JP2022031038A - METHOD FOR DETECTING NUMBER OF BACILLUS COAGULANS INCLUDED IN COMPLICATED MICROORGANISM SPECIMEN (VESPOtech(TM)), AND APPLICATION OF THE SAME - Google Patents

Abstract

To provide a method for detecting the number of bacillus coagulans included in a complicated microorganism specimen (VESPOtechTM), and an application thereof.SOLUTION: Disclosed are a method for detecting the number of bacillus coagulans included in a complicated microorganism specimen (VESPOtechTM), and an application thereof. In the method for detecting the number of bacillus coagulans included in a complicated microorganism specimen, after improved culture medium is provided and a sample to be measured is culture under a prescribed temperature, the total number of bacillus coagulans can be calculated by identifying bacillus coagulans or a colony including their spores on the basis of changes of color on the improved culture medium, and the number of spores of bacillus coagulans can be estimated. The detection method can be used for detecting the presence of activation of bacillus coagulans in the sample or the quality of its activation, and evaluating the function of promoting intestinal health of a bacillus coagulans strain.SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Act Bioscience, Biotechnology, and Biotechnology, Volume 83, Issue 12, Reiwa, published on August 12, 1327, pp. 2327-2333

The present invention relates to an in vitro measurement method for probiotics and its use, and particularly to a method for detecting the quantity of spore-forming lactic acid bacteria contained in a complicated microbial sample (VESPOtech ^TM ) and its use.

Spore-forming lactic acid bacteria (Bacillus coagulans), also called Bacillus coagulans, have the characteristics of lactic acid bacilli and the genus Bacillus, and have the property of forming spores. Studies have shown that spore-forming lactic acid bacteria are considered a type of probiotic because they have a variety of health-promoting effects, including gastrointestinal disease regulation, immune regulation, and cholesterol lowering. However, spore-forming lactic acid bacteria are existing non-intestinal bacterial species and must be ingested by the human body in order to acquire them.

Spore-forming spore-forming lactic acid bacteria can withstand harsh environments such as high temperature or acidity depending on the spore morphology. I often do it. However, if spores of spore-forming lactic acid bacteria do not germinate in the intestinal tract after being administered to an individual, conversion to vegetative cells and intestinal colonization in the intestinal tract become impossible. However, this means that spore-forming lactic acid bacteria cannot exert the physiological activity of vegetative cells in the body of an individual.

Current studies do not fully understand the growth mechanism of spore-forming lactic acid bacteria in the intestinal tract. In addition, conventional detection and analysis methods cannot isolate and culture spore-forming lactic acid bacteria and other microorganisms in feces, and a dedicated medium for screening or isolating Bacillus coagul from feces has not yet been provided. .. In addition, although research is being conducted to calculate the number of spore-forming spore-forming lactic acid bacteria in the spore form by removing other microorganisms through a short heat treatment such as treatment at 75 ° C. for 30 minutes, the heat treatment is "heat activation". Because of this phenomenon, spore germination and proliferation may be promoted, resulting in erroneous results.

Therefore, it is difficult to determine whether or not spore-forming lactic acid bacteria have germinated in the intestinal tract by the prior art, and a composition containing spores of spore-forming lactic acid bacteria or spores of spore-forming lactic acid bacteria is active in the individual. It turns out that it is difficult to know whether or not it is demonstrating.

The main object of the present invention is to provide a method (VESPOtech ^TM ) for detecting the number of spore-forming lactic acid bacteria contained in a complex microbial sample, and pretreated and / or untreated fecal samples are used. The number of spore-forming lactic acid bacteria or vegetative cells in the fecal sample is calculated directly or indirectly by culturing in the improved medium.

Another object of the present invention is to provide an application of a method (VESPOtech ^TM ) for detecting the quantity of spore-forming lactic acid bacteria contained in a complex microbial sample, and the method disclosed by the present invention is used in a fecal sample. A non-invasive method by detecting the efficacy of spore-forming lactic acid bacteria and / or their spores in the body and the number of spores thereof, and estimating the number of spores of spore-forming lactic acid bacteria converted into vegetative cells in the body. The composition containing spore-forming lactic acid bacteria is accurately and quickly evaluated, and the effect of constructing an intestinal environment advantageous for the growth of good bacteria is achieved.

In order to achieve the above-mentioned object, the present invention provides a method (VESPOtech ^TM ) for detecting the number of spore-forming lactic acid bacteria contained in a complex microbial sample, which is to inoculate a stool sample into a medium and culture it. Then, the colonies whose surrounding colors have changed are selected, it is determined whether or not the colonies are spore-forming lactic acid bacteria, and then the colonies are counted to obtain the quantity of the colonies.

In the examples, in order to allow the vegetative cells of the spore-forming lactic acid bacteria in the stool sample to proliferate on the medium, it is necessary to treat the stool sample with sodium hypochlorite before inoculating the medium.

Of these, the time for treating the stool sample with sodium hypochlorite is about 30 to 60 seconds, for example, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, but the treatment time is 30 seconds. It is preferable to do.

Among them, the concentration of sodium hypochlorite is 1000 ppm or less, for example, 100, 200, 300, 400, 500, 600, 700, 800, 900 ppm, but as an example, when the sodium hypochlorite concentration is 500 ppm. However, the number of spores of spore-forming lactic acid bacteria in the fecal sample before and after the treatment can be stably maintained.

In another example, in the method for detecting the number of spore-forming lactic acid bacteria contained in the complex microbial sample disclosed by the present invention (VESPOtech ^TM ), a stool sample and a stool sample treated with sodium hypochlorite are used, respectively. After culturing, the total number of spore-forming lactic acid bacteria and the number of spores thereof are calculated to estimate the number of vegetative cells of the spore-forming lactic acid bacteria. Specifically, a part of the stool sample is taken, treated with sodium hypochlorite, and the stool sample treated with sodium hypochlorite and the stool sample not treated with sodium hypochlorite are inoculated into the medium. Then, culture is performed. After the culture is completed, the color change around the colony on the medium is observed, the colony having the color change around the medium is selected, and the number of the colonies is counted, and the colony is next. If it is from a stool sample that has not been treated with sodium hypochlorite, the count result of the colony is the total number of spore-forming lactic acid bacteria, and the colony is from a stool sample that has been treated with sodium hypochlorite. If so, the count result of the colony is the number of spores of spore-forming lactic acid bacteria.

Among them, the concentration of sodium hypochlorite is less than 1000 ppm, for example, 500 ppm. The time for treating the stool sample with sodium hypochlorite is preferably less than 60 seconds, for example, 30 seconds or 45 seconds.

Further, in the examples, by subtracting the number of spores of the spore-forming lactic acid bacteria from the total number of the spore-forming lactic acid bacteria, the number of vegetative cells of the spore-forming lactic acid bacteria can be obtained.

In the examples of the present invention, the medium is a glucose yeast extract agar medium (GYEA medium), and the pH value is about 5.5.

In the examples of the present invention, the pH indicator is bromocresol green.

In the examples of the present invention, the culture temperature is about 55 ° C.

In the examples of the present invention, the donor of the fecal sample is administered with a spore-forming lactic acid bacterium or a composition containing the spore-forming lactic acid bacterium at least 5 days before sampling.

Further, the method for detecting the number of spore-forming lactic acid bacteria contained in the complex microbial sample disclosed in the present invention (VESPOTech ^TM ) is an acid-producing ability in one individual of spore-forming lactic acid bacteria spores in the sample to be measured. It can be used to detect or assess acid production, that is, the subject was administered the sample to be measured at least 5 days in advance, and the fecal sample from the subject was cultured in medium to spore-forming lactic acid bacteria and / or. The number of spores is calculated, and the larger the difference in the number of spore-forming lactic acid bacteria and their spores, the better the acid-producing ability or the acid-producing rate in the subject of the measurement target sample. An assessment of the intestinal health-promoting function of spore-forming lactic acid strains is achieved.

In FIG. 1, A is a spore-forming lactic acid bacterium cultured in an improved GYEA medium and the colony morphology observed with the naked eye. FIG. 1B is the result of culturing spore-forming lactic acid bacteria in the improved GYEA medium and observing the colony morphology under a microscope on the 4th day of the second stage culture. C in FIG. 1 is the result of culturing spore-forming lactic acid bacteria in the improved GYEA medium and observing the colony morphology under a microscope on the 5th day of the second stage culture. Spores. D in FIG. 1 is the result of culturing the spore-forming lactic acid bacterium in the improved GYEA medium and observing the colony morphology with a microscope on the 6th day of the second stage culture. Spores. Percentage of spore-bearing spore-forming lactic acid bacteria colonies by culture days (Duncan's test, p <0.05). It is the relative ratio of the number of spores of spore-forming lactic acid bacteria in the samples treated with different concentrations of sodium hypochlorite, and the number of spores of spore-forming lactic acid bacteria not treated with sodium hypochlorite is used as a comparison standard. .. Relative ratio of the number of spores of spore-forming lactic acid bacteria in samples treated with sodium hypochlorite for different times, of which the number of spores of spore-forming lactic acid bacteria treated with sodium hypochlorite for 0 seconds is used as a comparison standard. There is. The proportions of spore-forming lactic acid bacteria spores and vegetative cells in different intestinal parts, of which there is a significant difference (p <0.05) between a and b, and a significant difference between c and d (Duncan'. There is s test, p <0.05). The content of short-chain fatty acids in the feces of each rat group, of which * indicates a significant difference (Duncan's test, p <0.05).

The present invention discloses a method for detecting the quantity of spore-forming lactic acid bacteria contained in a complex microbial sample (VESPOtech ^TM ) and its use, which provides a medium having an improved predetermined pH value. In addition, by combining the pretreatment technology of the stool sample, the stool sample is inoculated into the medium, cultured at a predetermined temperature, and then the colony containing spore-forming lactic acid bacteria is selected by the color change around the colony on the medium. Judgment is made, and the total number of spore-forming lactic acid bacteria and / or the number of spores of spore-forming lactic acid bacteria are calculated. In addition, after obtaining the total number of spore-forming lactic acid bacteria and the number of spores of spore-forming lactic acid bacteria, the difference between the total number of spore-forming lactic acid bacteria and the number of spores of spore-forming lactic acid bacteria can be calculated. If it is possible to evaluate the acid-producing ability or acid-producing rate in an individual of spore-forming lactic acid bacteria in the sample to be measured, and to stably maintain a weakly acidic environment in the intestinal tract. , It is advantageous for the growth of good bacteria and the growth of bad bacteria is suppressed. Therefore, the method disclosed in the present invention estimates the presence or absence of activation in an individual in the sample to be measured and the quality of the activation. Therefore, the effect of determining the effectiveness of the sample to be measured can be achieved.

The "judgment" in the present invention refers to confirming that the colony is a spore-forming lactic acid bacterium by screening, and can be performed manually or by equipment.

The "vegetative cells" in the present invention refer to a state in which spore-forming bacteria are reactivated from a dormant state of spore form under an appropriate growth environment, and the vegetative cells have a proliferative ability in the growth state. ..

Hereinafter, in order to explain the present invention and its effects, some examples will be given, and the results of the examples and the drawings will be described in more detail.

The spore-forming lactic acid bacterium used in the present invention is a "spore-shaped pure Bacillus strain (about 5x109 ⁹ spores / g)", which is a commercial bacterium provided by Biotechnology Technology Co., Ltd., 4 It was stored in an environment of ℃. The selection of this strain is merely an example and does not limit the scope of protection of the present invention. The disclosed effect can be achieved.

All of the animal experiments in the following examples have been approved by the National Chung Hsing University and the Use Committee, and were conducted in compliance with the relevant ethical code.

All the data in the following examples were statistically analyzed by Duncan's new multiplex test (Duncan's test).

Confirmation of spore-forming lactic acid bacteria in complex microbial specimens

Bacterial samples to be measured were first taken and cultured in an improved GYEA (glucose yeast extract agar) medium. As for the improvement conditions, the pH value was adjusted to 5.5, and bromocresol green was used as a pH indicator. The cells were cultured in an aerobic environment at 55 ° C. for 2 days, and bacteria that could not survive at a temperature of 55 ° C. or higher were removed depending on the temperature, leaving spore-forming lactic acid bacteria colonies (as shown in A in FIG. 1). Due to the growth of spore-forming lactic acid bacteria, the pH value in the culture environment dropped to about pH 3.8, so that the color changed from blue to yellow around the spore-forming lactic acid bacteria colonies, and the spore formation status of the colonies under a microscope. It was found that the blue colonies having yellow rings and spores around them were spore-forming lactic acid bacteria colonies. The second stage culture was performed, that is, after culturing for a specific number of days in a constant temperature environment of 55 ° C., spore-forming lactic acid bacterium colonies having a yellow ring were selected, and malachite green and autumn crocus were used. It was stained and observed under a microscope. The results are as shown in B to D in FIG. The results of counting the colonies having spores are as shown in FIG.

B in FIG. 1 shows that after 4 days of the second stage culture, only proliferative vegetative cells (vegetative form) appear in the spore-forming lactic acid bacteria colonies, but from the 5th day of the second stage culture. It can be seen that spores began to appear in the spore-forming lactic acid bacterium colonies and increased as the number of culture days increased (as shown in C and D in FIG. 1). Furthermore, the results shown in FIG. 2 show that the proportion of spore-bearing spore-forming lactic acid bacteria colonies increased significantly on the 6th day of the second stage culture, and on the 7th day of the culture, almost all colonies had spores. It shows that it was doing.

Therefore, the environment for detecting and culturing spore-forming lactic acid bacteria needs to include the following conditions. The culture temperature is 55 ° C.; spore formation can be observed at least 5 days after the culture, but the effect is good when observed on the 7th day of the culture; the medium used is the above-mentioned improved GYEA medium. As such, it is necessary to allow the lactic acid-producing bacteria to grow, and it is preferable to have a pH value indicator.

Test of sample pretreatment parameters (1)

Samples containing spore-forming lactic acid bacteria were treated with different concentrations (0, 500, 750, 1000 ppm) of sodium hypochlorite for 30 seconds, then inoculated into the improved GYEA medium and cultured at 55 ° C. for 2 days. , Colonies with a yellow ring were selected and the number of colonies was counted to obtain the number of spores of spore-forming lactic acid bacteria. The results are shown in Table 1. Among them, there was a remarkable difference (Duncan's test, p <0.05) between each data. Furthermore, the relative proportion of spores of spore-forming lactic acid bacteria treated with different concentrations of sodium hypochlorite was calculated based on the number of spores of spore-forming lactic acid bacteria not treated with sodium hypochlorite. The results are as shown in FIG.

The purpose of treating the sample with sodium hypochlorite is to remove the spore-forming spore-forming spore-forming spores by removing the spore-forming spore-forming spores and maintaining a stable spore count of the spore-forming spore-forming spores. The number of spores of spore-forming lactic acid bacteria in the sample treated with sodium hypochlorite is the number of spores of spore-forming spores in the sample not treated with sodium hypochlorite because the number of spores is to be accurate. The closer it is, the more suitable it is.

From the results in Table 1 and FIG. 3, it can be seen that the higher the concentration of sodium hypochlorite treatment on the sample, the lower the number of spores of spore-forming lactic acid bacteria. This means that high concentrations of sodium hypochlorite cannot maintain the spore count of spore-forming lactic acid bacteria in the sample in a stable manner, which is not much different from that before the treatment. Therefore, from the results of Table 1 and FIG. 3, the hypochlorite used to simultaneously remove the spore-forming lactic acid bacteria vegetative cells in the sample and simultaneously achieve the maintenance of the spore count of the spore-forming lactic acid bacteria. It can be seen that the sodium concentration should not exceed 1000 ppm and a concentration of about 500 ppm is suitable.

Test of sample pretreatment parameters (2)

Spore-forming lactic acid bacteria samples were treated with sodium hypochlorite at a concentration of 500 ppm for different times (0, 30, 60, 180 seconds), then inoculated into the improved GYEA medium and cultured at 55 ° C. for 2 days. , Colonies with a yellow ring were selected and the number of colonies was counted to obtain the number of spores of spore-forming lactic acid bacteria. The results are shown in Table 2. Among them, there was a remarkable difference (Duncan's test, p <0.05) between each data. Furthermore, the relative proportion of spores of spore-forming lactic acid bacteria after undergoing sodium hypochlorite treatment for different times was calculated based on the number of spores of spore-forming lactic acid bacteria not treated with sodium hypochlorite. The results are as shown in FIG. As described above, the difference between the number of spore-forming spores in the sample after sodium hypochlorite treatment and the number of spore-forming spores in the sample not treated with sodium hypochlorite is The larger the value, the more difficult it is to maintain a stable spore count of spore-forming lactic acid bacteria.

From the results of Table 2 and FIG. 4, the number of spores of spore-forming lactic acid bacteria in the sample after being treated with sodium hypochlorite solution for 30 seconds was close to that without sodium hypochlorite treatment, and the sample was After 60 seconds of sodium hypochlorite treatment, the number of spores of spore-forming lactic acid bacteria was lower than that of the sample not treated with sodium hypochlorite, and the sample was hypochlorite for 180 seconds. When treated with sodium hypochlorite, the number of spores of spore-forming lactic acid bacteria was significantly higher than the number of spores of spore-forming lactic acid bacteria in the sample not treated with sodium hypochlorite. The above results show that the time to treat the sample with sodium hypochlorite is to simultaneously achieve the removal of spore-forming lactic acid spore-free cells in the sample and the maintenance of the spore count of the spore-forming spores. It should be 60 seconds or less, and the processing time is preferably about 30 seconds.

Animal experimentation

16 8-week-old male SD rats (purchased from BioLASCO Ltd., Taiwan) were taken, each weighing 336.6 ± 16.7 g, 22 ± 1 ° C, humidity 60 ± 5%, and a 12-hour light-dark cycle control environment. Raised in the house.

SD rats were randomly divided into two groups, one of which was the control group and the other was the spore-forming lactic acid bacterium group (hereinafter abbreviated as BC bacterium group), and the same food and drink was given to each rat group during the test period. The BC group was supplied with spores of spore-forming lactic acid bacteria ( ^5x107 CFU / g) daily. During the test period, food intake and body weight of each SD rat group were recorded daily, and fresh feces were collected on the 28th day of the test. The collected feces were stored in an environment of −20 ° C. After the test was completed, each SD rat group was sacrificed.

According to the results of the recording, the average food intake of SD rats was 28.4 to 28.9 g / day, and the average final body weight at the end of the test was 434.3 to 439.0 g.

Detection of bacterial morphology in the intestinal tract and feces

Feces of each rat group were collected, and the water content, weight and pH value of the feces were detected. The results are shown in Table 3.

Rat cecal fluid, colon contents and feces were collected and suspended in sterile phosphate buffered saline at a ratio of 1:10 (w / v), respectively. It was continuously diluted 10-fold with phosphate buffered saline to give the concentration required for bacterial count.

To count the spores of spore-forming lactic acid bacteria (spore counts, SCs), take the feces of each rat group before applying to the improved GYEA medium, and first use a solution of sodium hypochlorite (NaOCl) at a concentration of 500 ppm. Processed for 30 seconds.

The stools that had not been treated with sodium hypochlorite solution and the stools that had been treated with sodium hypochlorite solution of each rat group were cultured on the improved GYEA medium at 55 ° C. for 2 days, and colonies having a yellow ring were selected. If the number of colonies is counted and the colonies are from stool that has not been treated with sodium hypochlorite solution, the counted quantity is the total count of spore-forming lactic acid bacteria (TCs). And containing vegetative cells and spores, but if the colonies are from stool treated with sodium hypochlorite solution, the counted quantity is the spore count of spore-forming lactic acid bacteria, then spore-forming. Subtracting the spore count of spore-forming lactic acid bacteria from the total count of lactic acid bacteria gives the number of vegetative cells. The results are shown in Table 4 and FIG. In Table 4, the quantity less than 1x10 ² CFU / g was not detected.

All colonies have undergone the treatment of the method shown in Example 1, and 16S rRNA has been confirmed by the following RNA primer pair. Based on the results, the colonies detected and counted in this example and NCBI The similarity to spore-forming lactic acid bacteria in the BLAST database could be determined to be 99.8%, indicating that all colonies cultured in this example were spore-forming lactic acid bacteria colonies. rice field.

Forward primer (F): 5'-AGAGTTTGATCCTGGCTCAG-3';

Reverse primer (R): 5'-ACGGTTACCTTGTTACGACTT-3'.

From the results in Table 3, compared with the control group, administration of spore-forming lactic acid bacteria not only increases the humidity and weight of feces, but also the spore-forming lactic acid bacteria produce lactic acid. Therefore, it can be seen that the pH value of feces is lowered.

From the results in Table 4, the presence of spores of spore-forming lactic acid bacteria was not detected in the control group, and the BC bacterium group was subjected to an oral test of spores of spore-forming lactic acid bacteria for 28 consecutive days and then in the feces of SD rats. We were able to find spores of 3.64x10 ⁵ CFU / g and vegetative cells of 6.31x10 ⁵ CFU / g, respectively.

Furthermore, from the results of FIG. 5, the ratio of spores of spore-forming lactic acid bacteria to vegetative cells of spore-forming lactic acid bacteria in the cecal fluid, colon contents and feces of SD rats of the BC bacteria group was 2.3: 7, respectively. It was 0.7, 2.1: 7.9 and 3.9: 6.1. This result indicates that vegetative cells of spore-forming lactic acid bacteria appear in both the intestinal tract and feces, and the proportion in the cecum and colon is higher than that in feces, and the spore regeneration of spore-forming lactic acid bacteria. It can be inferred that this occurs distal to the colon, before defecation.

From the above results, when stool is cultured by the method disclosed by the present invention, the total number of spore-forming lactic acid bacteria and the number of spore-forming spore-forming spores in the stool can be known. The number of vegetative cells can be estimated accurately and quickly, and if the number of spore-forming lactic acid cells increases or is relatively high, or if the number of spore-forming spores decreases or decreases, the spore-forming lactic acid strain. It has been shown that spores can germinate in the intestinal tract and exert a health-promoting effect on vegetative cells, for example, having a good acid-producing ability. On the contrary, when the number of spore-forming lactic acid bacteria is decreased or relatively low, or when the number of spore-forming lactic acid bacteria is increased or relatively high, the health promoting effect of the spore-forming lactic acid bacterium is not good. Shows.

Therefore, the method disclosed in the present invention can be used to know the ability of spore-forming lactic acid bacteria to germinate and become vegetative cells in the gastrointestinal tract, whereby the spore-forming lactic acid bacteria administered and entered into an individual can be used. It can be used to assess whether a strain can help improve or maintain intestinal health.

Detection of short-chain fatty acids in feces

First, the fecal sample is homogenized with 0.9% (w / v) cold salt water at a ratio of 1:10 (w / v), centrifuged at 1006 g for 10 minutes, then 2 mL of the supernatant is taken and 10 μL of isocaproic acid is taken. (Internal standard) and 20 μL of sulfuric acid 50% (w / v) are mixed, extracted with ethyl ether, and then gas chromatographed using a column (Agilent J & W HP-INNO Wax GC Volume, 30 m, 0.25 mm. 0.25 μm). 1 μL of the ether layer was analyzed with a Tograph / Agilent ionized detector, of which helium gas was provided as a carrier gas at a flow rate of 7 mL / min. The analysis conditions are as follows. Initial oven temperature 80 ° C. for 1 minute, 20 ° C./min rate to 140 ° C., then 140 ° C. for 1 minute, then 20 ° C./min rate to 220 ° C., 220 ° C. 2 Minutes. The temperatures of the sample injector and the detector were 140 ° C and 250 ° C, respectively. The detection results of short-chain fatty acids in feces of each rat group are as shown in FIG.

From the results shown in FIG. 6, SD rats in the BC group were treated with acetic acid (96.1 to 147.7 mol / g feces) and butyric acid (34.9 to 99.0 mol) due to the administration of spores of spore-forming lactic acid bacteria. / G Feces) increased sharply, but the content of propionic acid did not increase significantly. Overall, after administration of spores of spore-forming lactic acid bacteria, germination and proliferation of spores can increase the total concentration of short-chain fatty acids by about 1.7 times (169.3 to 279.2 mol / g feces). did it.

When this result is combined with the results of the above-mentioned Examples, when the spores of spore-forming lactic acid bacteria are activated in the gastrointestinal tract and become vegetative cells, substances such as lactic acid are produced in the gastrointestinal tract and originally reside in the gastrointestinal tract. It is reasonably understood that acid-producing bacteria can grow and produce acid, thereby increasing the total concentration of short-chain fatty acids in the gastrointestinal tract.

Combining the results of the above-mentioned examples, the method for detecting the number of spore-forming lactic acid bacteria contained in the complex microbial sample disclosed in the present invention (VESPOtech ^TM ) is a method for detecting spore-forming lactic acid bacteria contained in a fecal sample. By detecting the quantity and the number of spores of spore-forming lactic acid bacteria, it is possible to know whether or not the spore-forming lactic acid bacteria that entered the body of the donor of the fecal sample could be activated or re-emergent to become vegetative cells. When the amount of spores of spore-forming lactic acid bacteria that have entered the body is high, the composition containing spore-forming lactic acid bacteria or spore-forming lactic acid bacteria administered into the body has good physiological activity. It has been shown that it can improve the intestinal environment and promote intestinal health.

Claims (8)

The process of obtaining a stool sample and
A step of treating the stool sample with sodium hypochlorite for 30 to 60 seconds, and
The process of preparing the medium and
A step of inoculating each of the stool sample treated with sodium hypochlorite and the stool sample not treated with sodium hypochlorite into a medium and culturing at a culture temperature of 50 ° C. to 60 ° C.
After the culture is completed, the step of observing the color change around the colony on the medium, selecting the colony having the color change in the surroundings on each medium, and counting the number of bacteria in the colony. Including,
The medium has a pH value of 5.0-6.0 and has a nutritional component provided for the growth of lactic acid-producing bacteria and bromocresol green used as a pH indicator.
When the colony is from the stool sample that has not been treated with sodium hypochlorite, the count result of the colony is the total number of spore-forming lactic acid bacteria, and the colony is the stool that has been treated with sodium hypochlorite. When it is from a sample, the count result of the colony is the number of spores of spore-forming lactic acid bacteria, which is a method for detecting the number of spore-forming lactic acid bacteria contained in a complex microbial sample (VESPOtech ^TM ). The method for detecting the quantity of spore-forming lactic acid bacteria contained in the complex microbial sample according to claim 1, wherein the concentration of sodium hypochlorite is 500 ppm (VESPOtech ^TM ). The method for detecting the number of spore-forming lactic acid bacteria contained in the complex microbial sample according to claim 1, wherein the time for treating the stool sample with sodium hypochlorite is 30 seconds (VESPOtech ^TM ). The spore-forming lactic acid bacterium contained in the complex microbial sample according to claim 1, wherein the number of spores of the spore-forming lactic acid bacterium is subtracted from the total number of the spore-forming lactic acid bacteria to obtain the number of vegetative cells of the spore-forming lactic acid bacterium. A method for detecting a quantity (VESPOtech ^TM ). The method for detecting the number of spore-forming lactic acid bacteria contained in the complex microbial sample according to claim 1, wherein the medium is a glucose yeast extract agar medium and the pH value is about 5.5 (VESPOtech ^TM ). The method for detecting the quantity of spore-forming lactic acid bacteria contained in the complex microbial sample according to claim 1, wherein the culture temperature is about 55 ° C. (VESPOtech ^TM ). The spores contained in the complex microbial sample according to claim 1, wherein the donor of the fecal sample is administered with a composition containing spore-forming lactic acid bacteria or spore-forming lactic acid bacteria at least 5 days before sampling. A method for detecting the quantity of sex lactic acid bacteria (VESPOtech ^TM ). The complex microbial sample according to any one of claims 1 to 7, which can be used to evaluate the acid-producing ability of spores of spore-forming lactic acid bacteria in a sample to be measured. A method for detecting the quantity of spore-forming lactic acid bacteria (VESPOtech ^TM ).

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