promoting effect by a candidate probiotic or candidate prebiotic is to be evaluated.

[0012] It is also preferred that the useful intestinal bacteria in the screening method of the present invention are butyric acid-producing bacteria, and that these butyric acid-producing bacteria belong to Clostridium cluster IV or Clostridium cluster XIVa. This allows the selection of a cluster into which many butyric acid-producing bacteria are classified among the useful intestinal bacteria, thereby enabling efficient evaluation of the growth-promoting effect of candidate probiotics or candidate prebiotics on butyric acid-producing bacteria.

[0013] Furthermore, in the screening method of the present invention, the growth state of useful enterobacteria in the liquid medium after anaerobic culture is preferably evaluated by quantitative PCR using primers specific to the Erec or Clep gene of butyric acid-producing bacteria as a target gene. This allows for the selection of a suitable means for evaluating the growth state of butyric acid-producing bacteria present in the intestine of a subject when butyric acid-producing bacteria are selected as useful enterobacteria.

[0014] Furthermore, in the screening method of the present invention, the growth state of useful intestinal bacteria in the liquid medium after anaerobic culture is preferably evaluated by quantitative PCR using the 16S rRNA gene of Bifidobacterium bacteria as a target gene and g-Bifid primers specific to this target gene. This allows for the selection of a suitable means for evaluating the growth state of Bifidobacterium bacteria present in the intestine of a subject when Bifidobacterium bacteria are selected as useful intestinal bacteria.

[0015] In addition, in the screening method of the present invention, it is also preferable to evaluate the growth state of useful intestinal bacteria in a liquid medium after anaerobic culture by detecting the number of useful intestinal bacteria in the liquid medium after anaerobic culture and the number of useful intestinal bacteria in the liquid medium after anaerobically culturing a fecal sample in a liquid medium without adding a candidate probiotic or prebiotic, and comparing the two. This allows for a simple evaluation of the growth-promoting effect of a candidate probiotic or prebiotic on useful intestinal bacteria.

[0016] Furthermore, in the screening method of the present invention, it is also preferred that the probiotics are lactic acid bacteria or butyric acid-producing bacteria, and the prebiotics are oligosaccharides or dietary fiber, thereby allowing the selection of suitable materials as probiotics or prebiotics.

[0017] Furthermore, the screening medium of the present invention is a liquid medium used in the anaerobic culture step of the above-mentioned screening method, and the concentrations of the components of this liquid medium are 1 / 3 to 1 / 6 of the concentrations typically used in the culture of useful intestinal bacteria. This prevents the liquid medium from quickly becoming saturated with fecal-derived viable bacteria during anaerobic culture, allowing the useful intestinal bacteria to grow in the liquid medium due to the added probiotics or prebiotics, while allowing room for the fecal-derived viable bacteria to grow slowly, making it possible to evaluate the growth-promoting effect of the added candidate material on useful intestinal bacteria.

[0018] The screening medium of the present invention is a liquid medium selected from the group consisting of reinforced clostridia (RCM) medium, GAM bouillon medium, modified GAM (mGAM) bouillon medium, Bryant-Berkey (BB) medium, TOS propionic acid medium, and a mixture thereof, and the concentrations of the components of this liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in the cultivation of useful enterobacteria. This allows the selection of a liquid medium that can be suitably and easily prepared as a liquid medium for anaerobic cultivation and that can also be used for screening.

[0019] According to the present invention, it is possible to provide a screening method for probiotics or prebiotics and a screening medium having the following excellent effects: (1) The effectiveness of probiotics and prebiotics on useful intestinal bacteria present in the intestines of individual subjects can be individually evaluated. (2) Because the test is conducted using a fecal sample provided by the subject, the burden on the subject is reduced and the screening test can be carried out safely and easily. (3) When evaluating the growth state of useful intestinal bacteria in a liquid medium after anaerobic culture, it is also possible to identify the species of useful intestinal bacteria by performing analysis using a next-generation sequencer, real-time PCR, or the like.

[0020] 1 is a graph showing the results of measuring the turbidity (OD value) of the culture solution when various strains of bifidobacteria were cultured using RCM medium and its diluted medium in Example 1. 2 is a graph showing the results of measuring the turbidity (OD value) of the culture solution when various strains of bifidobacteria were cultured using GAM bouillon medium and its diluted medium in Example 2. 3 is a graph showing the results of measuring the turbidity (OD value) of the culture solution when various strains of bifidobacteria were cultured using a 4-fold diluted medium of mGR medium (a mixed medium of mGAM:RCM=50:50), a 4-fold diluted medium of BG medium (a mixed medium of BB:mGAM=70:30), and a 4-fold diluted medium of TOR medium (a mixed medium of TOS:RCM=50:50) in Example 3. 1 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when lactic acid bacteria (Latilactobacillus sakei) were cultured using a diluted medium of GAM bouillon medium in Example 4. 2 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when various lactic acid bacteria were cultured using mGR medium (a mixed medium of mGAM:RCM=50:50) and its diluted medium, and BG medium (a mixed medium of BB:mGAM=70:30) and its diluted medium in Example 5. 3 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when lactic acid bacteria (Lacticaseibacillus paracasei) were cultured using MRS medium and its diluted medium in Example 6. 4 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when lactic acid bacteria (Lacticaseibacillus paracasei) were cultured using Subjects No. 1 to No. 2 in Example 8. 1 is a graph showing the results of quantitative PCR of butyric acid-producing bacteria in a culture medium obtained by co-culturing a fecal supernatant sample and a candidate probiotic according to Example 4. FIG. 1 is a graph showing the results of quantitative PCR of butyric acid-producing bacteria in a culture medium obtained by co-culturing a fecal supernatant sample and a candidate probiotic according to Example 8 from subjects No. 5 to No. 7. FIG. 1 is a graph showing the results of relative quantification by a next-generation sequencer of useful intestinal bacterial species in a culture medium obtained by co-culturing only a fecal supernatant sample (control) from subject No. 1 and a culture medium obtained by co-culturing a fecal supernatant sample and a lactic acid bacterium (Latilactobacillus sakei) according to Example 9. FIG. 10 is a graph showing the results of quantitative PCR of bifidobacteria in a culture medium obtained by adding a candidate prebiotic to fecal supernatant samples from subjects A and B according to Example 10.

[0021] The screening method according to the present invention and the screening medium used therein are described in detail below. The present invention is a method for screening probiotics or prebiotics that promote the growth of useful intestinal bacteria present in the intestines of a subject or test animal.

[0022] In the present invention, probiotics generally refer to microorganisms that improve the balance of intestinal flora and have beneficial effects on the human body. Examples of probiotics include lactic acid bacteria, bifidobacteria (Bifidobacterium bacteria), butyric acid-producing bacteria, and saccharifying bacteria. Among these, lactic acid bacteria include, but are not limited to, bacteria belonging to the genus Lactocaseibacillus (Lactobacillus), Latilactobacillus (Lactobacillus), Lactococcus, Enterococcus, Streptococcus, and Pediococcus. Examples of butyric acid-producing bacteria include bacteria belonging to the genus Clostridium, such as Clostridium butyricum, and bacteria belonging to the genus Butyrivibrio and Faecalibacterium. Examples of saccharifying bacteria include Bacillus subtilis, such as Bacillus subtilis (natto).

[0023] In the present invention, prebiotics generally refer to substances that selectively promote the growth of beneficial intestinal bacteria. More specifically, they are not decomposed or absorbed in the digestive tract, but serve as a selective nutrient source for beneficial intestinal bacteria, promoting their growth and improving the composition of the intestinal flora to a healthy balance. Examples of prebiotics include oligosaccharides and dietary fiber. Among these, oligosaccharides include, but are not limited to, fructooligosaccharides, kestose, galactooligosaccharides, maltooligosaccharides, isomaltooligosaccharides (branched oligosaccharides), xylooligosaccharides, milk oligosaccharides, mannan oligosaccharides, gentiooligosaccharides, lactosucrose, nigerooligosaccharides, soybean oligosaccharides, lactulose, and raffinose. Examples of dietary fiber include inulin, polydextrose, indigestible dextrin, and guar gum.

[0024] In the present invention, the intestinal flora refers to bacteria present in the large intestine of a host, and useful intestinal bacteria refers to bacteria that are beneficial to health. Examples of useful intestinal bacteria include, but are not limited to, Bifidobacterium bacteria, lactic acid bacteria, and butyric acid-producing bacteria. Any bacteria that contribute to improving or enhancing the health of the host or maintaining good health can be selected as useful intestinal bacteria. Therefore, in the present invention, useful intestinal bacteria does not mean only one type of bacterium belonging to a specific species, but also includes multiple bacteria belonging to a specific genus, or multiple bacteria belonging to multiple species or genera.

[0025] The present invention includes an anaerobic culturing step in which a fecal sample collected from a subject or test animal and a candidate probiotic or prebiotic are added to a liquid medium and anaerobically cultured, and an evaluation step in which the growth state of useful intestinal bacteria in the liquid medium after the anaerobic culturing is evaluated. Of these steps, the anaerobic culturing step will be described in detail below.

[0026] [Anaerobic Cultivation Step—Fecal Sample—] In this step, feces collected from a subject or test animal is first prepared. The collected feces is preferably anaerobically preserved using a predetermined preservative solution to prevent changes in the intestinal bacterial flora in the feces until the start of the anaerobic culture test. For example, the Amies transport medium used in the Examples described below can be used as the preservative solution. Furthermore, a commercially available fecal collection kit (fecal collection kit for bacterial isolation, Techno Suruga Lab Co., Ltd.) can also be used to collect the feces.

[0027] The collected feces is preferably suspended in a buffer solution such as PBS buffer, physiological saline, or the preservative solution used for feces storage, and then allowed to stand to separate into a supernatant and a sediment. The supernatant containing floating fecal-derived enterobacteria is then recovered and used for anaerobic culture. By adding the fecal supernatant to a liquid medium as a fecal sample and performing anaerobic culture, the turbidity of the culture medium can be determined as the level of bacterial growth, making it easy to determine whether the anaerobic culture process is progressing smoothly. When preparing a fecal supernatant sample from feces, the feces are preferably suspended in a liquid with a weight of 10 to 1,000 times the weight of the feces, more preferably 30 to 300 times the weight of the feces, and even more preferably 50 to 200 times the weight of the feces. Examples of the liquid used for suspension include buffer solutions such as PBS buffer, physiological saline, the preservative solution used for feces storage, and mixtures thereof. After dispersing and suspending the feces in these liquids, a fecal supernatant containing suspended enterobacteria can be obtained by leaving the feces to stand or spinning it down for a few seconds using a tabletop centrifuge, etc. Note that the fecal sample is not limited to a fecal supernatant, and it is also possible to add the feces in a state containing solid matter as it is or in a state of suspension in a preservative solution, etc. to a liquid medium and perform anaerobic culture.

[0028] [Anaerobic Cultivation Step—Liquid Medium (Screening Medium)] In this step, the fecal sample and the candidate probiotic or prebiotic material are added to a liquid medium and cultured anaerobically. The type of liquid medium used for this anaerobic culture is extremely important. This is because, when a fecal sample is cultured in a liquid medium typically used for culturing useful intestinal bacteria, the fecal-derived viable bacteria rapidly grow, resulting in saturation of the liquid medium. Therefore, even if a fecal sample and a candidate probiotic or prebiotic material are added to a liquid medium at a typically used concentration and cultured, there may be no room for the beneficial intestinal bacteria to grow due to the added candidate material, or the overall growth of the intestinal bacteria in the fecal sample may be too rapid. This makes it difficult to clearly evaluate the beneficial intestinal bacteria growth-promoting effect of the candidate material, making it difficult to distinguish whether the effect is actually due to the candidate material. Therefore, in the present invention, a liquid medium used for anaerobic culture, i.e., a screening medium, is used that is prepared so that the concentrations of the constituents of the liquid medium are 1 / 3 to 1 / 6 (33% to 17% concentration) of the concentrations normally used in the cultivation of useful enterobacteria. Of these, it is more preferable to use a medium prepared so that the concentrations are 1 / 3 to 1 / 5 (33% to 20% concentration) of the concentrations normally used in the cultivation of useful enterobacteria, and it is particularly preferable to use a medium prepared so that the concentrations are 1 / 4 (25% concentration) of the concentrations normally used in the cultivation of useful enterobacteria. Here, the concentrations normally used in the cultivation of useful enterobacteria refer to the concentrations of each constituent normally used when culturing bacteria selected as useful enterobacteria using that liquid medium, and more specifically, include the concentrations of each constituent shown in the formulations of commercially available media and conventionally known media, for example.

[0029] For example, while the amounts of each component of a reinforced clostridia (RCM) medium typically used for culturing useful enterobacteria are 10 g peptone, 10 g meat extract, 3 g yeast extract, 5 g glucose, 5 g sodium chloride, 3 g sodium acetate, 1 g soluble starch, and 0.5 g L-cysteine ​​hydrochloride per liter, the present invention uses a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this amount. Therefore, the amounts of each component of the screening medium used in the present invention are preferably, for example, 1.7 to 3.3 g peptone, 1.7 to 3.3 g meat extract, 0.5 to 1.0 g yeast extract, 0.8 to 1.7 g glucose, 0.8 to 1.7 g sodium chloride, 0.5 to 1.0 g sodium acetate, 0.17 to 0.33 g soluble starch, and 0.08 to 0.17 g L-cysteine ​​hydrochloride per liter.

[0030] Furthermore, the amounts of each component of GAM bouillon medium at concentrations typically used in the cultivation of useful enterobacteria are 10 g of peptone, 3 g of soybean peptone, 10 g of proteose peptone, 13.5 g of digested serum powder, 2.2 g of meat extract, 1.2 g of liver extract, 5 g of yeast extract, 3 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3 g of sodium chloride, 5 g of soluble starch, 0.3 g of L-cysteine ​​hydrochloride, and 0.3 g of sodium thioglycolate per liter. In the present invention, a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this concentration is used. Therefore, the amounts of each component of the screening medium used in the present invention are preferably, for example, 1.7 to 3.3 g of peptone, 0.5 to 1.0 g of soybean peptone, 1.7 to 3.3 g of proteose peptone, 2.25 to 4.5 g of digested serum powder, 0.37 to 0.73 g of meat extract, 0.2 to 0.4 g of liver extract, 0.8 to 1.7 g of yeast extract, 0.5 to 1.0 g of glucose, 0.42 to 0.83 g of potassium dihydrogen phosphate, 0.5 to 1.0 g of sodium chloride, 0.8 to 1.7 g of soluble starch, 0.05 to 0.1 g of L-cysteine ​​hydrochloride, and 0.05 to 0.1 g of sodium thioglycolate per liter.

[0031] Furthermore, the amounts of each component of modified GAM (mGAM) medium at concentrations typically used in the cultivation of useful enterobacteria are 5 g of peptone, 3 g of soybean peptone, 5 g of proteose peptone, 10 g of digested serum powder, 2.2 g of meat extract, 1.2 g of liver extract, 2.5 g of yeast extract, 0.5 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3 g of sodium chloride, 5 g of soluble starch, 0.3 g of L-cysteine ​​hydrochloride, 0.3 g of sodium thioglycolate, 0.2 g of L-tryptophan, 0.1 g of L-arginine, 5 mg of vitamin K1, and 10 mg of hemin per liter. In the present invention, a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this amount is used. Therefore, the amounts of each component of the screening medium used in the present invention may be, for example, per liter, 0.8 to 1.7 g of peptone, 0.5 to 1 g of soybean peptone, 0.8 to 1.7 g of protease peptone, 1.7 to 3.3 g of digested serum powder, 0.37 to 0.73 g of meat extract, 0.2 to 0.4 g of liver extract, 0.42 to 0.83 g of yeast extract, 0.08 to 0.17 g of glucose, 0.42 to 0.83 g of potassium dihydrogen phosphate, 0.5 to 1.0 g of sodium chloride, 0.8 to 1.7 g of soluble starch, 0.05 to 0.1 g of L-cysteine ​​hydrochloride, 0.05 to 0.1 g of sodium thioglycolate, 0.03 to 0.06 g of L-tryptophan, 0.017 to 0.033 g of L-arginine, and 0.017 to 0.033 g of vitamin K1. Preferably, the amount is 0.83 to 1.7 mg and hemin 1.7 to 3.3 mg.

[0032] Furthermore, while the amounts of each component of Bryant-Berkey (BB) medium at concentrations typically used for culturing useful enterobacteria are 15 g of casein peptone, 7.5 g of meat extract, 5 g of yeast extract, 5 g of sodium acetate, 0.5 g of L-cysteine ​​hydrochloride, 5 g of sodium lactate, and 5 mg of resazurin per liter, the present invention uses a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this amount. Therefore, the amounts of each component of the screening medium used in the present invention are preferably, for example, 2.5 to 5 g of casein peptone, 1.25 to 2.5 g of meat extract, 0.8 to 1.7 g of yeast extract, 0.8 to 1.7 g of sodium acetate, 0.08 to 0.17 g of L-cysteine ​​hydrochloride, 0.8 to 1.7 g of sodium lactate, and 0.8 to 1.7 mg of resazurin per liter.

[0033] Furthermore, the amounts of each component of the TOS propionic acid medium at concentrations typically used in the cultivation of useful enterobacteria are 10 g of peptone, 1 g of yeast extract, 4.8 g of dipotassium monohydrogen phosphate, 3 g of potassium dihydrogen phosphate, 3 g of ammonium sulfate, 0.2 g of magnesium sulfate heptahydrate, 10.5 g of L-cysteine ​​hydrochloride, 15 g of sodium propionate, and 10 g of galactooligosaccharides per liter. In the present invention, a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this concentration is used. Therefore, the amounts of each component of the screening medium used in the present invention are preferably, for example, 1.7 to 3.3 g of peptone, 0.17 to 0.33 g of yeast extract, 0.8 to 1.6 g of dipotassium hydrogen phosphate, 0.5 to 1.0 g of potassium dihydrogen phosphate, 0.5 to 1.0 g of ammonium sulfate, 0.03 to 0.06 g of magnesium sulfate heptahydrate, 0.08 to 0.17 g of L-cysteine ​​hydrochloride, 2.5 to 5 g of sodium propionate, and 1.7 to 3.3 g of galactooligosaccharides per liter.

[0034] As an example of a mixed medium, a mixed medium (mGR medium) obtained by mixing the above-mentioned mGAM medium and RCM medium in a volume ratio of, for example, 50:50 will be described. The amounts of each component at concentrations normally used in culturing useful enterobacteria are as follows per liter: peptone 7.5 g, soybean peptone 1.5 g, protease peptone 2.5 g, digested serum powder 5 g, meat extract 6.1 g, liver extract 0.6 g, yeast extract 2.75 g, glucose 2.75 g, potassium dihydrogen phosphate 1.25 g, sodium chloride 4 g, sodium acetate 1.5 g, soluble starch 3 g, L-cysteine ​​hydrochloride 0.4 g, sodium thioglycolate 0.15 g, L-tryptophan 0.1 g, L-arginine 0.5 g, vitamin K1 In the present invention, a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this amount is used. Therefore, the amounts of each component of the screening medium used in the present invention may be, for example, per liter, 1.25 to 2.5 g of peptone, 0.25 to 0.5 g of soybean peptone, 0.42 to 0.83 g of protease peptone, 0.8 to 1.7 g of digested serum powder, 1 to 2 g of meat extract, 0.1 to 0.2 g of liver extract, 0.46 to 0.92 g of yeast extract, 0.46 to 0.92 g of glucose, 0.21 to 0.42 g of potassium dihydrogen phosphate, 0.7 to 1.3 g of sodium chloride, 0.25 to 0.5 g of sodium acetate, 0.5 to 1 g of soluble starch, 0.067 to 0.13 g of L-cysteine ​​hydrochloride, 0.025 to 0.05 g of sodium thioglycolate, 0.017 to 0.033 g of L-tryptophan, 0.08 to 0.17 g of L-arginine, and 0.17 to 0.17 g of vitamin K1. It is preferable that the amount of erythritol is 0.41 to 0.83 mg and hemin is 0.8 to 1.7 mg.

[0035] Another example of a mixed medium is a mixed medium (BG medium) obtained by mixing the above-mentioned BB medium and mGAM medium in a volume ratio of, for example, 70:30. The amounts of each component at concentrations typically used in culturing useful enterobacteria are as follows per liter: peptone 12 g, soybean peptone 0.9 g, protease peptone 1.5 g, digested serum powder 3 g, meat extract 5.91 g, liver extract 0.36 g, yeast extract 4.25 g, glucose 0.15 g, potassium dihydrogen phosphate 0.75 g, sodium chloride 0.9 g, sodium acetate 3.5 g, soluble starch 1.5 g, L-cysteine ​​hydrochloride 0.44 g, sodium thioglycolate 0.09 g, L-tryptophan 0.06 g, L-arginine 0.3 g, vitamin K1 The concentrations of the components of the screening medium used in the present invention are, for example, 2 to 4 g of peptone, 0.15 to 0.3 g of soybean peptone, 0.25 to 0.5 g of protease peptone, 0.5 to 1 g of digested serum powder, 0.985 to 1.97 g of meat extract, 0.06 to 0.12 g of liver extract, 0.7 to 1.4 g of yeast extract, and 0.025 to 0.0 g of glucose per liter. 0.5 g, potassium dihydrogen phosphate 0.125 to 0.25 g, sodium chloride 0.15 to 0.3 g, sodium acetate 0.58 to 1.17 g, soluble starch 0.25 to 0.5 g, L-cysteine ​​hydrochloride 0.07 to 0.15 g, sodium thioglycolate 0.015 to 0.03 g, L-tryptophan 0.01 to 0.02 g, L-arginine 0.05 to 0.1 g, vitamin K1 0.25 to 0.5 mg, hemin 0.5 to 1 mg, sodium lactate 0.58 to 1.17 g, and resazurin 0.58 to 1.17 mg.

[0036] Another example of a mixed medium is a mixed medium (TOR medium) obtained by mixing the above-mentioned TOS propionic acid medium and RCM medium in a volume ratio of, for example, 50:50. The amounts of each component at concentrations typically used in the cultivation of useful enterobacteria are 10 g peptone, 5 g meat extract, 2 g yeast extract, 2.5 g glucose, 2.4 g dipotassium monohydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 1.5 g ammonium sulfate, 2.5 g sodium chloride, 1.5 g sodium acetate, 0.1 g magnesium sulfate heptahydrate, 0.5 g soluble starch, 0.5 g L-cysteine ​​hydrochloride, 7.5 g sodium propionate, and 5 g galactooligosaccharides per liter. In the present invention, a medium prepared to have a concentration of 1 / 3 to 1 / 6 of this amount is used. Therefore, the amounts of each component of the screening medium used in the present invention are preferably, for example, 1.67 to 3.33 g of peptone, 0.83 to 1.67 g of meat extract, 0.33 to 0.67 g of yeast extract, 0.41 to 0.83 g of glucose, 0.4 to 0.8 g of dipotassium monohydrogen phosphate, 0.25 to 0.5 g of potassium dihydrogen phosphate, 0.25 to 0.5 g of ammonium sulfate, 0.41 to 0.83 g of sodium chloride, 0.25 to 0.5 g of sodium acetate, 0.016 to 0.033 g of magnesium sulfate heptahydrate, 0.083 to 0.167 g of soluble starch, 0.083 to 0.167 g of L-cysteine ​​hydrochloride, 1.25 to 2.5 g of sodium propionate, and 0.83 to 1.67 g of galactooligosaccharides per liter.

[0037] More specifically, the liquid medium is a medium selected from the group consisting of reinforced clostridia (RCM) medium, GAM bouillon medium, modified GAM (mGAM) bouillon medium, Bryant-Berkey (BB) medium, TOS propionic acid medium, and a mixture thereof. It is preferable to use a medium prepared by diluting the above-mentioned medium 3 to 6 times so that the concentrations of the components of the above-mentioned medium are 1 / 3 to 1 / 6 of the concentrations normally used in culturing useful enterobacteria, more preferably a medium prepared by diluting the above-mentioned medium 3 to 5 times, and particularly preferably a medium prepared by diluting the above-mentioned medium 4 times. The mixed medium is not particularly limited, but examples include a mixed medium (mGR medium) obtained by mixing mGAM medium and RCM medium in a volume ratio of 30:70 to 70:30, a mixed medium (BG medium) obtained by mixing BB medium and mGAM medium in a volume ratio of 30:70 to 70:30, and a mixed medium (TOR medium) obtained by mixing TOS propionic acid medium and RCM medium in a volume ratio of 30:70 to 70:30. Here, the RCM liquid medium can be a product of Becton Dickinson, while the GAM bouillon medium and modified GAM bouillon medium can be products of Nissui Pharmaceutical Co., Ltd., and the Bryant-Berkey medium can be a product of Sigma. Regarding TOS propionic acid medium, since commercially available media are agar media (TOS propionic acid agar media, a product of Yakult Pharmaceutical Co., Ltd.), it is preferable to prepare a medium with a composition that does not contain only agar.

[0038] Furthermore, when selecting butyric acid-producing bacteria as useful intestinal bacteria and conducting a screening test to evaluate the growth state of butyric acid-producing bacteria using candidate materials, it is particularly preferable to use, as the liquid medium, a medium prepared by diluting 3 to 6 times a mixed medium (mGR medium) obtained by mixing GAM medium, mGAM medium, and RCM medium in a volume ratio of 50:50, or a mixed medium (BG medium) obtained by mixing BB medium and mGAM medium in a volume ratio of 70:30. Furthermore, when selecting bifidobacteria as useful intestinal bacteria and conducting a screening test to evaluate the growth state of bifidobacteria using candidate materials, it is particularly preferable to use, as the liquid medium, a medium prepared by diluting 3 to 6 times the following: RCM medium, GAM medium, a mixed medium obtained by mixing mGAM medium and RCM medium in a volume ratio of 50:50 (mGR medium), a mixed medium obtained by mixing BB medium and mGAM medium in a volume ratio of 70:30 (BG medium), or a mixed medium obtained by mixing TOS propionic acid medium and RCM medium in a volume ratio of 50:50 (TOR medium).

[0039] [Anaerobic Cultivation Step - Anaerobic Cultivation] In this step, the fecal sample and candidate probiotic or prebiotic material are added to the liquid medium and anaerobically cultured. The liquid medium is prepared anaerobically, and the fecal sample and candidate probiotic or prebiotic material are added anaerobically using a syringe or the like. The amount of the fecal sample added to the liquid medium is preferably 0.1 to 5 v / v % of the liquid medium, and more preferably 0.5 to 2 v / v %.

[0040] When adding candidate probiotics to a liquid medium, it is preferable to pre-culture each of the candidate probiotics in advance and add the pre-culture solution to the liquid medium. ６００又は６６０The culture medium is pre-cultured until the turbidity reaches approximately 1, and this pre-culture solution is added to the liquid medium in an amount of preferably 0.1 to 5 v / v%, more preferably 0.5 to 2 v / v%. Examples of candidate probiotics include lactic acid bacteria, bifidobacteria, butyric acid-producing bacteria, and saccharifying bacteria. Examples of candidate prebiotics include oligosaccharides and dietary fiber, and the concentrations added to the liquid medium can be freely set. For example, the concentrations of candidate prebiotics added based on the recommended daily intake of each prebiotic can be set as follows: galactooligosaccharides (GOS): 0.1%, fructooligosaccharides (FOS): 0.3%, xylooligosaccharides (XOS): 0.1%, milk oligosaccharides (milk): 0.5%, inulin: 0.5%, and kestose: 0.2%. Candidate probiotic or prebiotic materials can be added individually or in combination. It is also possible to add probiotics and prebiotics in combination.

[0041] In anaerobic culture, the culture temperature is preferably a temperature that mimics the intestinal environment of the host, and is therefore preferably about 32 to 40°C, and more preferably 35 to 37°C. Furthermore, the culture time is preferably at least 6 hours or more in order to evaluate the growth state of useful intestinal bacteria due to the candidate probiotic or prebiotic material added to the liquid medium. Specifically, the culture time is preferably 6 to 96 hours, more preferably 12 to 72 hours, and particularly preferably about 1 to 2.5 days.

[0042] Next, the evaluation step for evaluating the growth state of useful enterobacteria in the liquid medium after anaerobic culture will be described in detail below.

[0043] [Evaluation Step] In this step, the growth state of useful intestinal bacteria in the liquid medium after the anaerobic culture step described above is evaluated. The growth state can be evaluated by detecting the number or amount of the useful intestinal bacteria in the liquid medium, or by measuring the amount of substances (e.g., organic acids such as butyric acid) produced by metabolism of the useful intestinal bacteria in the liquid medium and the associated physical properties (e.g., pH) of the liquid medium. The growth state can be evaluated by using a detection value or measurement value obtained from a liquid medium in which a fecal sample is anaerobically cultured without the addition of a candidate probiotic or prebiotic as a control, and comparing this control value with the detection value or measurement value obtained from a liquid medium in which a fecal sample is anaerobically cultured with the addition of the candidate probiotic or prebiotic. In this case, among the candidate probiotic or prebiotic materials tested, probiotics or prebiotics that showed a value greater (higher) than the control are selected as probiotics or prebiotics that promote the growth of useful intestinal bacteria. It is also possible to set a predetermined set value or a rankable level for the detection or measurement value obtained from the liquid culture medium, and evaluate the growth state by comparing the detection or measurement value obtained from the liquid culture medium to which the candidate probiotic or the candidate prebiotic has been added and the fecal sample has been anaerobically cultured with this set value or level.

[0044] Here, the useful intestinal bacteria to be evaluated in this evaluation step can be a single bacterium belonging to a specific species (e.g., Clostridium butyricum), multiple bacteria belonging to a specific genus (e.g., Bifidobacterium), or multiple bacteria belonging to multiple species or genera (e.g., butyric acid-producing bacteria). More specifically, useful intestinal bacteria to be evaluated include, for example, butyric acid-producing bacteria, Bifidobacterium, or lactic acid bacteria. Although not particularly limited, when bacteria belonging to a specific species or genus are selected as useful intestinal bacteria to be evaluated, the number or amount of the bacteria in the liquid medium after anaerobic culture can be detected by quantitative PCR analysis targeting a gene specific to the selected bacteria. Similarly, when multiple bacteria belonging to multiple species or genera are selected, the number or amount of the bacteria in the liquid medium after anaerobic culture can be detected by quantitative PCR analysis targeting a gene specific to the selected bacteria. Quantitative PCR analysis can be performed by extracting DNA from the liquid medium after anaerobic culture using a known method, and performing real-time PCR or the like using the extracted DNA.

[0045] In this evaluation step, an evaluation method when butyric acid-producing bacteria are selected as the useful intestinal bacteria to be evaluated will be described. Most butyric acid-producing bacteria, which are useful intestinal bacteria, are classified into two clusters: Clostridium cluster IV or Clostridium cluster XIVa. Therefore, by performing quantitative PCR analysis targeting genes specific to butyric acid-producing bacteria belonging to Clostridium cluster IV or Clostridium cluster XIVa, the bacterial number or amount of butyric acid-producing bacteria in a liquid medium after anaerobic culture can be efficiently detected. Examples of genes specific to butyric acid-producing bacteria belonging to Clostridium cluster IV include Clep and Fprsn but, which are involved in butyric acid production. Furthermore, examples of genes specific to butyric acid-producing bacteria belonging to Clostridium cluster XIVa include Erec, Rrec, or Ehal, which are involved in butyric acid production. Of these, from the viewpoint of excellent detection sensitivity, it is preferable to select Clep for Clostridium cluster IV, and it is preferable to select Erec for Clostridium cluster XIVa. Furthermore, in this evaluation process, DNA extracted from the liquid medium after anaerobic culture is used to analyze the genes targeted in the quantitative PCR analysis using a next-generation sequencer, making it possible to identify butyrate-producing bacteria at the species level. This makes it possible to select probiotics or prebiotics that promote the growth of butyrate-producing bacteria present in the subject's intestine, and also to identify the species of butyrate-producing bacteria whose growth is actually promoted. Although not particularly limited, examples of primer sequences used in quantitative PCR analysis for each of the above-mentioned target genes are shown in Table 1 below.

[0046]

[0047] Next, an evaluation method will be described when bacteria of the genus Bifidobacterium (bifidobacteria) are selected as useful intestinal bacteria to be evaluated. Genes specific to bacteria belonging to the genus Bifidobacterium include the 16S rRNA gene and the GroEL gene. Among these, an example of a primer specific to the 16S rRNA gene is g-Bifid. Thus, quantitative PCR analysis using g-Bifid primers or the like can detect the number or amount of bifidobacteria in a liquid medium after anaerobic culture. Furthermore, in this evaluation step, DNA extracted from the liquid medium after anaerobic culture can be used to analyze the genes targeted in the quantitative PCR analysis with a next-generation sequencer, thereby enabling identification of bifidobacteria at the species level. This makes it possible to select probiotics or prebiotics that promote the growth of bifidobacteria present in the intestine of a subject, and also to identify the species of bifidobacteria whose growth is actually promoted.

[0048] As described above, the screening method and screening medium of the present invention enable the effectiveness of probiotics or prebiotics according to the intestinal flora of each individual to be individually evaluated, making it possible to propose probiotics or prebiotics suited to each individual and to prepare supplements, etc. Furthermore, since the screening only requires the subject to provide feces, the test can be conducted safely, and the test period is short, on the order of a few days, making it possible to provide the service safely and easily.

[0049] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0050] When fecal samples collected from subjects are bulk cultured in vitro, fecal-derived viable bacteria grow very actively in liquid media at concentrations typically used for culturing beneficial intestinal bacteria. Therefore, even if fecal samples and candidate probiotic or prebiotic materials are added to such liquid media, the liquid media quickly becomes saturated with fecal-derived viable bacteria. This can leave no room for beneficial intestinal bacteria to grow, or the overall growth of the intestinal bacteria in the fecal sample may be too rapid, making it difficult to clearly evaluate the growth-promoting effect of beneficial intestinal bacteria. Therefore, the present inventors came up with the idea of ​​diluting a liquid medium typically used for culturing beneficial intestinal bacteria to lower the concentrations of its components, in order to evaluate the growth-promoting effect of the added candidate material on beneficial intestinal bacteria while suppressing the vigorous growth of fecal-derived viable bacteria to some extent.

[0051] Regarding the degree of dilution of the liquid medium, if the dilution ratio is too high, the concentration of the medium's components will be too low, hindering the growth of the added probiotic or the useful intestinal bacteria to be evaluated, making it impossible to evaluate the growth-promoting effect of the candidate material on useful intestinal bacteria. On the other hand, if the dilution ratio is too low, the culture medium will be saturated with fecal-derived live bacteria immediately after the start of culture, making it impossible to evaluate the growth-promoting effect of the added candidate material on useful intestinal bacteria. Therefore, the inventors of the present application have found, through the studies shown in the following examples, that when a candidate probiotic or a useful intestinal bacterium to be evaluated is cultured alone, the growth-promoting effect of the candidate material on useful intestinal bacteria can be evaluated by adjusting the dilution ratio of the liquid medium so that the turbidity OD value of the single culture medium after 1 to 3 days from the start of culture is 0.5 or less, preferably 0.4 or less, and more preferably in the range of approximately 0.25±0.1. The OD value is measured at a wavelength of 600 nm or 660 nm.

[0052] Example 1 1. Investigation of Liquid Media (1) In this example, a culture test of the following three species of bifidobacteria was carried out using a stock solution (×1) and diluted media of RCM liquid medium (Reinforced Clostridial Medium, product of Becton, Dickinson and Company) at a concentration normally used: Bifidobacterium longum subsp. longum JCM1217, Bifidobacterium pseudocatenulatum JCM1200, and Bifidobacterium adolescentis JCM1275.

[0053] First, each bifidobacterium was streaked from a glycerol stock onto a TOS propionic acid agar medium (product of Yakult Pharmaceutical Co., Ltd.) and cultured at 37°C under anaerobic conditions to form colonies. The colonies of each bacterium were inoculated into RCM liquid medium (stock solution) with the composition shown in Table 2 below and cultured anaerobically at 37°C to obtain preculture solutions.

[0054]

[0055] The OD of each bifidobacterial preculture solution was ６６０ 50 μL of the prepared pre-culture solution was added to 5 mL of the liquid medium containing the original solution (×1) and 2 to 5 times diluted (×1 / 2 to ×1 / 5) as shown in Table 2, and anaerobic culture was carried out at 37°C. The OD of the culture solution was measured 24 hours, 48 ​​hours, and 72 hours after the start of culture. ６６０ The values ​​of were measured. The results are shown in Table 3 below and the graphs in Figures 1(A) to (C). In each graph in Figure 1, the vertical axis represents OD, and the horizontal axis represents the culture time (h). The value of x1 (undiluted solution) is represented by a solid line and an X-shaped marker, the value of x1 / 2 by a dotted line and a circle marker, the value of x1 / 3 by a dashed dotted line and a square marker, the value of x1 / 4 by a solid line and a triangle marker, and the value of 1 / 5 by a dotted line and a diamond marker.

[0056]

[0057] From these results, bifidobacteria (OD ６６０When a 1% volume of RCM liquid medium containing B. longum and B. adolescentis was inoculated and cultured, the OD values ​​of the culture solutions exceeded 1.0 when the undiluted RCM liquid medium (×1) was used. Furthermore, the OD value of B. pseudocatenulatum was 0.72 after 24 hours and reached 0.94 after 48 hours. On the other hand, when bifidobacteria were cultured using diluted medium, the OD value of the culture solution decreased compared to the undiluted RCM liquid medium, but growth was consistent with the culture time, indicating that this did not affect the growth of bifidobacteria. It was found that the OD value of the culture solution after 48 hours from the start of culture could be adjusted to 0.5 or less, preferably 0.4 or less, by adjusting the concentrations of the components of RCM liquid medium to 1 / 3 to 1 / 5 of the normal concentrations.

[0058] Example 2 2. Liquid Media Study (2) In this example, a culture test of the three bifidobacteria used in Example 1 was performed using liquid media of GAM bouillon medium (product of Nissui Pharmaceutical Co., Ltd.) at concentrations typically used (undiluted (x1) and 4-fold diluted (x1 / 4)). First, each bifidobacterium strain was streaked from a glycerol stock onto TOS propionic acid agar medium (product of Yakult Pharmaceutical Co., Ltd.) and cultured at 37°C under anaerobic conditions to form colonies. Colonies of each strain were inoculated into GAM bouillon medium (undiluted) with the composition shown in Table 4 below, and then cultured anaerobically at 37°C to obtain preculture solutions.

[0059]

[0060] The OD of each bifidobacterial preculture solution was ６６０ The OD of the culture medium was adjusted to a turbidity of 1.0 ... ６６０The values ​​of OD were measured. The results are shown in Table 5 below and the graphs in Figures 2(A) and (B). The vertical axis of each graph represents OD, and the horizontal axis represents the culture time (h). The values ​​for B. longum are represented by a solid line and X-shaped marker, the values ​​for B. pseudocatenulatum by a dotted line and circle marker, and the values ​​for B. adolescentis by a solid line and triangle marker.

[0061]

[0062] From these results, bifidobacteria (OD ６６０ When a 1% volume of 1% of the liquid medium was inoculated and cultured using the undiluted GAM liquid medium (x1), the OD value of the culture medium for B. pseudocatenulatum exceeded 1.0 even after 24 hours of culture. Furthermore, the OD values ​​of B. longum and B. adolescentis exceeded 0.6 after 24 hours of culture. On the other hand, when bifidobacteria were cultured using a 4-fold diluted medium, the OD value of the culture medium decreased compared to the undiluted medium, but growth was observed in accordance with the culture time, indicating that this did not affect the growth of the bifidobacteria themselves. Furthermore, it was found that the OD value of the culture medium after 48 hours from the start of culture could be adjusted to a range of 0.2 to 0.3 for all bacterial species.

[0063] [Example 3] 3. Investigation of Liquid Media (3) In this example, three types of liquid media were prepared by mixing known liquid media, and liquid media were prepared by 4-fold dilution (×1 / 4) to carry out a culture test of bifidobacteria shown in Table 6. The circles in the table indicate the bifidobacteria for which a culture test was carried out in the corresponding liquid medium.

[0064]

[0065] From the glycerol stock of each bifidobacterium, each bacterium was streaked onto TOS propionic acid agar medium (product of Yakult Pharmaceutical Co., Ltd.) and cultured at 37°C under anaerobic conditions to form colonies. Colonies of each bacterium were inoculated into mGR liquid medium (stock solution) shown in Table 7 below, BG liquid medium (stock solution) shown in Table 8 below, and TOR liquid medium (stock solution) shown in Table 9 below, and cultured anaerobically at 37°C to obtain preculture solutions.

[0066] The mGR liquid medium stock solution (x1) shown in Table 7 is a liquid medium prepared by mixing the RCM liquid medium used in Example 1 with mGAM liquid medium (modified GAM bouillon medium, product of Nissui Pharmaceutical Co., Ltd.) in a volume ratio of 50:50. Table 7 shows the composition of the mGR liquid medium obtained by mixing the mGAM liquid medium and the RCM liquid medium.

[0067]

[0068] The BG liquid medium stock solution (x1) shown in Table 8 is a liquid medium prepared by mixing Bryant-Berkey Broth (BB medium, a product of Sigma) and mGAM liquid medium (modified GAM bouillon medium, a product of Nissui Pharmaceutical Co., Ltd.) in a volume ratio of 70:30. Table 8 shows the composition of the BG liquid medium obtained by mixing Bryant-Berkey medium and mGAM liquid medium.

[0069]

[0070] Furthermore, the stock solution of TOR liquid medium (x 1) shown in Table 9 is a liquid medium prepared by removing only the agar from the TOS propionic acid agar medium (a product of Yakult Pharmaceutical Industry Co., Ltd.) used for colony formation from a glycerol stock, and mixing a TOS propionic acid medium (liquid medium) prepared separately for this test with the RCM liquid medium used in Example 1 in a volume ratio of 50:50. Table 9 shows the composition of the TOR liquid medium obtained by mixing the TOS propionic acid medium and the RCM liquid medium.

[0071]

[0072] The OD of each bifidobacterial preculture solution was ６６０ The OD of the culture medium was adjusted to a turbidity of 1 / 4. 50 μL of the prepared pre-culture medium was added to 5 mL of each of the 4-fold diluted (×1 / 4) liquid media shown in Tables 7, 8, and 9, and anaerobic culture was performed at 37° C. The OD of the culture medium was adjusted to 24 hours and 48 hours after the start of culture for the mGR liquid medium and BG liquid medium, and to 24 hours and 65 hours after the start of culture for the TOR liquid medium. ６６０The values ​​were measured. The results are shown in Table 10 below and the graphs in Figures 3(A), (B), and (C). The vertical axis of each graph represents OD, and the horizontal axis represents culture time (h). In Figures 3(A) and (B), the values ​​for B. longum are represented by a solid line and an X-shaped marker, the values ​​for B. pseudocatenulatum by a dotted line and a circle marker, the values ​​for B. breve by a dashed line and a square marker, and the values ​​for B. bifidum by a solid line and a triangle marker. In Figure 3(C), the values ​​for B. longum are represented by a solid line and an X-shaped marker, the values ​​for B. pseudocatenulatum by a dotted line and a circle marker, and the values ​​for B. adolescentis by a solid line and a triangle marker.

[0073]

[0074] From these results, bifidobacteria (OD ６６０ = 1) was inoculated into a liquid medium in an amount of 1% and cultured, it was found that by adjusting the concentrations of each component of mGR liquid medium, BG liquid medium, and TOR liquid medium to 1 / 4 of the concentrations normally used, the OD value of the culture medium after 48 hours of culturing bifidobacteria could be adjusted to 0.35 or less.

[0075] [Example 4] 4. Investigation of Liquid Culture Media (4) In this example, a lactic acid bacteria culture test was carried out using liquid media obtained by diluting the GAM bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) used in Example 2 described above at 2-fold (×½), 4-fold (×¼), and 6-fold (×⅙) dilutions.

[0076] First, a glycerol stock of lactic acid bacteria (Latilactobacillus sakei HS-1; Ravien Sante Co., Ltd.) was streaked onto MRS agar medium (Merck) and cultured under aerobic conditions at room temperature for 3 days to form colonies. The resulting colonies were inoculated into GAM bouillon medium (stock solution) shown in Table 4 and cultured aerobically at 37°C to obtain a preculture solution.

[0077] The OD value of the preculture solution was calculated as OD ６００The turbidity of the culture was adjusted to 1.0, and 50 μL of the prepared pre-culture solution was added to 5 mL of liquid medium diluted 2-fold (×1 / 2), 4-fold (×1 / 4), and 6-fold (×1 / 6) with the original GAM bouillon medium solution, and the culture was anaerobic cultured at 37°C. ６００ The values ​​of the 2-fold diluted (×1 / 2) medium were measured. The results are shown in Table 11 below and Figure 4. The values ​​of the 2-fold diluted (×1 / 2) medium are indicated by a solid line and an X-shaped marker, the values ​​of the 4-fold diluted (×1 / 4) medium are indicated by a dotted line and a circle marker, and the values ​​of the 6-fold diluted (×1 / 6) medium are indicated by a dashed line and a square marker.

[0078]

[0079] From these results, lactic acid bacteria (OD ６００ When lactic acid bacteria (GAM) was inoculated into a liquid medium at a 1% volume and cultured, it was found that when a 2-fold diluted (×1 / 2) medium of GAM liquid medium was used, the OD value of the culture solution exceeded 0.5 after 24 hours of culture. On the other hand, when lactic acid bacteria were cultured using a 4-fold diluted medium and a 6-fold diluted medium, the OD value of the culture solution decreased compared to a 2-fold diluted medium, but growth was observed in accordance with the culture time, indicating that increasing the dilution ratio did not affect the growth of the lactic acid bacteria themselves. Furthermore, it was found that the OD value of the culture solution after 48 hours of culture could be adjusted to a range of 0.2 to 0.35 by using a 4-fold diluted medium and a 6-fold diluted medium.

[0080] [Example 5] 5. Investigation of Liquid Media (5) In this example, two types of liquid media were prepared by mixing known liquid media, and liquid media were prepared as an undiluted solution (×1) and a 4-fold diluted solution (×1 / 4), and culture tests were carried out on the following two types of lactic acid bacteria: Lacticaseibacillus paracasei YK130220 (Ravien Sante Co., Ltd.) and Latilactobacillus sakei HS-1 (Ravien Sante Co., Ltd.).

[0081] Glycerol stocks of each lactic acid bacterium were streaked onto MRS agar medium (Merck) and cultured under aerobic conditions at room temperature for 3 days to form colonies. The resulting colonies of each bacterium were inoculated into mGR liquid medium (stock solution) shown in Table 7 and BG liquid medium (stock solution) shown in Table 8, respectively, and cultured aerobically at 37°C to obtain preculture solutions.

[0082] The OD value of the preculture solution was calculated as OD ６００ The turbidity was adjusted to 1.0, and 50 μL of the adjusted culture solution was added to 5 mL of the liquid medium containing the undiluted mGR medium (×1) and its 4-fold diluted (×1 / 4) shown in Table 7, and 5 mL of the liquid medium containing the undiluted BG medium (×1) and its 4-fold diluted (×1 / 4) shown in Table 8, respectively, and anaerobically cultured at 37 ° C. The OD values ​​of the culture solutions were measured 24 hours and 48 hours after the start of culture. The results are shown in Table 12 and Figure 5 below. In the graph of Figure 5, the X-shaped marker indicates the value for L. sakei, the solid line indicates the value for the undiluted solution (×1), and the dotted line indicates the value for the 4-fold diluted (×1 / 4). The square marker indicates the value for L. paracasei, the solid line indicates the value for the undiluted solution (×1), and the dotted and open square marker indicates the value for the 4-fold diluted (×1 / 4).

[0083]

[0084] These results showed that when lactic acid bacteria were inoculated into a liquid medium at a volume of 1% and cultured, the OD value of the culture solution exceeded 1 after 24 hours of culture if the undiluted mGR medium (x1) was used. It was also found that the OD value of the culture solution reached nearly 0.5 after 24 hours of culture if the undiluted BG medium (x1), which tends to be more difficult to grow than the mGR medium, was used. On the other hand, it was found that by using a 4-fold diluted liquid medium, the OD value of the culture solution after 48 hours of culture could be adjusted to a range of 0.25±0.1 for all culture solutions.

[0085] Example 6 6. Liquid Medium Study (6) In this example, a lactic acid bacteria culture test was performed using stock and diluted MRS liquid medium (a product of Merck) at concentrations commonly used. First, a glycerol stock of lactic acid bacteria (Lacticaseibacillus paracasei YK130220; Ravien Sante Co., Ltd.) was streaked onto MRS agar medium (a product of Merck) and cultured under aerobic conditions at room temperature for 3 days to form colonies. The resulting colonies were inoculated into the MRS liquid medium (stock solution) shown in Table 13 below and cultured aerobically at 37°C to obtain a preculture solution.

[0086]

[0087] The OD value of the preculture solution was calculated as OD ６００ The turbidity of the culture was adjusted to 1.0, and 50 μL of the prepared pre-culture solution was added to 5 mL of the undiluted (×1) or 2- to 5-fold diluted (×1 / 2 to ×1 / 5) MRS liquid medium shown in Table 13, and aerobic culture was carried out at 37°C. ６００ The values ​​of the undiluted (x1) liquid medium were measured. The results are shown in Figure 6. The values ​​of the undiluted (x1) liquid medium are indicated by a solid line and an X-shaped marker, the values ​​of the 2-fold diluted (x1 / 2) medium by a dotted line and a circle marker, the values ​​of the 3-fold diluted (x1 / 3) medium by a dashed line and a square marker, the values ​​of the 4-fold diluted (x1 / 4) medium by a solid line and a triangle marker, and the values ​​of the 5-fold diluted (x1 / 5) medium by a dotted line and a diamond marker.

[0088] From these results, it was found that the lactic acid bacteria (L. paracasei) was added at a concentration of 1% (OD ６００ = 1.0) and cultured, the OD of the culture medium reached 1.0 in about 2 days when the undiluted MRS liquid medium (×1) was used. ６００ It was found that the OD value increased to approximately 2. In addition, when diluted MRS liquid medium was used, the OD ６００ Although the value slightly decreased, the OD of the culture solution after 48 hours of culture was ６００It was found that the value did not fall below 0.5 and exceeded 1. The reason for this is that the MRS medium contains a higher amount of glucose in its constituent components than other media, at 20 g / L in the undiluted solution, and even in a 5-fold diluted medium, the glucose content is still 4 g / L, suggesting that this promotes active bacterial growth. For these reasons, it is preferable to use a liquid medium for anaerobic culture in the present invention that contains approximately 2 to 5 g of glucose per liter at a commonly used concentration, and it is preferable to use such a medium diluted 3 to 6 times.

[0089] Example 7 7. Co-culture of human fecal sample and probiotics In this example, a human fecal sample and probiotics were co-cultured in a dilution medium, and the OD of the culture solution was measured.

[0090] [Pre-culture of probiotics] The probiotics used in this example were the following four strains: Lactic acid bacteria: Lacticaseibacillus paracasei YK130220 (Ravien Sante Co., Ltd.) Lactic acid bacteria: Latilactobacillus sakei HS-1 (Ravien Sante Co., Ltd.) Butyric acid-producing bacteria: Clostridium butyricum NBRC 13949 Butyric acid-producing bacteria: Clostridium butyricum KO (Ravien Sante Co., Ltd.)

[0091] For the lactic acid bacteria among the probiotics, the glycerol stock of each lactic acid bacterium was scraped with a platinum loop and inoculated into 4 mL of MRS liquid medium (stock solution, product of Merck) shown in Table 13, followed by pre-preculture under aerobic conditions at 25°C for 3 days. 40 μL of this pre-preculture was inoculated into 4 mL of MRS liquid medium (stock solution) and cultured aerobically at 37°C for 17 hours to obtain a preculture solution.

[0092] For the butyric acid-producing bacteria among the probiotics, the glycerol stock of each butyric acid-producing bacterium was scraped with a platinum loop and inoculated into 4 mL of RCM liquid medium (stock solution, product of Becton Dickinson) shown in Table 2, and pre-preculture was carried out under anaerobic conditions at 35°C for 3 days. 40 μL of this pre-preculture was inoculated into 4 mL of RCM liquid medium (stock solution), and anaerobically cultured at 35°C for 16 hours to obtain a pre-culture solution.

[0093] [Preparation of Fecal Supernatant Samples] Seven healthy Japanese women in their twenties who were not taking antibiotics provided feces. Fecal supernatant samples were prepared within 24 hours and used for co-culture. The Amies transport medium shown in Table 14 below was used for transporting the feces. The feces and Amies transport medium were placed in a collection container, the lid was closed, and the container was sealed with an Anaero Pouch (manufactured by Mitsubishi Gas Chemical Company, Inc.) and transported at room temperature while maintaining anaerobic conditions. The fecal supernatant samples were prepared as follows: PBS buffer was added to the Amies transport medium to a volume 100 times the weight of the feces contained in the Amies transport medium, and the feces were diluted 100-fold. After vortexing well, the mixture was allowed to stand for 5 minutes, and the supernatant containing suspended enterobacteria was collected, taking care to avoid sediment. This was used as the fecal supernatant sample.

[0094]

[0095] [Co-culture of fecal supernatant samples and probiotics] The pre-culture solution of each probiotic was cultured at OD ６0０ The OD of the culture solution was adjusted to a turbidity of 0.05% (OD = 1), and 50 μL of the prepared pre-culture solution was added to 5 mL of a 4-fold diluted (×1 / 4) liquid medium of the GAM bouillon medium shown in Table 4. 50 μL of each prepared fecal supernatant sample was added to the liquid medium, and anaerobically cultured at 35°C for 48 hours. ６００ The results are shown in Table 15 below.

[0096]

[0097] These results revealed that by using a medium obtained by diluting GAM bouillon medium 4 times, when a fecal supernatant sample and various probiotics were inoculated and co-cultured, the OD value of the culture medium after 48 hours of culture could be adjusted to approximately 1. This showed that the viable bacteria or probiotics in the feces did not overgrow in the culture medium and become saturated, making it possible to evaluate the effect of the added probiotics in promoting the growth of useful intestinal bacteria.

[0098] Example 8 8. Evaluation of the Growth State of Useful Intestinal Bacteria in the Co-culture Solution of Fecal Supernatant Samples and Probiotics In this example, the abundance of butyric acid-producing bacteria (bacteria belonging to Clostridium cluster IV or Clostridium cluster XIVa) present in the co-culture solution of fecal supernatant samples and probiotics obtained in Example 7 above was quantified by real-time PCR. The target genes selected for the real-time PCR primers were the Erec gene specific to Clostridium cluster XIVa and the Clep gene specific to Clostridium cluster IV. The sequences of the PCR primers used are shown in Table 16 below, and examples of useful intestinal bacteria that can be detected by each target gene are shown in Table 17 below.

[0099]

[0100]

[0101] [DNA Extraction from Co-culture Solution] 4 mL of the co-culture solution of the fecal supernatant sample and probiotics obtained in Example 7 above was centrifuged (16,000 × g, room temperature, 5 minutes) to separate the supernatant and precipitate. DNA was extracted from the precipitate. DNA extraction was performed using a DNA extraction kit (ZymoBIOMICS DNA Miniprep Kit, Zymo Research) according to the attached protocol. The amount of extracted DNA was measured using a DNA quantification kit (Qubit dsDNA Quantification Assay Kit, Thermo Fisher Scientific).

[0102] [Real-time PCR reaction] Real-time PCR was performed using a SYBR (registered trademark) Green I detection system assay. A master mix reagent for real-time PCR (KOD SYBR (registered trademark) qPCR Mix, Toyobo Co., Ltd.) was used, and real-time PCR was performed according to the attached instruction manual. The real-time PCR device used was the Thermal Cycler Dice (registered trademark) Real Time System II, Takara Bio Inc.). A 25 μL reaction system was performed using 1 μL of DNA extracted from the co-culture solution as described above. DNA (1.0 μL) was mixed with KOD SYBR qPCR Mix (12.5 μL), PCR forward primer (10 μM; 0.5 μL), PCR reverse primer (10 μM; 0.5 μL), dH ２ 0 (10.5 μL) was added, and real-time PCR was performed to perform relative quantification of the target gene. The PCR reaction conditions were: 2 minutes at 98°C for polymerase activation, followed by 40 sets of [98°C for 10 seconds → 55°C or 60°C for 10 seconds → 68°C for 45 seconds], followed by 95°C for 15 seconds → 60°C for 30 seconds → 95°C for 15 seconds for the melting curve. The 16S rRNA gene was used as an internal standard to correct for DNA content between samples. After real-time PCR, the amplified product was subjected to agarose gel electrophoresis to confirm whether a band of the desired size was obtained. As a result, it was confirmed that the size of the band of the amplified product obtained by real-time PCR using the Erec and Clep primers shown in Table 16 matched the length of the target amplified product.

[0103] The proliferation of beneficial intestinal bacteria obtained after co-culturing fecal supernatant samples with various probiotics was evaluated as a multiplication factor, relative to the relative value of beneficial intestinal bacteria when only fecal supernatant samples were cultured (Control), which was set at 1. The results are shown in Figures 7 and 8. The error bars in each graph represent SEM (n = 3), and "*" indicates p < 0.05 in the Tukey test. According to these results, in subject No. 1, co-culturing fecal supernatant samples with L. sakei or C. butyricum NBRC, which has low butyrate-producing ability, significantly increased butyrate-producing bacteria possessing the Clep gene of Clostridium cluster IV. In subject No. 2, no increase in butyrate-producing bacteria was observed when co-culturing any of the probiotics tested. In subject No. 3, co-culturing fecal supernatant samples with L. paracasei, L. sakei, or C. By co-culturing with C. butyricum NBRC, an increase in butyrate-producing bacteria having the Clep gene of Clostridium cluster IV was observed. In subject No. 4, the addition of L. paracasei or L. sakei resulted in a significant increase in butyrate-producing bacteria having the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria having the Clep gene of Clostridium cluster IV. In subject No. 5, by co-culturing the fecal supernatant sample with L. paracasei, a significant increase in butyrate-producing bacteria having the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria having the Clep gene of Clostridium cluster IV was observed. Furthermore, by co-culturing with C. butyricum NBRC, a significant increase in butyrate-producing bacteria having the Erec gene was observed. Furthermore, L. Although no significant difference was observed when co-culturing with L. sakei, an increase in butyrate-producing bacteria having the Clep gene was observed. In subject No. 6, co-culturing the fecal supernatant sample with L. sakei resulted in a significant increase in butyrate-producing bacteria having the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria having the Clep gene of Clostridium cluster IV. In subject No. 7, co-culturing the fecal supernatant sample with L. paracasei resulted in an increase in butyrate-producing bacteria having the Clep gene. Furthermore, the addition of L. sakei resulted in a significant increase in butyrate-producing bacteria having the Erec gene.In none of the subjects was co-cultured with C. butyricum KO, which has strong butyrate-producing ability, an increase in beneficial intestinal bacteria was observed. Thus, it was revealed that the probiotics that increase beneficial intestinal bacteria such as butyrate-producing bacteria possessed by each subject vary from person to person, and that the evaluation method of the present invention can select probiotics suitable for the growth of beneficial intestinal bacteria possessed by each individual.

[0104] Example 9 9. Bacterial species analysis of useful intestinal bacteria in the co-culture solution of fecal supernatant sample and probiotics In this example, the butyric acid-producing bacteria (bacterial group belonging to Clostridium cluster IV or Clostridium cluster XIVa) present in the co-culture solution of fecal supernatant sample and probiotics obtained in Example 7 above were analyzed at the bacterial species level using a next-generation sequencer.

[0105] For the primers used in the real-time PCR of Example 8, in order to enable analysis using a next-generation sequencer ION PGM (Thermo Fisher Scientific), multiple forward primers with a barcode sequence attached to the A1 adapter to identify each sample and multiple reverse primers with a P1 adapter sequence attached were designed and synthesized. The expected PCR product size when using each primer was approximately 400 bp. PCR was performed using these primers and the extracted DNA obtained in Example 8. The PCR reaction was performed using a PCR reagent kit (Platinum PCR SuperMix High Fidelity, Thermo Fisher Scientific). The reaction conditions were: 94°C for 2 minutes to activate the polymerase, followed by 35 cycles of [94°C for 30 seconds → 55°C for 30 seconds → 68°C for 30 seconds]. The amplified product was subjected to agarose gel electrophoresis, and the band of the desired size was recovered from the gel and purified. The recovered DNA was processed according to the protocol of the next-generation sequencer ION PGM and analyzed using the next-generation sequencer ION PGM. Based on the obtained sequence results, de novo assembly was performed using sequence information multi-analysis software (Geneious, Tommy Digital Biology Co., Ltd.), and identification at the bacterial species level was performed using BLAST search. The analysis results for the butyric acid-producing bacterium (Clostridium cluster IV) containing the Clep gene of subject No. 1 are shown in Figure 9.

[0106] According to these results, in the control (fecal supernatant sample cultured only), the presence of non-butyric acid-producing bacteria, including Caproicproducens galactitolivorans, was observed, but by co-culturing the fecal supernatant sample with L. sakei, the proportion of non-butyric acid-producing bacteria decreased, and instead, a significant increase in the butyric acid-producing bacterium Faecalis prausnitzii was observed, and an increase in the butyric acid-producing bacterium Butyricicoccus faecihominis was also confirmed. Thus, it was confirmed that by co-culturing a fecal supernatant sample with various probiotics and performing next-generation sequencer analysis using DNA extracted from this co-culture solution, it is possible to identify the proliferated useful intestinal bacteria at the bacterial species level.

[0107] [Example 10] 10. Culturing of human fecal samples and prebiotics In this example, a human fecal sample and various prebiotics were co-cultured in a dilution medium to obtain a culture solution. The following six types of prebiotics were used in this example: Galactooligosaccharides (hereinafter also referred to as "GOS"), a product of Fujifilm Wako Pure Chemical Industries, Ltd. Fructooligosaccharides (hereinafter also referred to as "FOS"), a product of CanHeart Inc. Xylooligosaccharides (hereinafter also referred to as "XOS"), a product of Bussan Food Science Co., Ltd. Milk oligosaccharides (hereinafter also referred to as "Milk"), a product of Nichie Co., Ltd. Inulin, a product of Nippon Garlic Co., Ltd. Kestose, a product of Bussan Food Science Co., Ltd.

[0108] [Preparation of Fecal Supernatant Samples] Feces were provided by two healthy Japanese women in their twenties who were not taking antibiotics. Fecal supernatant samples were prepared within 24 hours and used in the culture test. The Amies transport medium shown in Table 14 was used for transporting the feces. The feces and Amies transport medium were placed in a stool collection container, the lid was closed, and the container was sealed with an Anaero Pouch (manufactured by Mitsubishi Gas Chemical Company, Inc.) and transported at room temperature while maintaining anaerobic conditions. The fecal samples were prepared as follows: PBS was added to the Amies transport medium to a volume 100 times the weight of the feces contained in the Amies transport medium, and the feces were diluted 100-fold. After vortexing well, the mixture was allowed to stand for 5 minutes, and the supernatant containing suspended enterobacteria was collected, taking care to avoid sediment. This was used as the fecal supernatant sample.

[0109] [Cultivation of fecal supernatant samples and prebiotics] 100 μL of the fecal supernatant sample prepared as described above was added to 10 mL of a 4-fold diluted (×1 / 4) liquid medium of the BG medium shown in Table 8. A filter-sterilized 10x stock solution of each prebiotic was aseptically added to the liquid medium to achieve the desired concentration, and the mixture was anaerobic cultured at 37°C for 48 hours. The final concentrations of each prebiotic in the 4x diluted BG medium were as follows: galactooligosaccharide (GOS): 0.1%, fructooligosaccharide (FOS): 0.3%, xylooligosaccharide (XOS): 0.1%, milk oligosaccharide (Milk): 0.1%, inulin: 0.5%, kestose: 0.2%. The final concentrations of each prebiotic were determined based on the recommended daily intake when consumed as a food.

[0110] [Evaluation of the growth state of useful intestinal bacteria] Next, the amount of bifidobacteria present in the culture solution after 48 hours of culture was quantified by real-time PCR. The real-time PCR primer used was the g-Bifid primer, which is a bifidobacteria-specific primer. The sequences of the PCR primers used are shown in Table 18 below, and the bifidobacteria that could be detected are shown in Table 19 below.

[0111]

[0112]

[0113] [DNA extraction from culture medium] 6 mL of culture medium was centrifuged (16,000 × g, room temperature, 5 minutes) to separate the supernatant and precipitate. DNA was extracted from the precipitate. DNA was extracted using a DNA extraction kit (ZymoBIOMICS DNA Miniprep Kit, Zymo Research) according to the attached protocol. The amount of extracted DNA was measured using a DNA quantification kit (Qubit dsDNA Quantification Assay Kit, Thermo Fisher Scientific).

[0114] [Real-time PCR reaction] Real-time PCR was performed using a SYBR (registered trademark) Green I detection system assay. A master mix reagent for real-time PCR (KOD SYBR (registered trademark) qPCR Mix, Toyobo Co., Ltd.) was used, and real-time PCR was performed according to the attached instruction manual. The real-time PCR device used was the Thermal Cycler Dice (registered trademark) Real Time System II, Takara Bio Inc.). A 25 μL reaction system was performed using 1 μL of DNA extracted from the co-culture solution as described above. DNA (1.0 μL) was mixed with KOD SYBR qPCR Mix (12.5 μL), PCR forward primer (10 μM; 0.5 μL), PCR reverse primer (10 μM; 0.5 μL), dH ２ 0 (10.5 μL) was added, real-time PCR was performed, and relative quantification of the target gene was performed. The PCR reaction conditions were: 2 minutes at 98 ° C for polymerase activation, followed by 40 sets of [98 ° C for 10 seconds → 55 ° C for 10 seconds → 68 ° C for 45 seconds], followed by 95 ° C for 15 seconds → 60 ° C for 30 seconds → 95 ° C for 15 seconds for the melting curve. In order to correct the amount of DNA between samples, the 16S rRNA gene was used as an internal standard. In addition, after performing real-time PCR, the amplified product was subjected to agarose gel electrophoresis to confirm whether a band of the desired size was obtained. As a result, it was confirmed that the size of the band of the amplified product obtained by real-time PCR using the g-Bifid primers shown in Table 18 matched the length of the desired amplified product.

[0115] The proliferation of bifidobacteria in cultures cultured with various prebiotics added to fecal supernatant samples was evaluated as a multiplication factor, with the relative number of bifidobacteria when fecal supernatant samples were cultured without prebiotics (Control) being set at 1. The results are shown in Figure 10. The error bars in each graph represent SEM (n = 3), and "**" indicates p < 0.05 in the Games-Howell test. According to these results, in subject A, a significant increase in bifidobacteria was observed by culturing fecal supernatant samples with the addition of xylooligosaccharides (XOS) or milk oligosaccharides (Milk). In subject B, a significant increase in bifidobacteria was observed by adding inulin, kestose, milk oligosaccharides (Milk), galactooligosaccharides (GOS), or xylooligosaccharides (XOS). Thus, it became clear that the prebiotic oligosaccharides that increase beneficial intestinal bacteria such as bifidobacteria possessed by each subject vary from person to person, and that the evaluation method according to the present invention makes it possible to select prebiotics that are suited to each individual.

[0116] The probiotics or prebiotics screening method and the screening medium used therein according to the present invention enable the selection of probiotics or prebiotics suited to an individual and the evaluation of their effectiveness, and are therefore useful for improving and enhancing the health status of individuals, and can be used in fields related to healthcare, medicine, functional foods, pharmaceuticals, etc.

Claims

1. A method for screening for probiotics or prebiotics that promote the growth of useful intestinal bacteria present in the intestines of a subject or test animal, comprising the steps of adding a fecal sample collected from the subject or test animal and a candidate probiotic or candidate prebiotic to a liquid medium and anaerobic culturing the sample and the candidate probiotic or candidate prebiotic, and evaluating the growth state of the useful intestinal bacteria in the liquid medium after anaerobic culturing, wherein the liquid medium used for anaerobic culturing is a medium prepared so that the concentrations of the components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in culturing the useful intestinal bacteria.

2. The screening method according to claim 1, characterized in that the liquid medium is a medium selected from the group consisting of Reinforced Clostridia (RCM) medium, GAM bouillon medium, modified GAM (mGAM) bouillon medium, Bryant-Berkey (BB) medium, TOS propionic acid medium, and a mixture thereof, and is prepared by diluting the selected medium 3 to 6 times so that the concentration of the components of the liquid medium is 1 / 3 to 1 / 6 of the concentration normally used in culturing the useful enterobacteria.

3. The screening method according to claim 1 or 2, characterized in that the useful intestinal bacteria are butyric acid-producing bacteria or Bifidobacterium bacteria.

4. The screening method according to claim 3, characterized in that the butyric acid-producing bacterium is a bacterium belonging to Clostridium cluster IV or Clostridium cluster XIVa.

5. The screening method according to claim 3, characterized in that the growth state of the useful enterobacteria in the liquid medium after the anaerobic culture is evaluated by quantitative PCR using primers specific to the target gene, the Erec gene or the Clep gene of butyric acid-producing bacteria as the target gene.

6. The screening method according to claim 3, characterized in that the growth state of the useful enterobacteria in the liquid medium after the anaerobic culture is evaluated by quantitative PCR using the 16S rRNA gene of Bifidobacterium bacteria as a target gene and g-Bifid primers specific to the target gene.

7. The screening method described in claim 1 or 2, characterized in that the evaluation of the growth state of the useful intestinal bacteria in the liquid medium after the anaerobic culture is performed by detecting the number of bacteria of the useful intestinal bacteria in the liquid medium after the anaerobic culture and the number of bacteria of the useful intestinal bacteria in the liquid medium after the fecal sample is anaerobically cultured in the liquid medium without adding the candidate probiotic or the candidate prebiotic, and comparing the two.

8. The screening method according to claim 1 or 2, characterized in that the probiotics are lactic acid bacteria or butyric acid-producing bacteria, and the prebiotics are oligosaccharides or dietary fiber.

9. A liquid medium used in the anaerobic culture step of the screening method described in claim 1, characterized in that the concentrations of the components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in the culture of the useful enterobacteria.

10. The screening medium according to claim 9, characterized in that the liquid medium is a medium selected from the group consisting of Reinforced Clostridia (RCM) medium, GAM bouillon medium, modified GAM (mGAM) bouillon medium, Bryant-Berkey (BB) medium, TOS propionic acid medium and a mixture thereof, and the concentrations of the components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations normally used in culturing the useful enterobacteria.

Images