JP7601332B2 - Constipation relief composition - Google Patents

Description

The present invention relates to a constipation relief composition, in particular a constipation relief composition that has the effect of improving the intestinal environment of obese people or people on a high-fat diet.

In Japan, the number of people with metabolic syndrome or at risk of developing it is said to be over 10 million, and the number of people affected has been increasing for five consecutive years, showing no signs of stopping. The Ministry of Health, Labor and Welfare has implemented "Health Japan 21" measures based on the Health Promotion Act, but there are no signs of improvement. Medical expenses are increasing along with the increase in metabolic syndrome, and according to data on national medical expenses by disease classification, "circulatory system diseases" caused by metabolic syndrome exceed 6 trillion yen, becoming a serious issue.
Generally, constipation is defined as a state in which one has not had a bowel movement for 3-5 days or more, but bowel movements vary from person to person. If one has not had a bowel movement for 2-3 days without feeling any discomfort, one is not considered constipated. If one has a bowel movement every day but feels discomfort or has a feeling of residual stool, one is considered constipated. In the case of constipation, one must make an effort and suffer from pain when defecating, and one may experience symptoms such as abdominal distension and abdominal pain, which often interfere with daily life. It is said that many young women suffer from constipation, and many laxatives are available on the market to alleviate this condition. However, if one uses a laxative too much, the body may become accustomed to it, and the effect may weaken or the dosage may increase, so a laxative that can be used stably for a long time has been desired.
Rhamnan sulfate extracted from Ulva gracilis growing in the seas off the coast of Japan is a sulfated polysaccharide whose main component is rhamnose, and is widely distributed in nature with a molecular weight of hundreds of thousands to millions. Rhamnan sulfate is known to have blood sugar level suppressing effects, antiviral effects, etc. (Patent Documents 1 and 2).

JP 2008-184390 A JP 2019-137644 A

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 effects of rhamnan sulfate on the body are not 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 laxative composition containing rhamnan sulfate.

As a result of extensive research, the present inventors have found that sulfated polysaccharides (particularly rhamnan sulfate) have the effect of improving the intestinal environment, and have basically completed the present invention.
Thus, the constipation relieving composition of the present invention is characterized by containing sulfated polysaccharides (particularly rhamnan sulfate). It has been found that the ingestion of rhamnan sulfate increases the number of lactic acid bacteria (Lactobacillus genus, Bacillus coagulans, etc.) in the intestine, improving the intestinal environment and relieving constipation.
In this case, the constipation relieving composition is preferably for obese people or people who take a high-fat diet.
An obese person is someone whose BMI (= weight (kg) / (height (m) x height (m)) is 25 or higher.
A high-fat diet means that the energy ratio of fat is 50% or more of the total energy intake from food. In a normal diet, the energy ratio from fat is about 30%, but if you consume a diet that is significantly higher than this, you are more likely to develop obesity or metabolic syndrome.

In addition, it is preferable that the composition has a blood glucose lowering effect. One of the factors that aggravate the symptoms of obesity and metabolic syndrome is the intestinal bacterium Oscillibacter valericigenes. It is said that this bacterium increases blood glucose levels in obese people and people who eat high-fat diets. In contrast, the constipation relieving composition of the present invention also has the effect of reducing blood glucose levels in people who eat high-fat diets by reducing Oscillibacter bacteria.
In the above invention, the intestinal environment improving effect is preferably a decrease in the F/B ratio, which is the ratio of intestinal bacteria Firmicutes to Bacteroidetes.
Furthermore, it is preferable that the intestinal environment improving effect is to increase lactic acid bacteria (e.g., Lactobacillus genus, Bacillus coagulans, etc.) in the intestines.
It is known that mice transplanted with intestinal bacteria from obese individuals and mice transplanted with intestinal bacteria from lean individuals have a significant weight gain in the former. It was found that the former had a higher proportion of bacteria belonging to the Firmicutes phylum, while the latter had a higher proportion of bacteria belonging to the Bacteroidetes phylum. This shows that the intestinal flora controls the lipid absorption rate. In addition, it is known that intestinal bacteria belonging to the Firmicutes phylum increase fat absorption efficiency and promote obesity. Thus, by examining the Firmicutes/Bacteroidetes phylum ratio (F/B ratio) for the intestinal flora of an individual, the degree of obesity can be understood to a certain extent, and the F/B ratio is called the obesity index. According to the present invention, by reducing the F/B ratio, the effect of improving the intestinal environment is shown and constipation can be relieved.

In addition, the present invention is preferably taken together with a high-fat diet. According to the present invention, the constipation relieving composition reduces the F/B ratio, and therefore is more likely to exert constipation relieving and anti-obesity effects, particularly when a high-fat diet is taken.
Moreover, the present invention is preferably a health food for oral administration. The toxicity of rhamnan sulfate when orally ingested is extremely low. However, it is difficult to ingest a large amount at once. Therefore, the dosage of rhamnan 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 an adult human.
The dosage form in this case can be appropriately determined, and examples thereof include solid preparations such as tablets, granules, capsules, powders, and dispersing agents, and liquid preparations such as liquids and suspensions.

In the food field, rhamnan sulfate can be added as a food ingredient in an effective amount to various foods to provide food compositions. Examples of food compositions include general foods, foods for specified health uses, foods with nutritional functions, foods with functional claims, foods for hospital patients, supplements, etc. In addition, it can be used as a food additive.
Examples of food compositions include processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, energy drinks, concentrated beverages, etc.), powdered beverages (powdered juice, powdered soup, etc.), confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chocolate, etc.), bread, cereals, seasonings, etc.

In the case of health foods, they can be provided in the form of capsules, troches, syrups, granules, powders, etc.
The present invention can also be provided as a food for specified health uses, a food with nutrient function claims, or a food with functional claims. A food for specified health uses is a food containing functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for a specific health use with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, the food is a food that is sold with a label indicating that it is for anti-obesity use. A food with nutrient function claims is a food used to supplement nutritional ingredients (vitamins, minerals), and displays the function of the nutritional ingredients. In order to be sold as a food with nutrient function claims, the amount of nutritional ingredients contained in the daily recommended intake must be within the range of the specified upper and lower limits, and not only the nutritional function labeling but also a warning labeling must be provided.

Foods with functional claims are foods that display functionality based on scientific evidence at the responsibility of the business operator. Information on the basis of safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale.
The present invention contains rhamnan sulfate as an active ingredient and is used as an anti-constipation food for specified health uses, an anti-constipation nutritional functional food, or an anti-constipation functional food that has an effect of improving the intestinal environment. In this case, the anti-constipation use can also be for anti-constipation and anti-obesity.
The present invention is used in health foods such as foods for specified health uses, foods with nutrient functions, and foods with functional claims that contain rhamnan sulfate as an active ingredient and have effects of improving the intestinal environment and relieving constipation, particularly in obese individuals or those who consume a high-fat diet.
Another aspect of the present invention is a lactic acid bacteria growth promoter that contains a sulfated polysaccharide. In this case, the sulfated polysaccharide is preferably rhamnan sulfate. When lactic acid bacteria are cultured in an in vitro system, for example, the lactic acid bacteria growth promoter can be added to the culture medium to shorten the growth time or improve the growth rate. The amount of rhamnan sulfate added to the culture 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.

According to the present invention, it is possible to provide a constipation relieving composition, particularly a constipation relieving composition that has the effect of improving the intestinal environment of obese people or people who consume high-fat diets. Furthermore, when culturing lactic acid bacteria in an in vitro system, the addition of the product of the present invention can shorten the growth time and improve the growth rate.

1 is a line graph showing the weight change in each group in a mouse study. 1 is a bar graph showing blood components of each group, (A) blood glucose level, (B) triglyceride level, and (C) total cholesterol level. 1 is a bar graph showing (A) fecal weight and (B) fecal calories for each group. Figure 1 shows the relative abundance of bacteria at the phylum level in feces, where NF means normal diet, HF means high-calorie diet, RS means rhamnan sulfate, 1w means 1 week after starting rhamnan sulfate administration, and 4w means 4 weeks after starting rhamnan sulfate administration. FIG. 1 is a bar graph showing (A) the relative abundance of Bacteroidetes in feces in groups fed a normal diet (NF) or a high-calorie diet (HF) at 1 or 4 weeks after the start of rhamnan sulfate administration, and (B) the F/B ratio (Firmicutes/Bacteroidetes). 1 is a bar graph showing the relative ratio of four types of bacteria in feces to the normal diet group: (A) Lactobacillus johnsonii, (B) Lactobacillus reuteri, (C) Lactobacillus intestinalis, and (D) Oscillibacter valericigenes. 1 is a graph showing the effect of rhamnan sulfate on the growth of JCM8130 (Lactobacillus paracasei), (A) the results in the presence or absence of 10 mg/ml rhamnox 100, (B) the results in the presence or absence of 0, 0.625, 1.25, 2.5, 5 and 10 mg/ml rhamnox 100. 1 is a graph showing the effect of rhamnan sulfate on the proliferation of JCM2257 (Bacillus coagulans), (A) the results in the presence or absence of 10 mg/ml rhamnox 100, (B) the results in the presence or absence of 0, 0.625, 1.25, 2.5, 5 and 10 mg/ml rhamnox 100. 1 is a graph showing the effect of rhamnan sulfate on the growth of JCM11046 (Lactobacillus gasseri), (A) the results in the presence or absence of 10 mg/ml rhamnox 100, (B) the results in the presence or absence of 0, 0.625, 1.25, 2.5, 5 and 10 mg/ml rhamnox 100. FIG. 1 shows the breakdown of test subjects in <Human Study 2>. This is a bar graph comparing the calculated values of bowel movements at 0W, 1W, and 2W between the test food group (white) and the control food group (gray) (data shown as "mean + standard deviation"). (A) Shows the results for the number of bowel movements, and (B) shows the results for the number of days with bowel movements. This is a bar graph comparing the change in bowel movement patterns at 0W, 1W, and 2W between the test food group (white) and the control food group (grey) (data shown as "average value + standard deviation"). (A) Shows the results for the number of bowel movements, and (B) shows the results for the number of days with bowel movements. This is a bar graph comparing the calculated values of bowel movements at 0 weeks, 1 week, and 2 weeks between the test food group (white) and the control food group (gray) in a stratified analysis of individuals with an average or higher number of intestinal bacteria species (data shown as "average value + standard deviation"). (A) Shows the results for the number of bowel movements, and (B) shows the results for the number of days with bowel movements. This is a bar graph comparing the change in bowel movement patterns at 0 weeks, 1 week, and 2 weeks between the test food group (white) and the control food group (grey) in stratified analysis of individuals with an average or higher number of intestinal bacteria species (data shown as "average + standard deviation"). (A) Shows the results for bowel movement frequency, and (B) shows the results for the number of days with bowel movements. This is a bar graph showing the results of amplicon sequencing of fecal microbiota and the occupancy rate of Clostridia at the class level. Data are shown as "mean ± standard deviation", with the test food group shown in white and the control food group in gray. This is a bar graph showing the results of amplicon sequencing of fecal microbiota and the occupancy rate of Negativecutes at the class level. Data are shown as "mean ± standard deviation", with the test food group shown in white and the control food group in gray. This is a bar graph showing the results of amplicon sequencing of fecal microbiota and the occupancy rate of Acidaminococcles at the order level. Data are shown as "mean ± standard deviation", with the test food group shown in white and the control food group in gray.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited to these embodiments, and the present invention can be embodied in various forms without changing the gist of the invention.
Preparation of rhamnan sulfates with different molecular weights
Any naturally occurring rhamnan sulfate can be used. In this embodiment, rhamnan sulfate obtained by hot water extraction from Monoeuca alternifolia was used. The process is briefly described as follows. Dried seaweed was washed with water and extracted with hot water (95°C to 100°C) for 6 hours. The resulting hot water extract was dialyzed against pure water and freeze-dried. The freeze-dried product was dissolved in water and then applied to anion exchange chromatography, where it was continuously eluted to purify rhamnan sulfate. This was used as the sample of the invention.
The sulfated polysaccharides (e.g., rhamnan sulfate) that can be used in the present invention can be prepared by various methods other than the above. In addition, the raw material is not limited to Ulva pertusa, and Ulva ribbonii can also be used.

Next, the intestinal bacteria improving effect and anti-obesity effect in mice were evaluated using the rhamnan sulfate obtained above by the following methods.
<Mouse test>
Oral administration tests were conducted using spontaneously type 2 diabetic mice (NYS/Hos male, 9 weeks old). They were fed diets containing the following components 1 to 4 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 + 0.25mg/g rhamnan sulfate added Test group 3: high fat diet Test group 4: high fat diet + 0.25mg/g rhamnan sulfate added The test was repeated 5 times. During the test period, food intake, body weight and feces were measured once a week, and blood biochemistry tests were performed at the end of the test.

Figure 1 shows the changes in body weight in each experimental group. Test group 3, which was fed only a high-fat diet, showed a significant (p<0.01) increase in body weight compared to the normal diet group. Test group 4, which was fed a high-fat diet with rhamnan sulfate, showed a tendency to suppress body weight gain compared to test group 3. Regarding food intake, no significant differences were observed between test groups 1 and 2, or between test groups 3 and 4.
Figure 2 shows the results of the analysis of blood components taken at the end of the test. It is known that the blood glucose level of the mice of the strain used in this test rises when they are fed a high-fat diet, even at a young age. In this test, the blood glucose level of the high-fat diet group (test group 3) also rose significantly (p<0.05). In contrast, the group in which rhamnan sulfate was added to the high-fat diet (test group 4) showed a tendency to reduce the blood glucose level that rose in test group 3. This shows that rhamnan sulfate has a blood glucose lowering effect, especially in those who consume a high-fat diet.
Furthermore, plasma triglycerides were significantly (p<0.05) reduced by the addition of rhamnan sulfate (Test Groups 2 and 4) in both the normal diet and high-fat diet groups. Plasma total cholesterol was also significantly increased in the high-fat diet group, Test Group 3, but was significantly (p<0.05) reduced by the addition of rhamnan sulfate (Test Group 4). Thus, it was found that rhamnan sulfate lowers elevated blood fat levels and contributes to the improvement of metabolic syndrome.

Figure 3 shows the daily fecal weight and calories. 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 fecal weight and calories increased significantly (p<0.05) with the addition of rhamnan sulfate (test group 4). This shows that the normal fecal weight increases and constipation can be relieved. In addition, the calorie content in the feces also increased significantly, which shows that the compound contributes to an anti-obesity effect.
Next, the sequences of 16s rRNA derived from bacteria contained in the collected feces were analyzed using Nanopore 16S Barcoding Kit (Oxford Nanopore). Figure 4 shows the relative abundance of bacteria at the phylum level. In NSY/HOS type 2 diabetic mice, Firmicutes dominated, followed by Bacteroidetes, Actinobacteria (approximately 1%), and Deferribateres (less than 1%). Figure 5 shows (A) the relative abundance of Bacteroidetes and (B) the F/B ratio (Firmicutes/Bacteroidetes) in the groups administered normal or high-calorie diets at 1 week or 4 weeks. In the group fed the high-calorie diet, administration of rhamnan sulfate increased Bacteroidetes and decreased the F/B ratio compared to 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. In other words, it is considered that a decrease in the F/B ratio indicates a shift toward reducing obesity.

Figure 6 shows a graph of bacteria that were significantly increased or decreased by administration of rhamnan sulfate. As shown in the figure, in both the high-fat diet and normal diet groups, administration of rhamnan sulfate increased lactic acid bacteria (Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus intestinalis, etc.). In addition, Oscillibacter valericigenes, one of the factors that aggravate symptoms of obesity and metabolic syndrome, was increased in the high-fat diet group (test group 3), but was reduced by rhamnan sulfate (test group 4). From the above, it was found that oral intake of rhamnan sulfate changes the composition of the intestinal bacterial flora. In particular, it was found that the obesity symptoms were improved in the group administered a high-fat diet (which is thought to be particularly common in obese people). There are reports that Oscillibacter bacteria are positively correlated with fasting blood glucose levels and the AUC of the oral glucose tolerance test (OGTT), and are reduced by DPP-4 inhibitors, which lower blood glucose levels (Non-Patent Documents 1 and 2). In other words, blood glucose levels can be reduced by reducing Oscillibacter bacteria. From the results in Figure 2 (A), rhamnan sulfate significantly reduces blood glucose levels that increased in the high-fat diet group. This effect was thought to be achieved by rhamnan sulfate reducing Oscillibacter.

<Confirmation test of the effect of rhamnan sulfate on microbial growth>
From the results of the above <Mouse Test>, it was found that rhamnan sulfate has a favorable effect on the proliferation of lactic acid bacteria. Therefore, the effect of rhamnan sulfate on the proliferation of microorganisms (particularly lactic acid bacteria) was confirmed in an in vitro test system.
1. Test method
(1) Cultivation of microorganisms
55 g of MRS powder medium (Difco Lactobacilli MRS Broth) was weighed out and diluted to 1000 mL with purified water. This was stirred and dissolved, and then sterilized in an autoclave at 2 atmospheres, 121°C, and 15 minutes. This was used as the MRS medium.
Rhamnan sulfate-supplemented medium was prepared by adding Rhamnox 100 (manufactured by Konan Kako Co., Ltd., a product that contains 100% Human Exoskeleton extract and 60% or more rhamnan sulfate). The same applies below.) to autoclaved MRS medium to a concentration of 10 mg/ml (or 0.625, 1.25, 2.5, 5, or 10 mg/ml). Rhamnan sulfate is difficult to dissolve, so the medium was dissolved in an autoclave at 105°C for 1 minute, and the resulting medium was used as rhamnan sulfate-supplemented MRS medium.
After cooling the MRS medium and the rhamnan sulfate-added MRS medium to room temperature, 100 mL of each was dispensed into a covered Erlenmeyer flask, to which 100 μL of the microorganism to be examined dissolved in the MRS medium was added, followed by incubation at 37°C.
The degree of microbial growth was examined by measuring the optical density at 660 nm (OD660). OD660 measurements were started when the microorganisms reached the logarithmic growth phase, and the measurement interval was set at 1 hour.
Furthermore, the lactic acid bacteria used were JCM8130 (Lactobacillus paracasei), JCM2257 (Bacillus coagulans), and JCM11046 (Lactobacillus gasseri) obtained from the Riken BioResource Research Center.

(2) Counting of microorganisms
Weighed out 24.6g of BCP-added plate agar medium (Eiken Chemical, for measuring lactic acid bacteria count) and made up to 1000mL with purified water. After stirring and dissolving, sterilized in an autoclave at 2 atmospheres, 121℃, and 15 minutes. This was used as the BCP-added plate agar medium.
After the incubation, 1 mL of the liquid microbial culture medium was taken and sterilized saline was added to create a 10-fold dilution sample. This process was repeated until a 1 billion-fold dilution was created.
1 mL of each of the lactic acid bacteria dilutions with dilution ratios of 1000 to 1 billion was taken and added to a petri dish for culture. Approximately 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 solidified, it was inverted and cultured at 37°C for 72 hours ± 3 hours. As a control, culture was performed in the same manner as above using the sterilized saline used for dilution. After culture, colonies that had turned yellow in the medium were counted as lactic acid bacteria. The count number was multiplied by the dilution ratio to obtain the number of lactic acid bacteria.

2. Test results As shown in Figures 7(A), 8(A) and 9(A), the growth of all three types of lactic acid bacteria was promoted by adding Rhamnox 100 to the medium at a concentration of 10 mg/ml (6 mg/ml or more as rhamnan sulfate). When the CFU (colony forming unit) was measured, it increased with the addition of rhamnan sulfate, as shown in Table 1, and it was found that the increase in the number of bacteria correlated with the turbidity.

Furthermore, to confirm whether rhamnan sulfate promotes growth in a concentration-dependent manner, rhamnox 100 was added to the medium at concentrations of 0.625, 1.25, 2.5, 5, and 10 mg/ml, and the growth-promoting effects were compared. As shown in Figures 7 (B), 8 (B), and 9 (B), growth was promoted in a concentration-dependent manner in JCM8130 (Lactobacillus paracasei) and JCM2257 (Bacillus coagulans) at concentrations of 0.625 mg/ml or more (0.375 mg/ml as rhamnan sulfate) and in JCM11046 (Lactobacillus gasseri) at concentrations of 1.25 mg/ml or more (0.75 mg/ml as rhamnan sulfate).
Thus, it was found that rhamnan sulfate has the effect of promoting the proliferation of lactic acid bacteria in vitro.

<Human Study 1>
Next, the effect of rhamnan sulfate was examined using several human volunteers from Konan Kako Co., Ltd. Three healthy volunteers were orally administered rhamnox (100 mg per day). Rhamnox (Konan Kako Co., Ltd.) was produced by hot water extraction of human Egusa at 95-100°C for 6 hours. Diatomaceous earth was added and the mixture was centrifuged. The supernatant was filtered under reduced pressure, and the filtrate was filtered through an ultrafiltration membrane (molecular weight cutoff: 10,000). The concentrated liquid was spray-dried to obtain a powder (rhamnan sulfate content: 87%).
As a result, the subjects reported that the frequency and amount of bowel movements had increased. At the same time, those who had experienced unpleasant symptoms such as abdominal bloating were relieved. No side effects such as diarrhea or vomiting were observed.
General constipation refers to a state in which a person does not have a bowel movement for 3 to 5 days or more. Also, constipation requires effort and pain when defecating, and symptoms include abdominal distension and abdominal pain. It has been found that the constipation relieving composition of this embodiment increases the number of bowel movements and the amount of stool by continuous ingestion. Thus, it has been found that the composition can significantly relieve constipation in humans as well.

According to the data of this embodiment, it was found that administration of rhamnan sulfate also relieves constipation symptoms in humans. In addition, taking into account the results of the above-mentioned <Mouse Test>, it was believed that this constipation relief effect is due to a change in the composition of the intestinal bacterial flora. Thus, it was found that administration of rhamnan sulfate can change the composition of the intestinal bacterial flora in a better direction, relieve constipation, and improve obesity, especially when a high-calorie diet is ingested.

<Human Study 2>
1. Test method
(1) Subjects A placebo-controlled, randomized, double-blind, parallel-group comparative study was conducted. Subjects were selected based on the following two criteria: (a) healthy men and women aged 20 to 65 years at the time of consent, and (b) subjects prone to constipation (funneling 3 to 5 times a week).
(2) Test items As test items, an intestinal bacteria test (amplicon sequence analysis) was performed on a stool test (collected from 5 days before visiting to the day before visiting in principle) at 0W, and an intestinal bacteria test (amplicon sequence analysis) was performed on a stool test (collected from 5 days before visiting to the day before visiting in principle) at 2W. In addition, a dietary survey form and a bowel movement diary (recording from the start of the previous observation period to the day before the end of 2W of this study) were administered as a web diary (life diary/bowel movement diary). At this time, the bowel movement status (number of bowel movements and date of collection, confirmation of which bowel movement date the stool was collected, Bristol scale (fecal characteristics), and stool volume were recorded as input items.
(3) Test Foods The test foods used were capsules containing 100 mg of rhamnox (manufactured by Konan Kako Co., Ltd., rhamnan sulfate content 87%) as the test food, and capsules containing no rhamnox as the control food.
The subjects were instructed to take one capsule of the above test food per day after breakfast with water or lukewarm water without chewing. They were instructed to open the test food immediately before ingestion.
(4) Allocation method: Study subjects selected based on the results of the preliminary test were stratified into two groups based on age, sex, and the online diary (number of bowel movements per week, and average Bristol scale (stool condition) value per bowel movement). The online diary values were used for the two weeks from the start of the observation period. Care was taken to avoid significant bias between the test food groups in the stratification factors.

(5) Data collection and analysis method
(i) Primary outcome: bowel movement status (stool frequency, number of days with bowel movement)
0W was defined as the week immediately prior to the 0W visit, 1W as the week from the start date of test food intake (0W visit), and 2W as the week immediately prior to the 2W visit, and the total number of bowel movements and days with bowel movements were calculated for each period. Comparisons between the control food group and the test food group using calculated values and changes from 0W were evaluated using unpaired t-tests. Comparisons over time between 0W, 1W, and 2W were evaluated using calculated values using Dunnett's test.

As an additional analysis, a stratified analysis was conducted based on the intestinal bacteria of the study subjects (the number of intestinal bacterial species detected by fecal bacterial flora (amplicon sequencing analysis) was categorized as "below average" and "above average" compared to the overall average value at 0W).
(ii) Secondary endpoint: Fecal microbiota (amplicon sequencing analysis)
(a) Library construction and sequencing methods
(a) DNA extraction After adding Lysis Solution F (Nippon Gene) to the sample, it was crushed for 2 minutes at 1500 rpm using Shake Master Neo bms. The crushed sample was left to stand at 65°C for 10 minutes. It was then centrifuged at 12000 x g for 1 minute, and the supernatant was separated. DNA was purified from the separated solution using the MPure 12 system and MPure Bacterial DNA Extraction Kit (MP Bio).
(a) 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
The library was constructed using the two-step tailed PCR method.
(D) Quantification of the library
The concentration of the constructed libraries was measured using Synergy H 1 (Bio Tek) and the QuantiFluor dsDNA System.
(E) Library quality check
The quality of the prepared libraries was confirmed using a 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) at 2 × 300 bp.

(b) Data analysis method
(a) Extraction of reads that match the primer sequence
Only the read sequences whose beginnings matched the primer sequences used were extracted using the fastq_barcode_spliltter of the Fastx toolkit (ver 0 0 14). When the primer sequence contained an N mix, this operation was repeated taking into account the number of N (a total of 36 types, consisting of 6 types of forward and 6 types of reverse combinations).
(a) Analysis using Qiime 2
After removing primer sequences, chimeric sequences, and noise sequences using the dada 2 plugin of Qiime 2 (ver 2020 2), representative sequences and an OTU table were output.
The number of paired reads was calculated for each bacterium, and the occupancy was calculated by calculating the ratio of the number of paired reads for the bacterium of interest to the number of paired reads for all bacteria. Comparisons between the control and test food groups using actual measurements and changes from 0W were evaluated using unpaired t-tests. Comparisons over time between 0W and 2W using actual measurements were evaluated using paired t-tests.

2. Test results
(1) Study Period The study was conducted from February 28, 2020 to April 23, 2020 (from the date of pre-test to the date when the investigator determined that further follow-up of adverse events was not necessary).
(2) Breakdown of test subjects The study started with 38 test subjects (19 subjects/group), although the target number of test subjects was 40 (20 subjects/group). The breakdown of test subjects is shown in Figure 10.
(3) Efficacy evaluation items Primary evaluation item: Bowel movement status (stool frequency, number of days with bowel movement)
The calculated values and the changes from 0 W when the analysis was divided into weekly intervals are shown in Tables 2 and 3 and Figures 11 and 12, respectively. In the tables, the data are shown as "mean ± standard deviation", and in between-group comparisons (unpaired t-test), "#" indicates significance at a risk level of less than 5% (p<0.05), and in time-dependent comparisons (Dunnett's test), "**" indicates significance at a risk level of less than 1% (p<0.01).

(i) Between-group comparison
(a) An intergroup comparison between the control food group and the test food group using calculated values revealed no statistically significant differences in any items.
(i) Comparison of the changes between the control food group and the test food group revealed statistically significant differences in the following items:
- Bowel movement frequency: At 2 weeks, the test food group showed a statistically significant higher value of 1.5±1.6 (times/week) compared to 0.3±1.7 (times/week) in the control food group (p<0.05).
(ii) Comparison over time Comparison over time at 0 W, 1 W, and 2 W using the calculated values revealed statistically significant changes in the following items.
-Frequency of bowel movements: In the test food group, there was a statistically significant increase from 4.1±1.5 (times/week) at 0W to 5.6±1.9 (times/week) at 2W (p<0.01).
- Number of days for bowel movements: In the test food group, there was a statistically significant increase from 3.8±1.0 (days/week) at 0W to 4.9±1.2 (days/week) at 2W (p<0.01).

(4) Stratified analysis: Average or higher number of enterobacteria species The calculated values and the changes from 0 weeks when the analysis was divided into weekly intervals are shown in Tables 4 and 5 and Figures 13 and 14, respectively. In the tables, the data are shown as "average value ± standard deviation", and in between-group comparisons (unpaired t-test), "#" indicates significance at a risk level of less than 5% (p<0.05), and in time-dependent comparisons (Dunnett's test), "**" indicates significance at a risk level of less than 1% (p<0.01).

(i) Between-group comparison
(a) Comparisons between the control food group and the test food group using calculated values revealed statistically significant differences in the following items:
- Number of days with bowel movements: At 2 weeks, the test food group showed a statistically significant higher number of days (5.1±1.1 days/week) compared to 3.7±1.6 days/week in the control food group (p<0.05).
(i) Comparison of the changes between the control food group and the test food group revealed statistically significant differences in the following items:
- Bowel movement frequency: At 2 weeks, the test food group showed a statistically significant higher value of 2.0±1.7 (times/week) compared to 0.0±2.1 (times/week) in the control food group (p<0.05).
- Number of days with bowel movements: At 2 weeks, the test food group showed a statistically significant higher value of 1.4±1.3 (days/week) compared to -0.2±1.8 (days/week) in the control food group (p<0.05).
(ii) Comparison over time Comparison over time at 0 W, 1 W, and 2 W using the calculated values revealed statistically significant changes in the following items.
-Frequency of bowel movements: In the test food group, there was a statistically significant increase from 3.7±1.1 (times/week) at 0W to 5.7±1.9 (times/week) at 2W (p<0.01).
- Number of days for bowel movements: In the test food group, there was a statistically significant increase from 3.7±1.1 (days/week) at 0W to 5.1±1.1 (days/week) at 2W (p<0.01).

(5) Secondary endpoint: Fecal bacterial flora (amplicon sequencing analysis)
The results of examining the occupancy rate at the class level are shown in Figures 15 and 16.
(i) Comparison over time Comparison over time between 0W and 2W using occupancy rate revealed statistically significant changes 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 results of examining the occupancy rate at the order level are shown in Figure 17. The items recognized in the "order" classification are 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. Discussion Clostridium is known to produce medium-length fatty acids, which increase water absorption, subsequently drying out feces and causing constipation. One of the mechanisms by which rhamnan sulfate relieves constipation may be the reduction of the Clostridium class.
Rhamnan sulfate selectively increased Negatiwictes and Acidaminococcales. Although the biological effect on constipation is unknown, there are reports that an increase in these bacteria is positively related to the improvement of constipation. Negatiwictes was increased by administration of psyllium husk (derived from the seeds of Plantago ovata) and bifidobacteria-based probiotic treatment during the improvement of constipation.
Acidaminococcus may be one of the main factors in curing constipation during fecal microbiota transplantation in clinical practice and administration of partially hydrolyzed guar gum (a soluble fiber) in children to improve constipation and rhamnan sulfate may increase this.
Thus, according to the present embodiment, a composition for relieving constipation that has an effect of improving the intestinal environment, particularly in obese people or people who consume high-fat foods, can be provided. In addition, when culturing lactic acid bacteria in an in vitro system, the addition of the product of the present invention can shorten the growth time and improve the growth rate.

Claims (8)

A composition for relieving constipation, comprising rhamnan sulfate derived from human einkorn . The constipation relieving composition according to claim 1, which is for obese people or people who consume a high-fat diet. The constipation relieving composition according to claim 1 or 2, which has a blood sugar level lowering effect. 4. The composition for relieving constipation according to claim 1, wherein the effect of improving the intestinal environment is to reduce the F/B ratio, which is the ratio of intestinal bacteria Firmicutes to Bacteroidetes. 5. The composition for relieving constipation according to claim 1, wherein the intestinal environment improving effect is to increase the Lactobacillus genus of intestinal bacteria. 6. The composition for relieving constipation according to any one of claims 1 to 5 , which is taken together with a high-fat meal. The composition for relieving constipation according to any one of claims 1 to 6 , which is a health food for oral administration. A lactic acid bacteria growth promoter comprising rhamnan sulfate derived from human einkorn .