KR102297982B1 - A method for predicting using kits capable of predicting inhibition of rumen microorganism using bile acid - Google Patents

Abstract

The present invention relates to a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids, and more particularly, to analyze the inhibitory activity of bile acids against microorganisms in vitro and predict the patient's response when applying bile acids therapeutic therapy. It relates to a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids that can

Description

A method for predicting inhibitory activity using a kit for predicting intestinal microbe inhibition using bile acids {A METHOD FOR PREDICTING USING KITS CAPABLE OF PREDICTING INHIBITION OF RUMEN MICROORGANISM USING BILE ACID}

The present invention relates to a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids, and more particularly, to analyze the inhibitory activity of bile acids against microorganisms in vitro and predict the patient's response when applying bile acids therapeutic therapy. It relates to a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids that can.

Microorganisms in the body are actually a part of our body and have an absolute influence on defense and digestion, including metabolism.

A disproportionate change in the composition of microorganisms in the gastrointestinal tract to alter the homeostasis of the host's intestinal environment and to distort physiology is called dysbiosis, and environmentally induced dysbiosis is often irreversible. It also causes chronic diseases by altering host metabolism, and contributes to the development of many diseases, including inflammatory bowel disease, autism, chronic fatigue syndrome, obesity, type 2 diabetes, and cancer.

Bile acids are used as major regulators for these microorganisms and play an important role in proliferation and inhibition of specific strains.

In particular, bile acids have two inhibitory effects on intestinal microbes, one is a direct antibacterial effect on the cell wall due to its hydrophobic properties, and the other is an inhibitory effect by a protein derived from a bile acid nuclear receptor. .

Since these properties are different for each bile acid, the inhibitory or accelerating effect on microorganisms for each type is very diverse.

Each individual has a characteristic microbial distribution and composition in the intestinal tract, which is presumed to be related to their characteristic bile acid composition. It is regulated by the bile acid composition and returns to the original intestinal microbial environment.

And there are documents that report that changes in bile acids in the intestinal tract cause dysbiosis, and artificial changes in the composition of bile acids can be an effective method to control the proliferation of target microorganisms.

Current application of the artificial bile acid composition change is urusa (UDCA; uresodeoxycholic acid), and only about 5% of bile acids in the body are excreted in feces every day, and 95% of bile acids are reabsorbed from the intestinal tract and moved to the liver. , because it is recycled, if it is continuously supplied to the intestinal tract, the proportion in the body gradually increases due to repeated reabsorption, taking up the largest fraction. Due to this, ursodeoxycholic acid, which is present in a small amount in the normal body, rises up to about 50% in the blood, hepatobiliary tract, and intestinal tract, thereby exerting a therapeutic effect.

Currently, antibiotic therapy, fecal transplantation, etc. are generally used as therapies for regulating the microbial environment in the intestinal tract.

In the case of the antibiotic therapy, there are various types of intravenous and oral drugs depending on the target strain, which act on the strains in the intestinal tract to inhibit the growth. There is a problem in using it as a short-term therapy.

And the stool transplantation therapy is a method of inhibiting the proliferation of pathogens by delivering the stool of a normal person without a specific disease into the intestinal tract for a disease caused by an imbalance of intestinal bacteria, caused by Clostridium difficile It is used as a treatment for pseudomembranous colitis.

As a prior art related to the fecal transplantation therapy, there is Korean Patent Application Laid-Open No. 10-2019-0004586 'Method for screening donor and donor feces for intestinal microbial transplantation (FMT)'. It is about a method for finding a donor or donor feces to have a healthy intestinal microbiome by analyzing and predicting the intestinal microbial community of

However, such fecal transplantation therapy may cause other diseases by supplying a large amount of microorganisms even if selected feces are used, and there is a problem in that there is no study in detail about the effect on the subject's immune system.

In addition, if the ratio in the body is increased by oral administration of a specific bile acid and the ratio is reduced by excreting the bile acids existing in the body, the microbial environment in the intestinal tract is expected to change. We need a way to predict in advance.

US 10-2019-0004586 A

The present invention has been devised in view of the above problems, and an object of the present invention is to use bile acids, but before supplying bile acids directly into the body, when bile acids are supplied to the microbial environment in the patient's intestine from outside the body, the microbial environment An object of the present invention is to provide a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids, which can be predicted in advance about what kind of change will be made.

According to the characteristics for achieving the object as described above, the microorganism is plated (plated) medium (plate); and a bile acid disk placed on the plate and treated with bile acid to which a buffer is added to a blank disk.

In this case, as the buffer, ethanol to tetrahydrofuran is used.

In addition, the bile acid is cholic acid (CA, cholic acid), lithocholic acid (LCA, lithocholic acid), deoxycholic acid (DCA, deoxycholic acid), keno deoxycholic acid (CDCA, chenodeoxycholic acid), ursode Oxycholic acid (UDCA, ursodeoxycholic acid) is used.

The method for predicting the inhibitory ability of bile acids against the microorganisms consists of the following steps.

Preparing a medium (plate) in response to the microorganism list and quantity; dispensing distilled water (DW) into each of the glass tubes corresponding to the number of microorganisms; using a sterilized cotton swab to scrape enough of the cultured bacteria in the source medium (plate), and then dissolving it in distilled water (DW) dispensed in the glass tube to make a solution; Wetting a cotton swab with the solution mixed with bacteria and plating on the plate; placing different types of bile acid disks on the plate using tweezers; culturing the medium (plate) on which the bile acid disk is mounted in an incubator; measuring the diameter of the bile acid inhibition region after the culture is completed; and supplying bile acid dissolved in sodium bicarbonate into the body, quantifying the number of microorganisms in the large intestine and comparing it with the diameter of the measured inhibition region.

At this time, in quantifying the number of microorganisms in the large intestine, RT-PCR (Real Time PCR) technique is used.

According to the inhibitory ability prediction method using the intestinal microbe inhibitory ability prediction kit using bile acids of the present invention, different types of bile acid discs are placed on a plate on which each microorganism is plated and then cultured so that each bile acid is absorbed into the microorganisms. In contrast, the inhibitory ability is exhibited against which microorganisms, and by quantifying the degree of such inhibitory activity, there is an effect of predicting in advance what kind of microbial environment will be created when bile acids are directly supplied to the patient's body.

Moreover, according to the present invention, by predicting the microbial environment to be created in the intestine of the patient in advance, there is an advantage that can cause an imbalance in the microbial environment due to the input of unspecified microorganisms and prevent the occurrence of other diseases in advance.

Moreover, according to the present invention, there is an advantage in that resistance does not occur by regulating the microbial environment control using bile acids and thus is effective.

In addition, according to the present invention, when a specific microorganism abnormally proliferates in the body or causes an infection, each bile acid analyzes the inhibitory ability to identify the cause of the growth of the target microorganism, and the advantage of administering a bile acid capable of inhibiting it. have.

1 is a view showing a region of inhibiting ability of microorganisms by bile acids;
Figure 2 is a graph showing the degree of inhibition of Figure 1 numerically,
3 is a graph showing the change in the number of microorganisms changed by supplying the intestinal bile acid to the mice.

Hereinafter, in conjunction with the accompanying drawings, a method for predicting inhibitory activity using a kit for predicting intestinal microbial inhibitory activity using bile acids according to the present invention will be described in detail.

1 is a diagram showing the inhibitory activity region of microorganisms by bile acids, FIG. 2 is a graph showing the degree of inhibitory activity in FIG. 1 digitized, and FIG. 3 is a change in the number of microorganisms changed by supplying the intestinal bile acid to mice. It is a graph.

As shown in FIG. 1 , the kit for predicting intestinal microbial inhibition ability using bile acids according to the present invention largely consists of a plate 1 and a bile acid disk 2 .

First, the medium (plate) 1 is a microorganism plated (plating), in the present invention, a common normal flora in the intestines of humans and mice, and a microorganism that can be purchased as a standard strain, E. coli (escherichia) coil; ATCC 25922), Proteus mirabilis (ATCC 12453), and Enterococcus faecalis (ATCC 29212) were used, and all microorganisms can be used according to the control goal.

And, the medium (plate) 1 used in the present invention is based on MHA medium, and when it is difficult to culture, any medium used for culturing microorganisms such as MacⅠ, BAPII, Chrome, which is a special medium suitable for each microorganism, can be used. .

The bile acid disk 2 is placed on a plate 1 on which the microorganism is plated, and a blank disc is treated with bile acid with a buffer added thereto. is made up of

At this time, the type of bile acid treated in the bile acid disk 2 is cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA, deoxycholic acid), kenodi. There are oxycholic acid (CDCA, chenodeoxycholic acid) and ursodeoxycholic acid (UDCA, ursodeoxycholic acid).

These bile acids are normally present in the human body, so bile acids less likely to cause an adverse reaction or immunological disturbance upon oral administration were adopted.

In addition, the buffer suitable for each of the bile acids is added, and the buffer has a property of dissolving the bile acids well, and volatile ethanol or tetrahydrofuran that does not remain after vaporization is used. it is preferable

The inhibitory activity prediction method using the intestinal microbial inhibitory activity prediction kit using bile acid according to the present invention having such a configuration consists of the following steps.

First, it starts with the step (S10) of preparing the medium (plate) 1 corresponding to the list and quantity of the microorganisms used in the present invention.

At this time, as described above, the medium (plate) (1) is based on MHA medium, and if culture is difficult, all mediums used for culturing microorganisms such as MacⅠ, BAPII, Chrome, which are special media suitable for each microorganism, can be used. have.

Next, a step 20 of dispensing distilled water (DW) into each of the glass tubes corresponding to the number of microorganisms is performed (20).

In dispensing the distilled water (DW) into a glass tube, the dispensed amount of the distilled water is 2 ml.

After the distilled water (DW) is dispensed into a glass tube, the bacteria cultured in the plate 1 are sufficiently scraped off using a sterile cotton swab, and then dispensed into the glass tube. It goes through a step (S30) of dissolving in distilled water (DW) to make a solution.

Thereafter, a cotton swab is wetted with the solution mixed with bacteria and plating is performed on the plate 1 ( S40 ). After the plating step, the A step (S50) of placing different types of bile acid disks 2 on a plate 1 on which a solution is plated is performed using tweezers.

In this case, the bile acid disk 2 is manufactured through the following process.

First, the five kinds of bile acids, cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA, chenodeoxycholic acid), ursode Measure 0.1 g to 0.2 g of powder of oxycholic acid (UDCA, ursodeoxycholic acid) using a scale, and transfer to a 1.5 ml tube.

Then, 1 ml of a buffer suitable for each bile acid is dispensed and dissolved well using a vortex mixer.

Then, 10 blank discs are prepared for each bile acid, 20 μl of each bile acid solution is treated on the blank disc, and dried for 10 minutes, and this process is repeated 3 times in total.

Thereafter, the dried blank disk is moved to a clean bench and sterilized for 30 minutes using a UV lamp.

After the step of placing the bile acid disc on the plate 1 using tweezers (S50), the culture plate 1 on which the bile acid disc is placed is placed in an incubator and culturing (S60). ) through

At this time, the temperature in the incubator is 37 ℃, it is preferable to incubate at this temperature for 24 hours.

When the culture is completed, a step (S70) of measuring the diameter of the bile acid inhibition region is performed, and through this step, it is possible to check to what extent the bile acid inhibits microorganisms.

In confirming the inhibitory ability, unlike the existing antibiotic susceptibility test that measures the diameter of the entire inhibition region in order to maintain the continuity of the values with the non-inhibited bile acid disk, the blank disk diameter is removed from the total measurement diameter and divided into two. Only the inhibition region in one direction was quantified.

Thereafter, bile acid dissolved in sodium bicarbonate is supplied to the body, and the number of microorganisms in the colon is quantified and compared with the diameter of the inhibition zone measured by the step (S80) to correspond to the diameter of the inhibition zone in the large intestine. It is possible to determine the number of microorganisms in the

The sodium bicarbonate (sodium bicarbonate) is a raw material for materials used for food, such as baking soda, and has the ability to dissolve bile acids, so it is a key material to be used in the case of future therapeutic application.

And in quantifying the number of microorganisms in the colon, RT-PCR (Real Time PCR) technique was used.

Hereinafter, it will be described to what extent each of the bile acids has inhibitory activity against microorganisms, but the plate 1 on which each bile acid disk 2 is placed will be referred to as a kit. .

1 shows each bile acid disk in the medium (plate) 1 on which the Escherichia coli, Proteus mirabilis, and Enterococcus faecalis microorganisms are plated. ) (2) is placed on it and shows the appearance of the kit in which the inhibitory ability was exerted.

As shown in FIG. 1 , it can be seen that the inhibitory ability of each of the bile acids is different according to each of the microorganisms, and numerical information regarding the degree of this inhibitory activity is shown in a graph in FIG. 2 .

First, looking at the graph shown in FIG. 2 (a), in the experimental results of the Escherichia coli microorganism, deoxycholic acid (DCA) showed the greatest inhibitory ability, ) and chenodeoxycholic acid (CDCA, chenodeoxycholic acid) showed the same inhibitory ability.

The Proteus mirabilis microorganism showed inhibitory ability in deoxycholic acid (DCA) as shown in the graph shown in FIG. 2(b), and was lower than that of deoxycholic acid (DCA, deoxycholic acid) Numerically, chenodeoxycholic acid (CDCA) showed inhibitory activity.

And, as shown in (c) of FIG. 2 , the Enterococcus faecalis microorganism showed a great inhibitory ability in chenodeoxycholic acid (CDCA), and the chenodeoxycholic acid (CDCA). Deoxycholic acid (DCA, deoxycholic acid) inhibitory ability was seen at a lower level.

Next, the comparative results of the inhibitory activity obtained in vitro through the kit and the inhibitory activity obtained when the bile acid is directly supplied to the body will be described.

3 is a graph showing the number of microorganisms in the colon when bile acid is directly supplied into the body, and RT-PCR (Real Time PCR) using a DNA primer for the target microorganism was used to obtain accurate quantification. .

At this time, the bile acid was dissolved in sodium bicarbonate (20 mg/ml) and supplied, and about 30 mg of each bicarbonate was dissolved in 10 ml.

As described above, the sodium bicarbonate (sodium bicarbonate) is a raw material for materials used for food, such as baking soda, and has the ability to dissolve bile acids, so it is a key material to be used in future therapeutic applications.

On the other hand, the control group used a vehicle (vehicle), that is, mice fed only a sodium bicarbonate (sodium bicarbonate) dilution solution containing no bile acid.

Looking at the graph shown in (a) of FIG. 3, with respect to the number of microbial populations of Escherichia coli present in the intestine, the deoxycholic acid (DCA, deoxycholic acid) was compared with the control vehicle (vehicle) It was shown that the number of individuals was suppressed, and the chenodeoxycholic acid (CDCA, chenodeoxycholic acid) showed a larger number of results when compared to the control, but it was found to be inhibited, and in the case of the cholic acid (CA, cholic acid) Results in populations almost similar to those of the control group were observed.

And in the result of the ursodeoxycholic acid (UDCA, ursodeoxycholic acid) and lithocholic acid (LCA, lithocholic acid) that do not show inhibitory ability against the Escherichia coli microorganism, the Escherichia coli proliferates and the number of individuals increases. phenomenon was observed.

With respect to the number of Proteus mirabilis microorganisms, as shown in FIG. 3 (b), compared to the control group deoxycholic acid (CDCA, chenodeoxycholic acid), deoxycholic acid (DCA, deoxycholic acid), The population was suppressed in ursodeoxycholic acid (UDCA, ursodeoxycholic acid) and lithocholic acid (LCA, lithocholic acid), and in cholic acid (CA, cholic acid) with low inhibitory ability, the Proteus mirabilis (proteus mirabilis) The number of individuals increased significantly compared to the control group, and an increase in the number of individuals was observed.

And with respect to the number of Enterococcus faecalis microorganisms, as shown in FIG. In the case of DCA, deoxycholic acid), the number of individuals was suppressed, and the result was almost similar to that of the control group, and ursodeoxycholic acid (UDCA, ursodeoxycholic acid) and lithocholic acid (LCA, lithocholic acid), which had weak inhibitory ability, were used in the control group. The comparison number increased, and in the case of the cholic acid (CA, cholic acid), a significantly larger number of individuals could be observed compared to the number of the control group.

Based on the above results, in comparison with the inhibitory activity results derived from the kit mentioned above, in the case of the Escherichia coli, cholic acid (CA, cholic acid) in the kit results tested in vitro, When compared with the results showing the inhibitory ability of chenodeoxycholic acid (CDCA, chenodeoxycholic acid) and deoxycholic acid (DCA, deoxycholic acid), similarly, the deoxycholic acid (DCA, deoxycholic acid) inhibited the number of individuals compared to the control group, the cholic acid ( CA, cholic acid) was similar to the control population, and the keno deoxycholic acid (CDCA, chenodeoxycholic acid) was decreased in number, but a result similar to that of the kit was observed through the result with a slightly larger number than the control group.

And in the case of the Proteus mirabilis, when compared with the results showing the inhibitory ability in deoxycholic acid (DCA, deoxycholic acid) and chenodeoxycholic acid (CDCA, chenodeoxycholic acid) in the kit results, the intestinal When the deoxycholic acid (DCA, deoxycholic acid) and chenodeoxycholic acid (CDCA, chenodeoxycholic acid) are supplied in similarity could be obtained.

Finally, in the case of the Enterococcus faecalis, when compared to the results of the kit, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA, chenodeoxycholic acid) showed inhibitory activity. , The population of Enterococcus faecalis present in the intestine is inhibited by the deoxycholic acid (DCA, deoxycholic acid) and chenodeoxycholic acid (CDCA, chenodeoxycholic acid) compared to the control group, resulting in a population similar to that of the control group showed

In conclusion, if a bile acid having a low inhibitory ability against the target microorganism is supplied to increase the occupancy rate in the body, the number of the target microorganism is increased, whereas when a bile acid having a high inhibitory ability against the target microorganism is supplied, a result similar to or more inhibited than that of the control group can be derived. there was.

And through the result of the number of microorganisms when the bile acids are supplied to the body corresponding to the result of the diameter size of the inhibition region derived from the kit, the inhibitory ability of bile acids against various microorganisms can be easily and effectively analyzed in vitro, , it can be seen that the patient's response can be predicted in advance when the bile acid is applied as a therapeutic therapy, and thus it can be seen that there is an effect that can be used in clinical practice.

The embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiment of the present invention and do not represent all the technical spirit of the present invention, so various equivalents that can be substituted for them at the time of the present application It should be understood that there may be variations and examples.

1: plate 2: bile acid disk

Claims (5)

delete delete delete Preparing a medium (plate) 1 corresponding to the microorganism list and quantity (S10);
dispensing distilled water (DW) into each of the glass tubes corresponding to the number of microorganisms (S20);
Using a sterile cotton swab to scrape the cultured bacteria in the source medium (plate) (1) sufficiently, and then dissolve in distilled water (DW) dispensed in the glass tube to make a solution (S30);
Wetting a cotton swab with the solution mixed with bacteria and plated on the plate 1 ( S40 );
placing different types of bile acid disks (2) on the plate (1) using tweezers (S50);
culturing the medium (plate) (1) on which the bile acid disk (2) is placed in an incubator (S60); and
measuring the diameter of the bile acid inhibition region after the culture is completed (S70);
A step (S80) of supplying bile acid dissolved in sodium bicarbonate to the body, quantifying the number of microorganisms in the large intestine and comparing it with the diameter of the measured inhibition region (S80);
The bile acid disk is treated with bile acid with a buffer added to the blank disk,
The buffer is ethanol to tetrahydrofuran,
The bile acid is cholic acid (CA, cholic acid), lithocholic acid (LCA, lithocholic acid), deoxycholic acid (DCA, deoxycholic acid), chenodeoxycholic acid (CDCA, chenodeoxycholic acid), ursodeoxycholic acid (UDCA, ursodeoxycholic acid),
In quantifying the number of microorganisms in the large intestine, RT-PCR (Real Time PCR) method is used. Inhibitory ability prediction method using a kit for predicting intestinal microorganism inhibition ability using bile acids. delete

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