JP2021191257A - Constipation resolution composition - Google Patents

Abstract

To provide a constipation resolution composition which comprises rhamnan sulfate.SOLUTION: Resolution is made by a constipation resolution composition which comprises a sulfated polysaccharide. Here, the composition preferably comprises a blood glucose level decreasing effect, the sulfated polysaccharide is preferably rhamnan sulfate, and the composition is preferably for an obese person or a person taking a high-fat meal.SELECTED DRAWING: Figure 3

Description

The present invention relates to a constipation relieving composition, particularly a constipation relieving composition having an effect of improving the intestinal environment of an obese person or a high-fat diet.

In Japan, the number of metabolic syndrome and its preliminary group is said to reach more than 10 million, and the number of affected people has increased for the fifth consecutive year, and the situation has not stopped. The Ministry of Health, Labor and Welfare has taken measures for "Health Japan 21" based on the Health Promotion Law, but no signs of improvement have been recognized. Medical expenses are increasing along with the increase in metabolic syndrome, and according to the data by disease classification of national medical expenses, "circulatory system diseases" caused by metabolic syndrome are on a scale of over 6 trillion yen, which is serious. It has become a challenge.
Generally, constipation refers to a state in which there is no bowel movement for 3 to 5 days or more, but there are individual differences in bowel movements, and if there is no discomfort even if there is no bowel movement for 2 to 3 days, it is not called constipation and every day. Constipation is when there is discomfort or residual stool even if there is defecation. In the case of constipation, it takes effort and pain when defecation, and there are symptoms such as bloating and abdominal pain, which often interferes with daily life. It is said that many young women, in particular, have constipation, and many laxatives to relieve this are on the market. However, if too much laxative is used, the body may get used to it, the effect may be diminished, or the dose may be increased. Therefore, a laxative that can be used stably for a long period of time has been desired.
By the way, ramnan sulfate extracted from sea lettuce growing in the sea near Japan is a sulfated polysaccharide whose main component is rhamnose, and is widely distributed naturally with a molecular weight of several hundred thousand to several million. There is. Ramnan sulfate is known to have an effect of suppressing an increase in blood glucose level and an antiviral effect (Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2008-184390 Japanese Unexamined Patent Publication No. 2019-137644

Marta Olivares et al. (2018) The DPP-4 inhibitor vildagliptin impacts the gut microbiota and prevents disruption of intestinal homeostasis induced by a Western diet in mice, Diabetologia 61: 1838-1848 Qian Zhang et al. (2017) Vildagliptin increases butyrate-producing bacteria in the gut of diabetic rats. PLoS ONE 12 (10): e0184735

However, the effect of ramnan sulfate on living organisms has not been fully known.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a constipation relieving composition containing ramnan sulfate.

As a result of diligent studies, the present inventor has found that sulfated polysaccharides (particularly, ramnan sulfate) have an effect of improving the intestinal environment, and have basically completed the present invention.
Thus, the constipation relieving composition according to the present invention is characterized by containing a sulfated polysaccharide (particularly, ramnan sulfate). It was found that ingestion of ramnan sulfate has the effect of increasing intestinal lactic acid bacteria (Lactobacillus, Bacillus coagulans, etc.), improving the intestinal environment, and relieving constipation. ..
At this time, the constipation relieving composition is preferably for obese people or for high-fat diet consumers.
An obese person means a person with a BMI (= weight (kg) / (height (m) x height (m)) of 25 or more.
A high-fat diet means that the energy ratio of fat is 50% or more of the total energy ingested from the diet. In a general diet, the energy ratio of fat is about 30%, but if you ingest a much higher energy ratio than this, you are more likely to develop obesity and metabolic syndrome.

Further, at this time, it is preferable that the blood glucose level is lowered. As one of the exacerbating factors of the symptoms of obesity / metabolic syndrome, there is Oscillibacter valericigenes, which is an intestinal bacterium. This bacterium is said to increase blood glucose levels by increasing in obese people and high-fat dieters. On the other hand, the constipation-relieving composition of the present invention also has the effect of reducing the blood glucose level of a high-fat diet ingestor by reducing the bacteria of the genus Osiribacta.
In the above invention, it is preferable that the effect of improving the intestinal environment is to reduce the F / B ratio, which is the ratio of Firmicutes and Bacteroidetes of the intestinal bacteria.
Further, it is preferable that the effect of improving the intestinal environment is to increase lactic acid bacteria (for example, Lactobacillus genus, Bacillus coagulans, etc.) of intestinal bacteria.
Comparing mice transplanted with gut microbiota in obese people with mice transplanted with gut microbiota in lean people, it is known that the former gains significant weight. In the former, it was found that the proportion of bacteria belonging to the phylum Firmicutes was high, and in the latter, the proportion of bacteria belonging to the phylum Bacteroides was high. From this, it was found that the intestinal flora controls the absorption rate of lipids. Intestinal bacteria belonging to the Firmicutes phylum are known to increase fat absorption efficiency and promote obesity. In this way, by examining the Firmicutes / Bacteroidetes phylum ratio (F / B ratio) of the intestinal flora of an individual, the degree of obesity can be understood to a certain extent, and the F / B ratio is the obesity index. It is called. According to the present invention, by reducing the F / B ratio, the effect of improving the intestinal environment can be exhibited and constipation can be eliminated.

In addition, the present invention is preferably taken in combination with a high-fat diet. According to the present invention, since the constipation relieving composition reduces the F / B ratio, it is easy to exert a constipation relieving effect and an anti-obesity effect, especially when a high-fat diet is ingested.
Further, the present invention is preferably a health food for oral administration. The toxicity of oral ingestion of ramnan sulfate is extremely low. However, it is difficult to take too much at once. Therefore, the dose of ramnan sulfate is not particularly limited, but is 50 mg to 400 mg, preferably 50 mg to 200 mg, and more preferably 100 mg to 200 mg per day for human adults.
The dosage form at this time can be appropriately set, and examples thereof include solid preparations such as tablets, granules, capsules, powders and powders, and liquid preparations such as liquids and suspensions.

In the food field, a food composition can be provided by blending an effective amount of ramnan sulfate capable of exerting an effect of improving the intestinal environment into various foods as a food material. Examples of the food composition include foods for specified health use, foods with nutritional function, foods with functional claims, foods for hospital patients, supplements and the like, in addition to general foods. In addition, it is used as a food additive.
Food compositions include, for example, processed livestock products, processed agricultural products, beverages (soft beverages, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, nutritional drinks, concentrated beverages, etc.), powdered beverages (powdered juice, etc.). Examples include powdered soup), confectionery (candy (throat candy), cookies, biscuits, gum, gummy, chocolate, etc.), bread, cereals, seasonings, and the like.

In the case of health food, it can be provided in the form of capsules, troches, syrups, granules, powders and the like.
The present invention can also be provided as foods for specified health use, foods with nutritional function, foods with functional claims, and the like. Specified health foods are foods containing health functional ingredients that affect physiological functions and the like, and can be labeled to be suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, the food is labeled as an anti-obesity use and sold. A nutritionally functional food is a food used for supplementing nutritional components (vitamins, minerals) and displays the function of the nutritional component. In order to sell as a nutritionally functional food, the amount of nutritional components contained in the daily intake guideline must be within the specified upper and lower limits, and not only the nutritional function label but also the warning label, etc. You also need to.

Foods with functional claims are foods with functional claims based on scientific evidence at the responsibility of the business operator. Information on the basis of safety and functionality was notified to the Commissioner of the Consumer Affairs Agency before the sale.
INDUSTRIAL APPLICABILITY The present invention is used as a food for specified health use for anti-constipation, a food with nutritional function for anti-constipation, and a food with functional claims for anti-constipation, which contains ramnan sulfate as an active ingredient and has an effect of improving the intestinal environment. At this time, anti-constipation can be used for anti-constipation / anti-obesity.
The present invention contains ramnan sulfate as an active ingredient, and is a health food such as a food for specified health use, a food with nutritional function, and a food with functional claims, which has an effect of improving the intestinal environment and an effect of relieving constipation, especially for obese people or high-fat diet consumers. Used for.
Further, the lactic acid bacterium growth promoter according to another invention is characterized by containing a sulfated polysaccharide. At this time, it is preferable that the sulfated polysaccharide is ramnan sulfate. This lactic acid bacterium growth promoter can accelerate the growth time or improve the growth rate by adding the product of the present invention, for example, when culturing lactic acid bacteria in an in vitro system. The amount added to the medium is 0.03 mg / ml to 100 mg / ml, preferably 0.1 mg / ml to 30 mg / ml, and more preferably 0.3 mg / ml to 10 mg / ml as ramnan sulfate.

According to the present invention, it is possible to provide a constipation relieving composition, particularly a constipation relieving composition having an effect of improving the intestinal environment of an obese person or a high-fat diet ingestor. Further, by adding the product of the present invention when culturing lactic acid bacteria in an in vitro system, the growth time can be shortened and the growth rate can be improved.

It is a line graph which showed the weight change of each group in a mouse test. It is a bar graph which shows the blood component of each group. The results of (A) blood glucose level, (B) triglyceride, and (C) total cholesterol are shown. It is a bar graph which shows (A) stool weight and (B) stool calorie of each group. It is a graph which shows the result of having examined the relative abundance of bacteria in feces at the gate level. NF means a normal diet, HF means a high-calorie diet, RS means ramnan sulphate, 1w means the first week from the start of ramnan sulphate administration, and 4w means the fourth week from the start of ramnan sulphate administration. Relative abundance of Bacteroidetes in the group receiving a normal diet (NF) or a high-calorie diet (HF) in the feces at 1 or 4 weeks after the start of ramnan sulfate administration, (B). ) It is a bar graph showing the F / B ratio (Firmicutes / Bacteroidetes). It is a bar graph which shows the relative ratio to the normal diet group about 4 kinds of bacteria in feces. (A) Lactobacillus johnsonii, (B) Lactobacillus reuteri, (C) Lactobacillus intestinalis, and (D) Oscillibacter valericigenes are shown, respectively. It is a graph which shows the result of confirming the influence of ramnan sulfate on the growth of JCM8130 (Lactobacillus paracasei). The results of (A) Ramnox 100, 10 mg / ml and (B) Ramnox 100, 0, 0.625, 1.25, 2.5, 5 and 10 mg / ml are shown. It is a graph which shows the result of confirming the influence of ramnan sulfate on the growth of JCM2257 (Bacillus coagulans). The results of (A) Ramnox 100, 10 mg / ml and (B) Ramnox 100, 0, 0.625, 1.25, 2.5, 5 and 10 mg / ml are shown. It is a graph which shows the result of confirming the influence of ramnan sulfate on the growth of JCM11046 (Lactobacillus gas seri). The results of (A) Ramnox 100, 10 mg / ml and (B) Ramnox 100, 0, 0.625, 1.25, 2.5, 5 and 10 mg / ml are shown. It is a figure which shows the breakdown of the test subject in <human test 2>. It is a bar graph comparing the calculated values of defecation status at 0 W, 1 W and 2 W between the test food group (white) and the control food group (gray) (data are shown by "mean value + standard deviation". ). (A) The result of the number of defecations and (B) the result of the number of defecation days are shown. It is a bar graph comparing the amount of change in defecation status at 0 W, 1 W and 2 W between the test food group (white) and the control food group (gray) (data is shown by "mean value + standard deviation". ). (A) The result of the number of defecations and (B) the result of the number of defecation days are shown. The calculated values of defecation status at 0 W, 1 W, and 2 W when stratified analysis of those with intestinal bacterial species above the average value were compared between the test food group (white) and the control food group (gray). It is a bar graph (data is shown by "mean value + standard deviation"). (A) The result of the number of defecations and (B) the result of the number of defecation days are shown. The changes in defecation status at 0 W, 1 W, and 2 W when stratified analysis was performed on those with an intestinal bacterial species above the average value were compared between the test food group (white) and the control food group (gray). It is a bar graph (data is shown by "mean value + standard deviation"). (A) The result of the number of defecations and (B) the result of the number of defecation days are shown. It is a bar graph showing the result of amplicon sequence analysis of the bacterial flora in feces and the occupancy rate of Clostridia at the class level. The data are shown as "mean ± standard deviation", the test food group is shown in white and the control food group is shown in gray. It is a bar graph showing the result of amplicon sequence analysis of the bacterial flora in feces and the occupancy rate of Negative cutes at the class level. The data are shown as "mean ± standard deviation", the test food group is shown in white and the control food group is shown in gray. It is a bar graph showing the result of amplicon sequence analysis of the bacterial flora in feces and the occupancy rate of Acidaminococcles at the eye level. The data are shown as "mean ± standard deviation", the test food group is shown in white and the control food group is shown in gray.

Next, embodiments of the present invention will be described with reference to figures and tables, but the technical scope of the present invention is not limited to these embodiments, and various embodiments are made without changing the gist of the invention. Can be carried out at.
<Preparation of ramnan sulfuric acid with different molecular weight>
As the ramnan sulfate, any naturally occurring material can be used. In this embodiment, the one obtained by extracting hot water from Monostroma nitidum was used. The brief explanation is as follows. The dried seaweed was washed with water and extracted with hot water (95 ° C to 100 ° C) for 6 hours. The obtained hot water extract was dialyzed against pure water and freeze-dried. After dissolving this freeze-dried product in water, it was applied to anion exchange chromatography and continuously eluted to purify ramnan sulfate. This was used as a sample of the invention.
The sulfated polysaccharide (for example, ramnan sulfate) that can be used in the present invention can be prepared by various methods other than the above method. Further, the raw material is not limited to Monostroma nitidum, and can be used such as Ana-Aosa and Ribbon-Aosa.

Next, using the ramnan sulfate obtained above, the effect of improving intestinal bacteria and the effect of anti-obesity in mice were evaluated by the following methods.
<Mouse test>
An oral administration test was performed using spontaneously-onset type 2 diabetic mice (NYS / Hos Male, 9 weeks old). The diet to which each component of the following test groups 1 to 4 was added was ingested for 4 weeks, and the intestinal flora was analyzed after 1 week and 4 weeks.
Test group 1: Normal diet Test group 2: Normal diet + ramnan sulfate 0.25 mg / g addition Test group 3: High-fat diet test group 4: High-fat diet + ramnan sulfate 0.25 mg / g addition The number of repeated tests was 5. .. During the test period, food intake measurement, body weight measurement and fecal collection were performed once a week, and a blood biochemical test was performed at the end of the test.

FIG. 1 shows the changes in body weight of each experimental group. In test group 3 fed only a high-fat diet, the body weight increased significantly (p <0.01) as compared with the normal diet group. In contrast to test group 3, test group 4 in which ramnan sulfate was added to a high-fat diet showed a tendency to suppress weight gain. Regarding the amount of food intake, no significant difference was observed between the test groups 1 and 2 and the test groups 3 and 4.
FIG. 2 shows the analysis results of the blood components collected at the end of the test. It is known that the strains of mice used in this study have elevated blood glucose levels due to high-fat dietary load even at a young age. In this study as well, the blood glucose level increased significantly (p <0.05) in the high-fat diet group (test group 3). On the other hand, in the group in which ramnan sulfate was added to the high-fat diet (test group 4), the elevated blood glucose level tended to decrease in the test group 3. From this, it was found that ramnan sulfate has an effect of reducing blood glucose level, especially for those who ingest a high-fat diet.
In addition, the neutral fat in plasma was significantly (p <0.05) decreased by the addition of ramnan sulfate (test groups 2 and 4) in both the normal diet group and the high-fat diet group. Total cholesterol in plasma was also significantly increased in test group 3 of the high-fat diet group, but was significantly (p <0.05) decreased by the addition of ramnan sulfate (test group 4). Thus, it was found that ramnan sulfate reduces the high blood fat and contributes to the improvement of metabolic syndrome.

FIG. 3 shows the weight and calories of feces per day. No difference was observed between the normal diet groups (test groups 1 and 2), but between the high-fat diet groups (test groups 3 and 4), the addition of ramnan sulfate (test group 4) was significantly (p <0.05). ) Fecal weight and calories were increased. From this, it was found that the normal amount of feces increased and constipation could be resolved. In addition, the calories in feces were also significantly increased, indicating that it contributes to the anti-obesity effect.
Next, the sequence of 16s rRNA derived from bacteria contained in the collected feces was analyzed using the Nanopore 16S Barcoding Kit (Oxford Nanopore). FIG. 4 shows the relative abundance of bacteria at the phylum level. Firmicutes accounted for most of the NSY / HOS type 2 diabetic mice, followed by Bacteroidetes, Actinobacteria (around 1%), and Deferribateres (1% or less). FIG. 5 shows the relative abundance of (Bacteroidetes) and (B) Firmicutes / Bacteroidetes in the group treated with (A) normal diet or high-calorie diet at week 1 or 4. )showed that. It was found that in the group fed a high-calorie diet, administration of ramnan sulfate increased Bacteroidetes and decreased the F / B ratio as compared with the control. It is generally known that obese people have more Firmicutes and less Bacteroidetes, and the F / B ratio is used as an obesity index. That is, it is considered that when the F / B ratio becomes smaller, obesity is resolved.

FIG. 6 shows a graph of bacteria significantly increased or decreased by administration of ramnan sulfate. As shown in the figure, an increase in lactic acid bacteria (Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus intestinalis, etc.) was observed by administration of ramnan sulfate in both the high-fat diet and the normal diet administration group. In addition, Oscillibacter valericigenes, which is one of the exacerbating factors of obesity / metabolic syndrome symptoms, was increased in the high-fat diet group (test group 3), but it was decreased by ramnan sulfate. (Test group 4). From the above, it was found that oral ingestion of ramnan sulfate changes the composition of the intestinal flora. In particular, it was found that the group treated with a high-fat diet (especially considered to be more common in obese people) improved various symptoms of obesity. Bacteria of the genus Osiribacta have a positive correlation with fasting blood glucose level and AUC of oral glucose tolerance test (OGTT), and it has been reported that they are reduced by DPP-4 inhibitor that lowers blood glucose level (Non-Patent Documents 1 and 2). That is, the blood glucose level can be reduced by reducing the bacteria of the genus Osiribacter. From the results of FIG. 2 (A), ramnan sulfate significantly lowers the increased blood glucose level in the high-fat diet group. This effect was thought to be obtained by the reduction of osiribacter by ramnan sulfate.

<Confirmation test of the effect of ramnan sulfate on the growth of microorganisms>
From the results of the above <mouse test>, it was found that ramnan sulfate has a good effect on the growth of lactic acid bacteria. Therefore, in an in vitro test system, the effect of ramnan sulfate on the growth of microorganisms (particularly lactic acid bacteria) was confirmed.
1. 1. Test method
(1) Culture of microorganisms
55 g of MRS powder medium (Difco Lactobacilli MRS Broth) was weighed and measured up to 1000 mL with purified water. This was stirred, and after dissolution, sterilization was performed in an autoclave under the conditions of 2 atm, 121 ° C., and 15 minutes. This was used as MRS medium.
The ramnan sulfate-added medium is an autoclaved MRS medium with a concentration of Ramnox 100 (Rhamnox 100 (manufactured by Gangnam Kako Co., Ltd., 100% human exo extract, 60% or more ramnan sulfate content). The same applies hereinafter) at a concentration of 10 mg / ml. (Or 0.625, 1.25, 2.5, 5 and 10 mg / ml) was added. Since ramnan sulfate is difficult to dissolve, the medium dissolved in an autoclave at 105 ° C for 1 minute was used as the ramnan sulfate-added MRS medium.
After cooling the MRS medium and the MRS medium supplemented with ramnan sulfate to room temperature, 100 mL each was dispensed into an Erlenmeyer flask with a lid. 100 μL of each of the microorganisms for examination dissolved in the MRS medium was added thereto, and the cells were cultured at 37 ° C.
The degree of microbial growth was examined by measuring the absorbance at 660 nm (OD660). The measurement of OD660 was started from the time when the microorganism entered the logarithmic growth phase, and the measurement time interval was 1 hour.
As lactic acid bacteria, JCM8130 (Lactobacillus paracasei), JCM2257 (Bacillus coagulans) and JCM11046 (Lactobacillus gas seri) were obtained from RIKEN BioResource Research Center and used.

(2) Microorganism count
Weighed 24.6 g of BCP-added plate agar medium (for measuring the number of lactic acid bacteria of Eiken Chemical), and increased the size to 1000 mL with purified water. This was stirred, and after dissolution, sterilization was performed in an autoclave under the conditions of 2 atm, 121 ° C., and 15 minutes. This was used as a BCP-added plate agar medium.
1 mL of the microbial liquid medium after culturing was taken, and sterilized physiological saline was added to prepare a 10-fold diluted sample. This work was repeated until it was diluted 1 billion times.
1 mL each of lactic acid bacteria diluted solutions having a dilution ratio of 1000 to 1 billion times was taken and added to a petri dish for culture. About 20 mL of BCP-added plate agar medium kept at 44 ° C was added to the petri dish containing the sample and mixed well. After the medium was coagulated, it was inverted and cultured at 37 ° C for 72 hours ± 3 hours. As a control, culturing was carried out in the same manner as above using the sterilized physiological saline used for dilution. After culturing, colonies whose medium turned yellow were counted as lactic acid bacteria. The number of lactic acid bacteria was calculated by multiplying the count number by the dilution ratio.

2. 2. Test Results As shown in FIGS. 7 (A), 8 (A) and 9 (A), all three types of lactic acid bacteria have Ramnox 100 at a concentration of 10 mg / ml (6 mg / ml or more as ramnan sulfate). Growth was promoted by adding to the medium. When CFU (colony forming unit) was measured, as shown in Table 1, it was found that all of them increased with the addition of ramnan sulfate, and the number of bacteria increased in correlation with turbidity.

Furthermore, Ramnox 100 was added to the medium at a concentration of 0.625, 1.25, 2.5, 5, 10 mg / ml in order to confirm whether or not the growth was promoted depending on the concentration of ramnan sulfate, and the growth promoting effects were compared. As shown in 7 (B), FIG. 8 (B) and FIG. 9 (B), at a concentration of 0.625 mg / ml (0.375 mg / ml as ramnan sulfate) or higher in JCM8130 (Lactobacillus paracasei) and JCM2257 (Bacillus coagulans). , JCM11046 (Lactobacillus gasseri) promoted growth in a concentration-dependent manner at a concentration of 1.25 mg / ml (0.75 mg / ml as ramnan sulfate) or higher.
Thus, it was found that ramnan sulfate has an effect of growing lactic acid bacteria in an in vitro system.

<Human test 1>
Next, the effect of ramnan sulfate was investigated by several human volunteers in Gangnam Kako Co., Ltd. Ramnox (100 mg / day) was orally administered to 3 healthy volunteers. Ramnox (manufactured by Gangnam Kako Co., Ltd.) manufactured Monostroma nitidum by hot water extraction at 95 ° C to 100 ° C for 6 hours. Diatomaceous earth was added and centrifuged, the supernatant was filtered under reduced pressure, and the filtrate was filtered through an ultrafiltration membrane (molecular weight cut off 10,000). The concentrate was spray-dried to obtain a powder (ramnan sulfuric acid content 87%).
As a result, I got the impression that the number of defecations and the amount of defecations increased. At the same time, those who had unpleasant symptoms such as hunger were resolved. No side effect symptoms such as diarrhea and vomiting were observed.
In general constipation, it means that there is no defecation for 3 to 5 days or more. In addition, constipation requires effort and pain during defecation, and there are symptoms such as bloating and abdominal pain. According to the constipation relieving composition of the present embodiment, it was found that continuous ingestion seems to increase the number of defecations and the amount of defecation. Thus, it was found that constipation was significantly resolved even in humans.

According to the data of this embodiment, it was found that the constipation symptom was resolved by the administration of ramnan sulfate even in humans. In addition, when combined with the results of the above <mouse test>, it was considered that this constipation-relieving effect was due to a change in the composition of the intestinal bacterial flora. Thus, it was found that administration of ramnan sulfate can change the composition of the intestinal flora in a better direction, relieve constipation, and improve obesity, especially when a high-calorie diet is ingested.

<Human test 2>
1. 1. Test method
(1) Subject A placebo-controlled randomized, double-blind, parallel-group comparative study was adopted. The selection criteria for the subjects are (a) healthy men and women aged 20 to 65 years old at the time of consent acquisition, and (b) constipation-prone people (those who satisfy the two conditions of defecation frequency 3 to 5 times a week). And said.
(2) Inspection items As inspection items, a stool test (amplicon sequence analysis) is performed at 0W in a stool test (a stool collected from 5 days before the visit to the day before the visit in principle), and a stool test (visit 5) is performed at 2W. An intestinal bacterial test (amplicon sequence analysis) was performed on the stools collected from the day before to the day before the visit in principle. In addition, as a WEB diary (life diary / bowel movement diary), a meal survey table / bowel movement diary (records from the start date of the previous observation period to the day before the end of 2W of this test) was performed. At this time, as input items, the bowel movement status (number of bowel movements, date of collection, confirmation of how many times the bowel movement was collected, Bristol scale (fecal properties), and amount of bowel movements were recorded.
(3) Test food As the test food, a capsule containing Ramnox (manufactured by Gangnam Kako Co., Ltd., Ramnan sulfate content 87%) and 100 mg was used as the test food, and a capsule containing no Ramnox was used as the control food.
For the above test food, 1 capsule daily was taken after breakfast with water or lukewarm water without chewing. The test food was instructed to be opened immediately before ingestion.
(4) Allocation method The test subjects selected based on the results of the preliminary examination, etc., are considered for their age, gender, and WEB diary (number of defecations per week, average value per defecation on the Bristol scale (fecal properties)). Then, the two groups were assigned to each layer. For the WEB diary, the value for 2 weeks from the start date of the previous observation period was used. Consideration was given so that the stratification factors were not significantly biased among the test food groups.

(5) Aggregation / analysis method
(i) Primary endpoint: Defecation status (number of defecations, number of days of defecation)
At 0W, it is one week immediately before the visit at 0W, at 1W it is one week from the test food intake start date (visit date at 0W), and at 2W it is one week immediately before the visit at 2W. The total number of each was calculated. A comparison between the control food group and the test food group using the calculated value and the amount of change from 0 W was evaluated by the unpaired t-test. The comparison over time at 0 W, 1 W, and 2 W using the calculated values was evaluated by Dunnett's test.

As an additional analysis, the number of gut microbiota detected by the test subject's gut microbiota (fecal flora (amplicon sequence analysis) is "less than average" and "mean" with respect to the overall average at 0 W. A stratified analysis was performed according to the value above the value).
(ii) Secondary endpoint: fecal bacterial flora (amplicon sequence analysis)
(a) Library preparation and sequencing method
(A) After adding Lysis Solution F (Nippon Gene) to the DNA extraction sample, it was pulverized at 1500 rpm for 2 minutes using Shake Master Neo bms. The crushed sample was allowed to stand at 65 ° C. for 10 minutes. Then, centrifugation was performed at 12000 × g for 1 minute, and the supernatant was separated. DNA was purified from the fractionated solution using the MPure 12 system and the MPure Bacterial DNA Extraction Kit (MP Bio).
(B) Quantitative measurement of DNA solution
The concentration of the DNA solution was measured using Synergy LX (Bio Tek) and QuantiFluor dsDNA System (Promega).
(C) Library creation
A library was prepared using the 2-step tailed PCR method.
(D) Quantification of library
The concentration of the prepared library was measured using Synergy H 1 (Bio Tek) and QuantiFluor dsDNA System.
(E) Checking the quality of the library
The quality of the prepared library was confirmed using Fragment Analyzer and dsDNA 915 Reagent Kit (Advanced Analytical Technologies).
(F) Sequencing analysis
Sequencing was performed using the MiSeq system and MiSeq Reagent Kit v 3 (Illumina) under the condition of 2 × 300 bp.

(b) Data analysis method
(A) Extraction of reads that match the primer sequence
Only the read sequence that exactly matches the primer sequence used at the beginning of reading the read sequence obtained by using fastq_barcode_spliltter of Fastx toolkit (ver 0 0 14) was extracted. When N mix was included in the primer sequence, this operation was repeated in consideration of the number of N (6 types on the forward side and 6 types on the reverse side for a total of 36 types).
(B) Analysis using Qiime 2
After removing the primer sequence, chimera sequence, and noise sequence with the dada 2 plug-in of Qiime 2 (ver 2020 2), the representative sequence and OTU table were output.
The occupancy rate was calculated by calculating the number of pair reads for each bacterium and calculating the ratio of the number of pair reads of the bacterium of interest to the number of pair reads of all the bacterium. A comparison between the control food group and the test food group using the measured values and the amount of change from 0 W was evaluated by the unpaired t-test. The time-dependent comparison between 0 W and 2 W using the measured values was evaluated by the paired t-test.

2. 2. Test results
(1) Study period The study was conducted from February 28, 2020 to April 23, 2020 (from the pre-examination date to the date when the investigator determined that further follow-up of adverse events was unnecessary).
(2) Breakdown of test subjects The test was started with 38 people (19 people / group) while the target number of test subjects was 40 (20 people / group). The breakdown of the test subjects is shown in FIG.
(3) Efficacy endpoint Primary endpoint: Defecation status (number of defecations, number of days of defecation)
Tables 2 and 3 and FIGS. 11 and 12 show the calculated values when the analysis category was set to weekly and the results of the amount of change from 0 W, respectively. In the table, the data are shown as "mean ± standard deviation", and "#" was significant at a risk rate of less than 5% (p <0.05) in the intergroup comparison (unpaired t-test). In a comparative comparison (Dunnett's test), "**" was significant at a risk rate of less than 1% (p <0.01).

(i) Comparison between groups
(A) As a result of comparing the control food group and the test food group using the calculated values, no statistically significant difference was observed in all items.
(B) As a result of comparison between the control food group and the test food group using the amount of change, a statistically significant difference was found in the following items.
-Number of defecations: At 2 W, the test food group 1.5 ± 1.6 (times / week) showed a statistically significant higher value than the control food group 0.3 ± 1.7 (times / week) (p). <0.05).
(ii) Comparison over time As a result of comparing over time at 0 W, 1 W, and 2 W using the calculated values, statistically significant changes were observed in the following items.
-Number of defecations: In the test food group, a statistically significant increase was observed at 5.6 ± 1.9 (times / week) at 2W compared to 4.1 ± 1.5 (times / week) at 0W (p). <0.01).
-Number of days of defecation: In the test food group, a statistically significant increase was observed at 4.9 ± 1.2 (days / week) at 2W compared to 3.8 ± 1.0 (days / week) at 0W (p). <0.01).

(4) Stratification analysis: Average value of intestinal bacterial species or more The calculated values when the analysis category is set to weekly and the results of the amount of change from 0 W are shown in Tables 4, 5 and 13 and 14, respectively. .. In the table, the data are shown as "mean ± standard deviation", and "#" was significant at a risk rate of less than 5% (p <0.05) in the intergroup comparison (unpaired t-test). In a comparative comparison (Dunnett's test), "**" was significant at a risk rate of less than 1% (p <0.01).

(i) Comparison between groups
(A) As a result of comparing the control food group and the test food group using the calculated values, statistically significant differences were found in the following items.
-Number of days of defecation: At 2 W, the test food group 5.1 ± 1.1 (days / week) showed a statistically significant higher value than the control food group 3.7 ± 1.6 (days / week) (p). <0.05).
(B) As a result of comparison between the control food group and the test food group using the amount of change, a statistically significant difference was found in the following items.
-Number of defecations: At 2 W, the test food group 2.0 ± 1.7 (times / week) showed a statistically significant higher value than the control food group 0.0 ± 2.1 (times / week) (p). <0.05).
-Number of days of defecation: At 2 W, the test food group 1.4 ± 1.3 (days / week) showed a statistically significant higher value than the control food group-0.2 ± 1.8 (days / week) (). p <0.05).
(ii) Comparison over time As a result of comparing over time at 0 W, 1 W, and 2 W using the calculated values, statistically significant changes were observed in the following items.
-Number of defecations: In the test food group, a statistically significant increase was observed at 5.7 ± 1.9 (times / week) at 2W compared to 3.7 ± 1.1 (times / week) at 0W (p). <0.01).
-Number of days of defecation: In the test food group, a statistically significant increase was observed at 5.1 ± 1.1 (days / week) at 2W compared to 3.7 ± 1.1 (days / week) at 0W (p). <0.01).

(5) Secondary endpoint: fecal bacterial flora (amplicon sequence analysis)
The results of examining the occupancy rate at the class level are shown in FIGS. 15 and 16.
(i) Comparison over time As a result of comparison over time between 0W and 2W using the occupancy rate, statistically significant changes were observed in the following items.
・ Bacteria-Firmicutes-Clostridia
In the test food group, a statistically significant decrease was observed at 31.56 ± 12.64% at 2W compared to 36.09 ± 12.96% at 0W (p <0.05).
・ Bacteria-Firmicutes-Negativicutes
In the test food group, a statistically significant increase was observed at 7.45 ± 5.02% at 2W compared to 4.62 ± 5.03% at 0W (p <0.05).
The result of examining the occupancy rate at the order level is shown in FIG. The items recognized in the "eye" classification were as follows.
(ii) Comparison over time ・ Bacteria-Firmicutes-Negativicutes-Acidaminococcales
In the test food group, a statistically significant increase was observed at 3.18 ± 3.64% at 2W compared to 1.93 ± 2.98% at 0W (p <0.01).

3. 3. Discussion Clostridium is known to produce medium-length fatty acids, increase water absorption, and subsequently dry feces, causing constipation. One of the mechanisms by which lambnan sulfate relieves constipation may be the reduction of Clostridium class.
Ramnan sulfate selectively increased Negativicutes and Acid Amino Coccares. Although the biological effect on constipation is unknown, there are reports that the increase in these bacteria has a positive relationship with the improvement of constipation. Negativicutes was increased by administration of psyllium husks (derived from Plantago ovata seeds) and bifidobacteria-based probiotic treatment during improvement of constipation.
Acidaminococcus is one of the main factors healing constipation during fecal microflora transplantation in the administration of partially hydrolyzed guar gum (water-soluble fiber) in clinical and pediatric to improve constipation. Possibly ramnan sulfate may increase this.
As described above, according to the present embodiment, it was possible to provide a constipation-relieving composition having an effect of improving the intestinal environment, particularly for obese people or high-fat diet consumers. In addition, by adding the product of the present invention when culturing lactic acid bacteria in an in vitro system, it was possible to accelerate the growth time and improve the growth rate.

Claims (10)

A constipation-relieving composition comprising a sulfated polysaccharide. The constipation-relieving composition according to claim 1, which is for obese people or high-fat diet consumers. The constipation-relieving composition according to claim 1 or 2, which has a blood glucose lowering effect. The constipation-relieving composition according to any one of claims 1 to 3, wherein the sulfated polysaccharide is ramnan sulfate. The constipation according to any one of claims 1 to 4, wherein the effect of improving the intestinal environment is to reduce the F / B ratio, which is the ratio of the intestinal bacteria Firmicutes and Bacteroidetes. Elimination composition. The constipation-relieving composition according to any one of claims 1 to 5, wherein the effect of improving the intestinal environment is to increase the genus Lactobacillus of an intestinal bacterium. The constipation-relieving composition according to any one of claims 1 to 6, which is to be ingested together with a high-fat diet. The constipation-relieving composition according to any one of claims 1 to 7, which is a health food for oral administration. A lactic acid bacterium growth promoter characterized by containing a sulfated polysaccharide. The lactic acid bacterium growth promoter according to claim 9, wherein the sulfated polysaccharide is ramnan sulfate.

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