Screening method for probiotics or prebiotics and screening medium used therefor

Abstract

To provide a method for screening probiotics and prebiotics that promote the growth of useful intestinal bacteria present in the intestines of individuals through simple and safe in vitro testing.SOLUTION: 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 a test animal, and includes the steps of: adding a fecal sample collected from the subject or the test animal and a candidate probiotic or candidate prebiotic to a liquid medium and anaerobic culturing the fecal sample; and evaluating a growth state of the useful intestinal bacteria in the liquid medium after anaerobic culturing. The liquid medium used for anaerobic culturing is a medium prepared so that the concentrations of components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations usually used in culturing useful intestinal bacteria.SELECTED DRAWING: None

Description

[Technical field]

[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. [Background technology]

[0002] It is known that the human intestinal flora differs from person to person due to dietary habits, lifestyle, stress, history of antibiotic use, etc. It is also known that health conditions can be improved or enhanced by adjusting the intestinal environment. For this reason, useful intestinal bacteria that are beneficial to 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 medicines. When these microorganisms or their metabolic substances are orally ingested, they reach the intestines and produce lactic acid and acetic acid. This lowers the pH level in the intestines, suppressing the growth of bad bacteria and activating the activity of lactic acid bacteria and bifidobacteria present in the host's intestines.

[0003] In recent years, so-called "butyrate-producing bacteria" that have the property of producing butyrate have been attracting attention as a further useful intestinal bacterium. Clostridium butyricum is well-known as a butyrate-producing bacterium, but other butyrate-producing bacteria belonging to various genera such as Agatho- bacterium, Anaerobutyricum, Anaerostipes, Coprococcus, Faecalibacterium, and Roseburia are known. Butyrate produced by butyrate-producing bacteria is not only used as the main energy source for colonic epithelial cells, but also plays a role in regulating the energy metabolism and immune systems and maintaining homeostasis through short-chain fatty acid receptors. Recently, it has been found that butyrate acts to enhance the host's immunity by acting on the intestinal immune system to promote IgA secretion, and also has the function of rapidly 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 other products. Summary of the Invention [Problem to be solved by the invention]

[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, due to the negative feedback caused by the excess butyrate produced by the bacteria.

[0005] Therefore, in order to enhance butyric acid production by butyric acid-producing bacteria present in the intestine of the host, probiotics or prebiotics that can promote the proliferation of butyric acid-producing bacteria are required. However, since the intestinal flora is different for each individual, the butyric acid-producing bacteria possessed by each individual are also different, and the probiotics or prebiotics that can promote the proliferation are also different for each individual. In such a case, a possible screening method is an in vivo test in which a subject orally ingests candidate probiotic and prebiotic materials for a certain period of time and examines the change in the intestinal environment of the subject before and after ingestion. However, in this method, if a test is performed for each type and dose of candidate material, in addition to the complicated ethical review procedures, the test takes time and places a heavy burden on the subject, and there are problems from the viewpoint of safety, making it extremely difficult to perform the test.

[0006] Therefore, the present invention has been made in consideration of the above-mentioned 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 each individual 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. [Means for solving the problem]

[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 and culturing the fecal sample in vitro in bulk, 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 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 a step 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 a step of evaluating the growth state of the useful intestinal bacteria in the liquid medium after anaerobic culturing, and the liquid medium used for anaerobic culturing is a medium prepared so that the concentration of the components of the liquid medium is 1 / 3 to 1 / 6 of the concentration usually used in culturing useful intestinal bacteria. This allows the useful intestinal bacteria to grow in the liquid medium due to the added probiotics or prebiotics during anaerobic culturing, and allows the fecal-derived live bacteria to grow slowly while the useful intestinal bacteria grow in the liquid medium, so that it is possible to evaluate the growth-promoting effect of the added candidate material on the 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, for example, the concentration of each component indicated in the recipe of a commercially available medium available on the market or a conventionally known medium.

[0010] The liquid medium 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 preferably a medium prepared by diluting the 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 usually used in the cultivation of useful enterobacteria. In this way, a medium that can be suitably and easily prepared as a liquid medium used for anaerobic cultivation and that can also be suitably used for screening is selected.

[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 are evaluated for the growth-promoting effect of a candidate probiotic or prebiotic.

[0012] It is also preferred that the useful enterobacteria 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 enterobacteria, making it possible to efficiently evaluate the growth-promoting effect of candidate probiotics or prebiotics on butyric acid-producing bacteria.

[0013] In addition, 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, thereby selecting 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] In addition, 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 the 16S rRNA gene of Bifidobacterium bacteria as a target gene and g-Bifid primers specific to this target gene. This allows 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 enterobacteria.

[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 the liquid medium without adding a candidate probiotic or prebiotic, and comparing the two. This allows the growth-promoting effect of the candidate probiotic or prebiotic on useful intestinal bacteria to be easily evaluated.

[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 the probiotics or prebiotics.

[0017] The screening medium of the present invention is a liquid medium used in the anaerobic culture step in the above-mentioned screening method, and the concentrations of the components of this liquid medium are 1 / 3 to 1 / 6 of the concentrations usually used in the culture of useful enterobacteria. This allows the useful enterobacteria to grow in the liquid medium due to the added probiotics or prebiotics while allowing the fecal-derived live bacteria to grow slowly during anaerobic culture, without the liquid medium being immediately saturated with fecal-derived live bacteria, making it possible to evaluate the growth-promoting effect of the added candidate material on useful enterobacteria.

[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 concentration of the components of this liquid medium is 1 / 3 to 1 / 6 of the concentration usually 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 used for anaerobic cultivation and that can also be suitably used for screening. Effect of the Invention

[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 the beneficial intestinal bacteria present in the intestines of each individual subject can be evaluated individually. (2) Since the test is conducted using a stool sample provided by the subject, the burden on the subject is reduced, and the screening test can be conducted safely and easily. (3) When evaluating the growth state of useful enterobacteria in liquid medium after anaerobic cultivation, it is also possible to identify the species of useful enterobacteria by performing analysis using next-generation sequencers or real-time PCR, etc. [Brief description of the drawings]

[0020] [Figure 1] 1 is a graph showing the measurement results of the turbidity (OD value) of the culture medium when various types of bifidobacteria were cultured using the RCM medium and its diluted medium in Example 1. [Diagram 2] 1 is a graph showing the measurement results of the turbidity (OD value) of the culture medium when various types of bifidobacteria were cultured using the GAM bouillon medium and its diluted medium in Example 2. [Diagram 3] 1 is a graph showing the measurement results of the turbidity (OD value) of the culture medium when various bifidobacteria were cultured using a 4-fold diluted mGR medium (a mixed medium of mGAM:RCM=50:50), a 4-fold diluted BG medium (a mixed medium of BB:mGAM=70:30), and a 4-fold diluted TOR medium (a mixed medium of TOS:RCM=50:50) in Example 3. [Figure 4] 1 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when lactic acid bacteria (Latilactobacillus sakei) was cultured using a diluted medium of the GAM bouillon medium in Example 4. [Diagram 5]1 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. [Figure 6] 1 is a graph showing the measurement results of the turbidity (OD value) of the culture solution when lactic acid bacteria (Lacticaseibacillus paracasei) was cultured using the MRS medium and its diluted medium in Example 6. [Figure 7] 1 is a graph showing the results of quantitative PCR of butyric acid-producing bacteria in culture media obtained by co-culturing fecal supernatant samples from subjects No. 1 to No. 4 and candidate probiotics in Example 8. [Figure 8] 1 is a graph showing the results of quantitative PCR of butyric acid-producing bacteria in culture media obtained by co-culturing fecal supernatant samples from subjects No. 5 to No. 7 and candidate probiotics in Example 8. [Figure 9] FIG. 13 is a graph showing the relative quantification results of useful intestinal bacterial species in a culture medium (control) in which only a fecal supernatant sample from subject No. 1 was cultured, and in a culture medium in which a fecal supernatant sample was co-cultured with lactic acid bacteria (Latilactobacillus sakei) in Example 9, using a next-generation sequencer. [Figure 10] 1 is a graph showing the results of quantitative PCR of bifidobacteria in the culture medium after adding candidate prebiotics to fecal supernatant samples from subjects A and B in Example 10 and culturing them. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 intestine of a subject or test animal.

[0022] In the present invention, probiotics generally refers to microorganisms that improve the balance of intestinal flora and are beneficial to 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 Lactocaceibacillus (Lactobacillus), Latilactobacillus (Lactobacillus), Lactococcus, Enterococcus, Streptococcus, Pediococcus, etc. In addition, 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. In addition, examples of saccharifying bacteria include Bacillus subtilis, such as Bacillus subtilis, such as Bacillus subtilis.

[0023] In the present invention, prebiotics generally refers to a substance that selectively grows useful intestinal bacteria, and more specifically refers to a substance that is not decomposed or absorbed in the digestive tract, but serves as a selective nutrient source for useful 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, raffinose, etc. Examples of dietary fiber include inulin, polydextrose, indigestible dextrin, guar gum, etc.

[0024] In the present invention, the intestinal flora refers to the bacteria present in the large intestine of the host, and the 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, and any bacteria that contributes to improving or enhancing the health of the host or maintaining a good health condition can be selected as useful intestinal bacteria. Therefore, in the present invention, the useful intestinal bacteria does not mean only one type of bacteria belonging to a specific species, but also includes a concept that 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 a test animal and a candidate probiotic or a candidate prebiotic are added to a liquid medium and cultured anaerobically, and an evaluation step in which the growth state of useful intestinal bacteria in the liquid medium after anaerobic culture is evaluated. Of these steps, the anaerobic culturing step will be described in detail below.

[0026] [Anaerobic culture process - fecal sample] In this step, first, feces collected from a subject or test animal is prepared. The collected feces is preferably preserved anaerobically until the start of the anaerobic culture test, for example, by using a predetermined preservation solution so that the intestinal bacterial flora in the feces does not change. As the preservation solution, for example, Amies transport medium used in the examples described below can be used. In addition, a commercially available feces collection kit (feces collection kit for bacteria 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, a preservation solution used for preserving the feces, etc., and then allowed to stand to separate into a supernatant and a precipitate, and the supernatant portion in which the feces-derived enterobacteria float is collected and used for anaerobic culture. By adding fecal supernatant to a liquid medium as a fecal sample and performing anaerobic culture, the turbidity of the culture solution can be grasped as the level of bacterial growth, making it easy to grasp whether the anaerobic culture process is progressing smoothly. When preparing a fecal supernatant sample from feces, it is preferable to suspend the feces in a liquid having a weight of 10 to 1000 times the weight of the feces, more preferably in a liquid having a weight of 30 to 300 times the weight of the feces, and even more preferably in a liquid having a weight of 50 to 200 times the weight of the feces. Examples of the liquid to be suspended include buffer solutions such as PBS buffer, physiological saline, a preservation solution used for preserving the feces, or a mixture thereof. After dispersing and suspending the feces in these liquids, a fecal supernatant in which the enterobacteria are suspended is obtained by leaving it 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 solids as it is or in a state of suspension in a preservation liquid, etc. to a liquid medium and perform anaerobic culture.

[0028] [Anaerobic culture process - Liquid medium (screening medium)] In addition, in this step, the above-mentioned fecal sample and the candidate probiotic or prebiotic material are added to a liquid medium and cultured anaerobically, but it is very important to select the type of liquid medium to be used for this anaerobic culture. This is because when a fecal sample is cultured in a liquid medium that is normally used to culture useful intestinal bacteria, the bacterial growth of fecal-derived live bacteria is very active, and the liquid medium is quickly saturated with fecal-derived live bacteria. Therefore, even if a fecal sample and a candidate probiotic or prebiotic material are added to a liquid medium of a normally used concentration and cultured, there may be no room for useful 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 fast, making it difficult to clearly evaluate the growth-promoting effect of the candidate material on useful intestinal bacteria, such as 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 in which the concentration of the components of the liquid medium is 1 / 3 to 1 / 6 (33% to 17% concentration) of the concentration usually used in the culture of useful enterobacteria. Of these, it is more preferable to use a medium prepared to have a concentration of 1 / 3 to 1 / 5 (33% to 20% concentration) of the concentration usually used in the culture of useful enterobacteria, and it is particularly preferable to use a medium prepared to have a concentration of 1 / 4 (25% concentration) of the concentration usually used in the culture of useful enterobacteria. Here, the concentration usually used in the culture of useful enterobacteria refers to the concentration of each component usually used when culturing bacteria selected as useful enterobacteria using that liquid medium, and more specifically, for example, the concentration of each component shown in the formulation of a commercially available medium or a conventionally known medium can be mentioned.

[0029] For example, the amounts of each component in the Reinforced Clostridia (RCM) medium at concentrations usually used in the cultivation of 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 1 L, whereas in the present invention, a medium prepared to have 1 / 3 to 1 / 6 of this concentration is used. Therefore, the amounts of each component in 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 1 L.

[0030] Furthermore, for GAM bouillon medium, the amounts of each component at concentrations typically used in the cultivation of useful enterobacteria are 10 g peptone, 3 g soybean peptone, 10 g proteose peptone, 13.5 g digested serum powder, 2.2 g meat extract, 1.2 g liver extract, 5 g yeast extract, 3 g glucose, 2.5 g potassium dihydrogen phosphate, 3 g sodium chloride, 5 g soluble starch, 0.3 g L-cysteine ​​hydrochloride, and 0.3 g sodium thioglycolate per 1 L, and in the present invention, a medium prepared to have 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 1 L.

[0031] In addition, for the modified GAM (mGAM) medium, the blending amounts of each component at concentrations normally used in the cultivation of useful enterobacteria are 5 g peptone, 3 g soybean peptone, 5 g proteose peptone, 10 g digested serum powder, 2.2 g meat extract, 1.2 g liver extract, 2.5 g yeast extract, 0.5 g glucose, 2.5 g potassium dihydrogen phosphate, 3 g sodium chloride, 5 g soluble starch, 0.3 g L-cysteine ​​hydrochloride, 0.3 g sodium thioglycolate, 0.2 g L-tryptophan, 0.1 g L-arginine, 5 mg vitamin K1, and 10 mg hemin per 1 L, and in the present invention, a medium prepared to have 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 may be, for example, 0.8 to 1.7 g of peptone, 0.5 to 1 g of soybean peptone, 0.8 to 1.7 g of proteose peptone, 1.7 to 3.3 g of digested serum powder, 0.37 g 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 0.027 to 0.033 g of vitamin K1 per 1 L. It is preferable that the amount of erythritol is 0.83 to 1.7 mg and hemin is 1.7 to 3.3 mg.

[0032] In addition, the amounts of each component of Bryant-Berkey (BB) medium at concentrations usually used in the cultivation of useful enterobacteria are 15 g casein peptone, 7.5 g meat extract, 5 g yeast extract, 5 g sodium acetate, 0.5 g L-cysteine ​​hydrochloride, 5 g sodium lactate, and 5 mg resazurin per liter, whereas in the present invention, a medium prepared to have 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, 2.5 to 5 g casein peptone, 1.25 to 2.5 g meat extract, 0.8 to 1.7 g yeast extract, 0.8 to 1.7 g sodium acetate, 0.08 to 0.17 g L-cysteine ​​hydrochloride, 0.8 to 1.7 g sodium lactate, and 0.8 to 1.7 mg resazurin per liter.

[0033] Furthermore, for TOS propionic acid medium, the amounts of each component at concentrations typically used in the cultivation of useful enterobacteria are 10 g peptone, 1 g yeast extract, 4.8 g dipotassium monohydrogen phosphate, 3 g potassium dihydrogen phosphate, 3 g ammonium sulfate, 0.2 g magnesium sulfate heptahydrate, 10.5 g L-cysteine ​​hydrochloride, 15 g sodium propionate, and 10 g galactooligosaccharides per 1 L. In the present invention, a medium prepared to have 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 usually used in the cultivation of useful enterobacteria are 7.5 g peptone, 1.5 g soybean peptone, 2.5 g proteose peptone, 5 g digested serum powder, 6.1 g meat extract, 0.6 g liver extract, 2.75 g yeast extract, 2.75 g glucose, 1.25 g potassium dihydrogen phosphate, 4 g sodium chloride, 1.5 g sodium acetate, 3 g soluble starch, 0.4 g L-cysteine ​​hydrochloride, 0.15 g sodium thioglycolate, 0.1 g L-tryptophan, 0.5 g L-arginine, 0.0025 g vitamin K1, and 0.005 g hemin per 1 L. In the present invention, a medium prepared to have 1 / 3 to 1 / 6 of these concentrations 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, 1.0 to 1.0 g of vitamin K1, and 1.0 to 1.0 g of L-arginine. It is preferable that the amount of niacin is 0.41 to 0.83 mg and that of hemin is 0.8 to 1.7 mg.

[0035] As another example of the mixed medium, 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 will be described. The amounts of each component at concentrations usually used in the cultivation of 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 screening medium used in the present invention contains 0.0015 g, 0.003 g of hemin, 3.5 g of sodium lactate, and 0.0035 g of resazurin, and the medium used in the present invention is prepared to have concentrations of 1 / 3 to 1 / 6 of these concentrations. Therefore, the amounts of each component of the screening medium used in the present invention per 1 L 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, 0.025 to 0.0 g of glucose, and 0.015 to 0.015 g of hemin, 0.003 g of sodium lactate, 3.5 g of sodium lactate, and 0.0035 g of resazurin. Preferably, the total amount of the soluble starch is 0.5 g, 0.125 to 0.25 g of potassium dihydrogen phosphate, 0.15 to 0.3 g of sodium chloride, 0.58 to 1.17 g of sodium acetate, 0.25 to 0.5 g of soluble starch, 0.07 to 0.15 g of L-cysteine ​​hydrochloride, 0.015 to 0.03 g of sodium thioglycolate, 0.01 to 0.02 g of L-tryptophan, 0.05 to 0.1 g of L-arginine, 0.25 to 0.5 mg of vitamin K1, 0.5 to 1 mg of hemin, 0.58 to 1.17 g of sodium lactate, and 0.58 to 1.17 mg of resazurin.

[0036] As another example of the mixed medium, 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 will be described. The amounts of each component at concentrations usually 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 1 L. In the present invention, a medium prepared to have 1 / 3 to 1 / 6 of these concentrations 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 1 L.

[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 mixed medium thereof. It is preferable to use a medium prepared by diluting the above-mentioned medium 3 to 6 times so that the concentration of the components of the above-mentioned medium is 1 / 3 to 1 / 6 of the concentration normally used in the cultivation of useful enterobacteria, it is more preferable to use a medium prepared by diluting the above-mentioned medium 3 to 5 times, and it is particularly preferable to use a medium prepared by diluting the above-mentioned medium 4 times. The mixed medium is not particularly limited, but examples thereof 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 Co., Ltd., the GAM bouillon medium and modified GAM bouillon medium can be a product of Nissui Pharmaceutical Co., Ltd., and the Bryant-Berkey medium can be a product of Sigma Co., Ltd. In addition, since the commercially available TOS propionic acid medium is an agar medium (TOS propionic acid agar medium, a product of Yakult Pharmaceutical Co., Ltd.), it is preferable to prepare a medium having a composition that does not contain only agar.

[0038] Furthermore, when selecting butyric acid-producing bacteria as useful intestinal bacteria and conducting screening tests to evaluate the growth state of butyric acid-producing bacteria using candidate materials, it is particularly preferable to use, as a 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 a 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 culture process - Anaerobic culture] In this step, the fecal sample and the candidate probiotic or prebiotic material are added to the liquid medium and cultured anaerobically. The liquid medium is prepared anaerobically, and the fecal sample and the 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 pre-culture is performed until the turbidity reaches about 1, and the pre-culture liquid 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 the candidate probiotics include lactic acid bacteria, bifidobacteria, butyric acid-producing bacteria, and saccharifying bacteria. Examples of the candidate prebiotics include oligosaccharides and dietary fiber, and the concentration of the candidate prebiotics added to the liquid medium can be freely set. As an example, the concentration of the candidate prebiotics added can be set based on the daily recommended intake of each prebiotic, and can be galactooligosaccharide (GOS): 0.1%, fructooligosaccharide (FOS): 0.3%, xylooligosaccharide (XOS): 0.1%, milk oligosaccharide (Milk): 0.1%, inulin: 0.5%, and kestose: 0.2%. The candidate probiotic or prebiotic materials can be added one by one, or multiple types can be added in combination. It is also possible to add probiotics and prebiotics in combination.

[0041] In the anaerobic culture, the culture temperature is preferably set to a temperature simulating the intestinal environment of the host, and is therefore preferably about 32 to 40° C., and more preferably 35 to 37° C. 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 process] In this step, the growth state of the useful intestinal bacteria in the liquid medium that has been subjected to the above-mentioned anaerobic culture step is evaluated. The growth state can be evaluated by detecting the number or amount of bacteria of the useful intestinal bacteria to be evaluated in the liquid medium, as well as by measuring the amount of substances (e.g., organic acids such as butyric acid) produced by metabolism in the liquid medium by the useful intestinal bacteria to be evaluated, and the physical properties of the liquid medium (e.g., pH) associated therewith. 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 adding a candidate probiotic or the candidate 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 a candidate probiotic or candidate prebiotic. In this case, among the candidate probiotic or prebiotic materials tested, a probiotic or prebiotic that shows a value larger (higher) than the control is selected as a probiotic or prebiotic that promotes 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 proliferation state by comparing the detection or measurement value obtained from the liquid culture medium in which a candidate probiotic or the candidate prebiotic has been added and a 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 may be one type of bacteria 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 bacteria, or lactic acid bacteria. Although not particularly limited, when a bacterium belonging to a specific species or genus is selected as a useful intestinal bacterium to be evaluated, the number or amount of the bacterium in the liquid medium after anaerobic culture can be detected by performing quantitative PCR analysis targeting a gene specific to the selected bacterium. 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 performing quantitative PCR analysis targeting a gene specific to the selected bacterial group. 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 useful intestinal bacteria to be evaluated will be described. Most of the 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 number or amount of bacteria of butyric acid-producing bacteria in the 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 Fprsnbut, which are involved in butyric acid production. 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. Among 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 step, the 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 the butyric acid-producing bacteria at the species level. This makes it possible to select probiotics or prebiotics that promote the growth of butyric acid-producing bacteria present in the intestine of the subject, and also to identify the species of butyric acid-producing bacteria whose growth has actually been promoted. Although not particularly limited, examples of primer sequences used in performing quantitative PCR analysis for each of the above-mentioned target genes are shown in Table 1 below.

[0046] [Table 1]

[0047] In addition, an evaluation method will be described when Bifidobacterium bacteria (bifidobacteria) are selected as useful intestinal bacteria to be evaluated. Examples of genes specific to bacteria belonging to the Bifidobacterium genus 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. In this way, by performing quantitative PCR analysis using g-Bifid primers or the like, it is possible to detect the number or amount of bifidobacteria in the liquid medium after anaerobic culture. Furthermore, in this evaluation process, it is possible to identify bifidobacteria at the species level by analyzing the genes targeted in the quantitative PCR analysis using DNA extracted from the liquid medium after anaerobic culture with a next-generation sequencer. This makes it possible to select probiotics or prebiotics that promote the growth of bifidobacteria present in the intestine of the subject, and also to identify the species of bifidobacteria whose growth has actually been promoted.

[0048] In this way, the screening method and screening medium of the present invention make it possible to individually evaluate the effectiveness of probiotics or prebiotics according to the intestinal flora of each individual, making it possible to propose probiotics or prebiotics suitable for each individual, and to prepare supplements, etc. Furthermore, since the subject only needs to provide feces for screening, the test can be performed safely, and the test period is short, about a few days, making it possible to provide the service safely and easily.

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

[0050] When a fecal sample collected from a subject is bulk cultured in vitro, the proliferation of fecal-derived live bacteria is very active in a liquid medium with a concentration normally used for the cultivation of useful intestinal bacteria. Therefore, even if a fecal sample and a candidate probiotic or prebiotic material are added to such a liquid medium and cultured, the liquid medium is quickly saturated with fecal-derived live bacteria, so that there is no room for the useful intestinal bacteria to proliferate due to the added candidate material, or the overall proliferation of the intestinal bacteria in the fecal sample may be too fast, making it difficult to clearly evaluate the proliferation-promoting effect of the useful intestinal bacteria, and so on. Therefore, the present inventor came up with the idea of ​​using a liquid medium that is normally used for the cultivation of useful intestinal bacteria, by diluting the liquid medium and adjusting the concentration of the components of the medium to be lower than usual, in order to evaluate the proliferation-promoting effect of the added candidate material on the useful intestinal bacteria while suppressing the vigorous proliferation of fecal-derived live bacteria to some extent.

[0051] Here, regarding the degree of dilution of the liquid medium, if the dilution ratio is large, the concentration of the components of the medium becomes too low, which hinders the growth of the added probiotic or the useful intestinal bacteria to be evaluated, and the growth-promoting effect of the useful intestinal bacteria by the candidate material cannot be evaluated. On the other hand, if the dilution ratio is small, the culture solution becomes saturated with live bacteria derived from feces immediately after the start of culture, and the growth-promoting effect of the useful intestinal bacteria by the added candidate material cannot be evaluated. Therefore, the inventor of the present application found, as a result of the study shown in the following examples, that when the candidate probiotic or the useful intestinal bacteria to be evaluated are cultured alone, the growth-promoting effect of the useful intestinal bacteria by the candidate material can be evaluated by adjusting the dilution ratio of the liquid medium so that the OD value of the turbidity of the single culture solution 1 to 3 days after the start of culture is 0.5 or less, preferably 0.4 or less, and more preferably in the range of about 0.25±0.1. The wavelength for measuring the OD value is 600 nm or 660 nm.

[0052] [Example 1] 1. Liquid medium (1) In this example, we performed a culture test of the following three types of bifidobacteria using the commonly used concentration of RCM liquid medium (Reinforced Clostridial Medium, a product of Becton Dickinson) in its original solution (x1) and diluted medium. ·Bifidobacterium longum subsp. longum JCM1217 ·Bifidobacterium pseudocatenulatum JCM1200 ·Bifidobacterium adolescentis JCM1275

[0053] First, each bifidobacterium was streaked from the 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. 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] [Table 2]

[0055] The OD of each bifidobacterial preculture was 660 50 μL of the preculture solution was added to 5 mL of the liquid medium containing the original solution (×1) and 2- to 5-fold diluted (×1 / 2 to ×1 / 5) shown in Table 2, and cultured anaerobically 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 OD were measured. The results are shown in Table 3 below and in the graphs in Figures 1(A) to (C). In each graph in Figure 1, the vertical axis indicates OD, and the horizontal axis indicates culture time (h). The values ​​of ×1 (undiluted solution) are indicated by a solid line and an X-shaped marker, the values ​​of ×1 / 2 by a dotted line and a circle marker, the values ​​of ×1 / 3 by a dashed line and a square marker, the values ​​of ×1 / 4 by a solid line and a triangle marker, and the values ​​of 1 / 5 by a dotted line and a diamond marker.

[0056] [Table 3]

[0057] From these results, Bifidobacteria (OD 660 When the liquid medium was inoculated with 1% of the original RCM liquid medium (x1), the OD value of the culture medium exceeded 1.0 for B. longum and B. adolescentis even after 24 hours of culture. 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 a diluted medium, the OD value of the culture medium decreased compared to the original solution, but the bifidobacteria showed growth according to the culture time, and it was found that this did not affect the growth of bifidobacteria. Of these, it was found that the OD value of the culture medium 48 hours after the start of culture can be adjusted to 0.5 or less, preferably 0.4 or less, by setting the concentration of the components of the RCM liquid medium to 1 / 3 to 1 / 5 of the normal concentration.

[0058] [Example 2] 2. Liquid medium (2) In this example, a culture test was carried out on the three types of bifidobacteria used in Example 1 using liquid media of the normally used concentration of GAM bouillon medium (product of Nissui Pharmaceutical Co., Ltd.) in the original solution (x1) and 4-fold diluted (x1 / 4). 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. A colony of each bacterium was inoculated into a GAM bouillon medium (original solution) having the composition shown in Table 4 below, and cultured anaerobically at 37°C to obtain a preculture solution.

[0059] [Table 4]

[0060] The OD of each bifidobacterial preculture was 660The OD of the culture medium was adjusted to 24 hours and 65 hours after the start of the culture for the undiluted solution (x1), and 24 hours and 48 hours after the start of the culture for the 4-fold diluted solution (x1 / 4). 50 μL of the preculture medium was added to 5 mL of the undiluted solution (x1) and 4-fold diluted solution (x1 / 4) shown in Table 4, respectively, and cultured anaerobically at 37°C. 660 The values ​​of OD and OD were measured. The results are shown in Table 5 below and in the graphs in Figures 2 (A) and (B). The vertical axis of each graph shows OD, and the horizontal axis shows the culture time (h). The values ​​for B. longum are shown with a solid line and X-shaped marker, those for B. pseudocatenulatum with a dotted line and circle marker, and those for B. adolescentis with a solid line and triangle marker.

[0061] [Table 5]

[0062] From these results, Bifidobacteria (OD 660 When B. pseudocatenulatum was inoculated into the liquid medium at 1% of the volume and cultured, the OD value of the culture medium exceeded 1.0 even after 24 hours of culture when the undiluted GAM liquid medium (x1) was used. The OD value 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 solution, but the bifidobacteria showed growth according to the culture time, and it was found that this did not affect the growth of the bifidobacteria themselves. It was also found that the OD value of the culture medium 48 hours after the start of culture could be adjusted to the range of 0.2 to 0.3 for all species of bacteria.

[0063] [Example 3] 3. Liquid medium (3) In this example, three types of liquid media were prepared by mixing known liquid media, and liquid media were prepared by diluting them four-fold (×1 / 4) to carry out a culture test of the 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] [Table 6]

[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, respectively, and cultured anaerobically at 37°C to obtain preculture solutions.

[0066] Here, the stock solution (x1) of mGR liquid medium shown in Table 7 is a liquid medium prepared by mixing the RCM liquid medium used in Example 1 and 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] [Table 7]

[0068] The BG liquid medium stock solution (x1) shown in Table 8 is a liquid medium prepared by mixing Bryant and Burkey Broth (BB medium, Sigma product) and mGAM liquid medium (modified GAM bouillon medium, 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 and Burkey medium and mGAM liquid medium.

[0069] [Table 8]

[0070] Furthermore, the stock solution (x1) of TOR liquid medium 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 Co., Ltd.) used for colony formation from the glycerol stock, and is a liquid medium prepared by 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] [Table 9]

[0072] The OD of each bifidobacterial preculture was 660 The OD of the culture medium was adjusted to 24 hours and 48 hours after the start of the culture for the mGR liquid medium and the BG liquid medium, and the OD of the culture medium was adjusted to 24 hours and 65 hours after the start of the culture for the TOR liquid medium. 660 The values ​​of OD 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 indicates OD, and the horizontal axis indicates the culture time (h). In Figures 3 (A) and (B), the values ​​of B. longum are indicated by a solid line and X-shaped marker, the values ​​of B. pseudocatenulatum by a dotted line and circle marker, the values ​​of B. breve by a dashed line and square marker, and the values ​​of B. bifidum by a solid line and triangle marker. In Figure 3 (C), the values ​​of B. longum are indicated by a solid line and X-shaped marker, the values ​​of B. pseudocatenulatum by a dotted line and circle marker, and the values ​​of B. adolescentis by a solid line and triangle marker.

[0073] [Table 10]

[0074] From these results, Bifidobacteria (OD660 =1) was inoculated into liquid medium at a volume 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 48 hours after cultivation of bifidobacteria could be adjusted to 0.35 or less.

[0075] [Example 4] 4. Liquid medium (4) In this example, lactic acid bacteria culture tests were carried out using liquid media diluted 2-fold (×1 / 2), 4-fold (×1 / 4), and 6-fold (×1 / 6) of the GAM bouillon medium (product of Nissui Pharmaceutical Co., Ltd.) used in Example 2 described above.

[0076] First, a glycerol stock of lactic acid bacteria (Latilactobacillus sakei HS-1; Ravien Sante Co., Ltd.) was streaked onto MRS agar medium (Merck product) and cultured for 3 days under aerobic and room temperature conditions to form colonies. The resulting colonies were inoculated into the 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 600 The turbidity of the culture was adjusted to OD = 1.0, and 50 μL of the preculture solution was added to 5 mL of liquid medium diluted 2-fold (× 1 / 2), 4-fold (× 1 / 4), and 6-fold (× 1 / 6) of the original GAM bouillon medium solution, and cultured anaerobically at 37°C. The OD of the culture solution was measured 24 hours and 48 hours after the start of culture. 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 shown by a solid line and an X-shaped marker, the values ​​of the 4-fold diluted (×1 / 4) medium are shown by a dotted line and a circle marker, and the values ​​of the 6-fold diluted (×1 / 6) medium are shown by a dashed line and a square marker.

[0078] [Table 11]

[0079] From these results, lactic acid bacteria (OD 600When lactic acid bacteria were inoculated into a liquid medium at a rate of 1% and cultured, if a 2-fold diluted (×1 / 2) 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 the 2-fold diluted medium, but the growth corresponded to the culture time, and it was found that increasing the dilution ratio did not affect the growth of the lactic acid bacteria itself. It was also found that the OD value of the culture solution after 48 hours of culture could be adjusted to the range of 0.2 to 0.35 by using a 4-fold diluted medium and a 6-fold diluted medium.

[0080] [Example 5] 5. Liquid medium (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 of two types of lactic acid bacteria were performed as described below. Lacticaseibacillus paracasei YK130220 (Ravien Sante Co., Ltd.) ·Latilactobacillus sakei HS-1 (Ravien Sante Co., Ltd.)

[0081] Glycerol stocks of each lactic acid bacterium were streaked onto MRS agar medium (Merck product) and cultured for 3 days under aerobic and room temperature conditions 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 600The turbidity was adjusted to 1.0, and 50 μL of the adjusted culture solution was added to 5 mL of the liquid medium of the original solution (×1) of mGR medium and its 4-fold dilution (×1 / 4) shown in Table 7 and the liquid medium of the original solution (×1) and 4-fold dilution (×1 / 4) of BG medium shown in Table 8, respectively, and anaerobic culture was performed at 37 ° C. The OD value of the culture solution was 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 of L. sakei, the solid line indicates the value of the original solution (×1), and the dotted line indicates the value of the 4-fold dilution (×1 / 4). In addition, the square marker indicates the value of L. paracasei, the solid line indicates the value of the original solution (×1), and the dotted line and the white square marker indicate the value of the 4-fold dilution (×1 / 4).

[0083] [Table 12]

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

[0085] [Example 6] 6. Liquid medium (6) In this example, a lactic acid bacteria culture test was performed using the stock solution and diluted medium of MRS liquid medium (Merck product) at the concentration normally used. First, a glycerol stock of lactic acid bacteria (Lacticaseibacillus paracasei YK130220; Ravien Sante Co., Ltd.) was streaked onto MRS agar medium (Merck product) and cultured for 3 days under aerobic and room temperature conditions 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] [Table 13]

[0087] The OD value of the preculture solution was calculated as OD 600 The preculture solution was adjusted to a turbidity of 0.05% (OD 0.05%), and 50 μL of the preculture solution was added to 5 mL of MRS liquid medium (×1) or 2- to 5-fold diluted (×1 / 2 to ×1 / 5) shown in Table 13, and aerobic culture was performed at 37°C. 600 The values ​​of 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 are indicated by a dotted line and a circle marker, the values ​​of the 3-fold diluted (x1 / 3) medium are indicated by a dashed line and a square marker, the values ​​of the 4-fold diluted (x1 / 4) medium are indicated by a solid line and a triangle marker, and the values ​​of the 5-fold diluted (x1 / 5) medium are indicated by a dotted line and a diamond marker.

[0088] From these results, the lactic acid bacteria (L. paracasei) was added at 1% (OD 600 = 1.0), the OD of the culture medium reached 1.0 in about 2 days when the undiluted MRS liquid medium (x1) was used. 600 The OD value of the culture medium increased to about 2. 600 The OD value of the culture after 48 hours of incubation was slightly decreased, but as with the other liquid media mentioned above, 600It was found that the value did not fall below 0.5, but exceeded 1. The reason for this is that the MRS medium contains a large amount of glucose in its constituent components, at 20 g / L in the original solution, compared to other media, and even in a 5-fold diluted medium, the amount of glucose is 4 g / L, suggesting that bacterial growth is active. For these reasons, it is preferable to use a liquid medium used for anaerobic culture in the present invention that contains about 2 to 5 g of glucose per 1 L at a concentration normally used, and it is preferable to use such a medium after diluting it 3 to 6 times.

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

[0090] [Probiotic pre-culture] 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 bacteria was scraped off with a platinum loop, inoculated into 4 mL of MRS liquid medium (undiluted, Merck product) shown in Table 13, and pre-precultured under aerobic conditions at 25°C for 3 days. 40 μL of this pre-preculture was inoculated into 4 mL of MRS liquid medium (undiluted), and aerobically cultured 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 bacteria was scraped with a platinum loop and inoculated into 4 mL of RCM liquid medium (undiluted, Becton Dickinson product) shown in Table 2, and pre-precultured under anaerobic conditions at 35°C for 3 days. 40 μL of this pre-preculture was inoculated into 4 mL of RCM liquid medium (undiluted) and cultured anaerobically at 35°C for 16 hours to obtain a preculture solution.

[0093] [Preparation of fecal supernatant samples] Seven healthy Japanese women in their twenties who were not taking antibiotics provided feces, and 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 stool collection container, the lid was placed on the container, and the container was sealed with an Anaero Pouch Kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.) and transported under room temperature conditions while maintaining anaerobic conditions. The fecal supernatant sample was prepared as follows. PBS buffer was added to the feces in an amount 100 times the weight of the feces contained in the Amies transport medium, and the feces were diluted 100 times. After stirring well with a vortex, the mixture was left to stand for 5 minutes, and the supernatant in which the intestinal bacteria were suspended was collected without any precipitate. This was used as the fecal supernatant sample.

[0094] [Table 14]

[0095] [Co-culture of fecal supernatant samples with probiotics] The OD of each probiotic preculture was 600 The OD of the culture solution was adjusted to 1, and 50 μL of the prepared preculture 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] [Table 15]

[0097] These results demonstrated that by using a medium in which the GAM bouillon medium was diluted 4-fold, when fecal supernatant samples were inoculated and co-cultured with various probiotics, the OD value of the culture medium after 48 hours of culture could be adjusted to approximately 1. This demonstrated that the viable bacteria or probiotics in the feces did not grow too much 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 status of beneficial intestinal bacteria in co-cultures of fecal supernatant samples and probiotics In this example, the amount of butyric acid-producing bacteria (bacteria belonging to Clostridium cluster IV or Clostridium cluster XIVa) present in the co-culture solution of the fecal supernatant sample and the probiotic obtained in Example 7 above was quantified by real-time PCR. As the target genes of the real-time PCR primers, the Erec gene specific to Clostridium cluster XIVa and the Clep gene specific to Clostridium cluster IV were selected. The sequences of the PCR primers used are shown in Table 16 below, and an example of useful enterobacteria that can be detected by each target gene is shown in Table 17 below.

[0099] [Table 16]

[0100] [Table 17]

[0101] [DNA extraction from co-culture fluid] 4 mL of the co-culture solution of the fecal supernatant sample and the probiotics obtained in Example 7 above was centrifuged (16,000×g, room temperature, 5 minutes) to separate the supernatant and the 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).

[0102] [Real-time PCR reaction] Real-time PCR was performed using a SYBR (registered trademark) Green I detection system assay. A real-time PCR master mix reagent (KOD SYBR (registered trademark) qPCR Mix, Toyobo Co., Ltd.) was used to perform real-time PCR according to the attached instruction manual. The real-time PCR device used was Thermal Cycler Dice (registered trademark) Real Time System II, Takara Bio Inc.) The real-time PCR was performed in a 25 μL reaction system using 1 μL of DNA extracted from the co-culture solution as described above. DNA (1.0 μL) was added with KOD SYBR qPCR Mix (12.5 μL), PCR forward primer (10 μM; 0.5 μL), PCR reverse primer (10 μM; 0.5 μL), and dH2O (10.5 μL), and real-time PCR was performed to perform relative quantification of the target gene. The PCR reaction conditions were as follows: 2 minutes at 98°C for polymerase activation, followed by 40 sets of [98°C 10 seconds → 55°C or 60°C 10 seconds → 68°C 45 seconds], followed by 95°C 15 seconds → 60°C 30 seconds → 95°C 15 seconds for melting curve. The 16S rRNA gene was used as an internal standard to correct the amount of DNA between samples. In addition, 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 by real-time PCR using the Erec and Clep primers shown in Table 16 was consistent with the length of the desired amplified product.

[0103] The proliferation of useful intestinal bacteria obtained after co-culturing fecal supernatant samples with various probiotics was evaluated by multiplying the relative value of useful intestinal bacteria when only fecal supernatant samples were cultured (Control) by 1. The results are shown in Figures 7 and 8. The error bars in each graph indicate SEM (n=3), and "*" indicates p<0.05 in Tukey's test. According to the results, in subject No. 1, a significant increase in butyrate-producing bacteria having the Clep gene of Clostridium cluster IV was observed by co-culturing the fecal supernatant sample with L. sakei or C. butyricum NBRC, which has a weak ability to produce butyrate. In subject No. 2, no increase in butyrate-producing bacteria was confirmed by co-culturing any of the probiotics tested in this study. In subject No. 3, an increase in butyrate-producing bacteria having the Clep gene of Clostridium cluster IV was observed by co-culturing the fecal supernatant sample with L. paracasei, L. sakei, or C. butyricum NBRC. In subject No. 4, the addition of L. paracasei or L. sakei significantly increased butyrate-producing bacteria with the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria with the Clep gene of Clostridium cluster IV. In subject No. 5, co-culturing the fecal supernatant sample with L. paracasei significantly increased butyrate-producing bacteria with the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria with the Clep gene of Clostridium cluster IV. In addition, co-culturing with C. butyricum NBRC significantly increased butyrate-producing bacteria with the Erec gene. In addition, co-culturing with L. sakei increased butyrate-producing bacteria with the Clep gene, although no significant difference was observed. In subject No. 6, co-culturing the fecal supernatant sample with L. sakei significantly increased butyrate-producing bacteria with the Erec gene of Clostridium cluster XIVa and butyrate-producing bacteria with the Clep gene of Clostridium cluster IV. In subject No. 7, co-cultivation of fecal supernatant samples with L. paracasei increased the number of butyric acid-producing bacteria carrying the Clep gene, and the addition of L. sakei significantly increased the number of butyric acid-producing bacteria carrying the Erec gene.In none of the subjects was there an increase in useful intestinal bacteria when co-cultured with C. butyricum KO, which has a strong butyric acid producing ability. Thus, it was revealed that the probiotics that increase the useful intestinal bacteria such as butyric acid producing bacteria possessed by each subject differ from person to person, and that the evaluation method according to the present invention can select probiotics suitable for the proliferation of useful intestinal bacteria possessed by each individual.

[0104] [Example 9] 9. Analysis of useful intestinal bacteria species in co-cultured broth of fecal supernatant samples and probiotics In this example, a species-level analysis of butyric acid-producing bacteria (bacteria belonging to Clostridium cluster IV or Clostridium cluster XIVa) present in the co-culture medium of the fecal supernatant sample and probiotics obtained in Example 7 above was performed using a next-generation sequencer.

[0105] For the primers used in the real-time PCR of Example 8, in order to enable analysis with a next-generation sequencer ION PGM (Thermo Fisher Scientific), a forward primer with a barcode sequence added to the A1 adapter that can identify each sample and a reverse primer with a P1 adapter sequence added were designed and synthesized. The expected size of the PCR product when each primer was used was about 400 bp. PCR was performed using this primer 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), and the reaction conditions were heating at 94°C for 2 minutes to activate the polymerase, and then [94°C 30 seconds → 55°C 30 seconds → 68°C 30 seconds] was repeated 35 times. 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 treated according to the protocol of the next-generation sequencer ION PGM and analyzed with 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 by BLAST search. The analysis results for the butyric acid-producing bacteria (Clostridium cluster IV) with the Clep gene of subject No. 1 are shown in Figure 9.

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

[0107] [Example 10] 10. Cultivation 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 prebiotics used in this example are the following six types: Galactooligosaccharides (hereinafter referred to as "GOS"), a product of Fujifilm Wako Pure Chemical Industries, Ltd. Fructooligosaccharides (hereinafter referred to as "FOS"), a product of CanHeart Xylooligosaccharides (hereinafter referred to as "XOS"), a product of Bussan Food Science Co., Ltd. Milk oligosaccharides (hereinafter referred to as "Milk"), a product of Nichiei Co., Ltd. Inulin, a product of Japan 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, and were prepared as fecal supernatant samples 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 placed on, and the container was sealed with an Anaero Pouch Kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.) and transported under room temperature conditions while maintaining anaerobic conditions. The fecal samples were prepared as follows. PBS was added to the feces in an amount 100 times the weight of the feces contained in the Amies transport medium, and the feces were diluted 100 times. After stirring well with a vortex mixer, the mixture was left to stand for 5 minutes, and the supernatant in which the intestinal bacteria were suspended was collected without any sediment. This was used as the fecal supernatant sample.

[0109] [Culture 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 10-fold concentrated stock solution of each prebiotic, which had been filter-sterilized, was aseptically added to the liquid medium to give a predetermined concentration, and anaerobically cultured at 37°C for 48 hours. The final concentrations of each prebiotic in the 4-fold 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 set based on the recommended daily intake amount when ingested as food.

[0110] [Evaluation of the growth status of beneficial intestinal bacteria] Next, the amount of bifidobacteria present in the culture solution cultured for 48 hours 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] [Table 18]

[0112] [Table 19]

[0113] [DNA extraction from culture medium] Six milliliters of the culture medium was centrifuged (16,000 x 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 real-time PCR master mix reagent (KOD SYBR (registered trademark) qPCR Mix, Toyobo Co., Ltd.) was used to perform real-time PCR according to the attached instruction manual. The real-time PCR device used was Thermal Cycler Dice (registered trademark) Real Time System II, Takara Bio Inc.) The real-time PCR was performed in a 25 μL reaction system using 1 μL of DNA extracted from the co-culture solution as described above. DNA (1.0 μL) was added with KOD SYBR qPCR Mix (12.5 μL), PCR forward primer (10 μM; 0.5 μL), PCR reverse primer (10 μM; 0.5 μL), and dH2O (10.5 μL), and real-time PCR was performed to perform relative quantification of the target gene. The PCR reaction conditions were as follows: 2 minutes at 98°C for polymerase activation, followed by 40 sets of [98°C 10 seconds → 55°C 10 seconds → 68°C 45 seconds], followed by 95°C 15 seconds → 60°C 30 seconds → 95°C 15 seconds for melting curve. The 16S rRNA gene was used as an internal standard to correct the amount of DNA between samples. 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 by real-time PCR using the g-Bifid primer shown in Table 18 was consistent with the length of the desired amplified product.

[0115] The proliferation of bifidobacteria in the culture medium obtained by adding various prebiotics to fecal supernatant samples and culturing them was evaluated by the multiplication factor of the relative value of bifidobacteria when fecal supernatant samples were cultured without adding prebiotics (Control) taken as 1. The results are shown in FIG. 10. The error bars in each graph indicate SEM (n=3), and "**" indicates p<0.05 in the Games-Howell test. According to the 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 was revealed 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. [Industrial Applicability]

[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 suitable for each individual and the evaluation of their effectiveness, and are therefore useful for improving and enhancing the health condition of each individual and can be used in fields related to health care, medicine, functional foods, pharmaceuticals, etc.

Claims

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

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 mixed medium thereof, and is prepared by diluting the 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 culturing the useful enterobacteria.

3. 3. The method for screening according to claim 1 or 2, wherein 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 a 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 a g-Bifid primer specific to the target gene, the 16S rRNA gene of a Bifidobacterium bacterium being used as a target gene.

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

8. The probiotic is a lactic acid bacteria or a butyric acid producing bacteria, 3. The screening method according to claim 1 or 2, wherein the prebiotics are oligosaccharides or dietary fiber.

9. The liquid medium used in the anaerobic culture step in the screening method according to claim 1, A screening medium, characterized in that the concentrations of the constituent components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations usually used in the cultivation 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 components of the liquid medium are 1 / 3 to 1 / 6 of the concentrations usually used in culturing the useful enterobacteria.

Images