KR20200082825A - Prebiotics composition comprising extract of Porphyra yezoensis as effective component and uses thereof - Google Patents

Abstract

The present invention relates to a prebiotics composition comprising a Pyropia yezoensis extract as an active ingredient and a use thereof. The Pyropia yezoensis extract of the present invention has excellent intestinal function improvement effects and can be used as food additives, functional health foods, animal feed additives, and the like, and thus is considered to be able to enhance utilization of Pyropia yezoensis.

Description

Prebiotics composition comprising extract of Porphyra yezoensis as effective component and uses thereof}

The present invention relates to a prebiotic composition containing a seaweed extract as an active ingredient and its use.

Since the early 2000s, research technology in the field of metagenomics has been developed, scientific research has been conducted on human gut bacteria, and research results show that the distribution of microorganisms in the gut is related to obesity, disease, immunity and even personality of humans. It continues to be introduced. Advances in research to analyze metagenomes do not only analyze existing cultivable microorganisms in a specific environment, but also perform various studies on the interaction of microorganisms by analyzing distributions at the DNA level, even absolute anaerobic bacteria that are difficult to culture. It became possible. The results of these studies are being conducted in various attempts to apply to the food, medicine, and pharmaceutical industries, and in particular, analyze the distribution of microorganisms in the intestine and prescribe suitable probiotics and prebiotics, which are probiotic accelerators according to the physical characteristics of the individual. As a result, it improves the intestinal environment and is ultimately used for research on the development of customized functional useful materials that are expected to prevent and treat diseases, and the animal resource industry has recently begun studies on the analysis of microbial intestinal microbes in livestock using metagenome research technology. have.

Prebiotics (prebiotics) is a type of dietary fiber that is not digested and absorbed, but is a substance that promotes intestinal movement while moving in the intestine. In particular, it serves as a prey for lactic acid bacteria, which helps the growth of lactic acid bacteria in the intestine. Oligosaccharides, linulin, and lactulose are typical prebiotics. It is known that ingestion of prebiotics is helpful in preventing intestinal shedding of bacteria that cause inflammation and toxins, and preventing overgrowth of harmful bacteria. When probiotics intake proliferates intestinal lactic acid bacteria, pH in the intestine decreases. This is because the organic acid produced by beneficial bacteria acidifies the intestine. This is because when it is lowered to pH 6 or less, ammonia production and absorption decrease, and fewer harmful bacteria generating ammonia are generated. Particularly, among harmful bacteria, permicutes, which cause obesity, are included, which can help with weight loss. A high level of ammonia in the body means that there are many gases and ammonia produced in the intestine, and at the same time there are many harmful bacteria. For example, the more severe the constipation, the higher the ammonia level tends to be, but the secret that the harmful bacteria are suppressed by prebiotics is also in acidity (pH). Bifidobacteria (pH4.5), which usually has an acidity of 6 or less, helps promote the killing of harmful bacteria in the intestine.

Among the seaweeds, Kim belongs to the red algae plant species (rhodophyta), red algae plant species (rhodophyceae), purple algae (bangiales), and purple algae (bangiacea). It is known that there are about 140 species worldwide and 16 species in Korea. The major domestic species are Pyropia yezoensis , P. dentata , P. seriata , and Sesame ( P. tenera ). Of these, radiated seaweed has the best taste and texture, and it is suitable for commercialization of seaweed and has the highest consumption worldwide.

On the other hand, Korean Registered Patent No. 1853687 discloses'Anti-inflammatory composition comprising a sterol fraction of a radioactive seaweed extract and a method of manufacturing a sterol fraction of a radioactive seaweed extract', and Korean Registered Patent No. 1805176 discloses Immuno-enhancing composition comprising', but has not been described for the prebiotic composition and its use containing the extract of the present invention as an active ingredient.

The present invention has been derived by the above-mentioned demands, and the present inventors feed the hot water extract of the radiated seaweed into experimental animals to develop a prebiotic material by utilizing polysaccharides abundantly contained in the seaweed. As a result of monitoring the ecological changes in the intestinal microflora, in the case of experimental animals fed with hot water extract of radiated seaweed, the bacteria of the genus Lactobacillus or Bifidobacterium, which are representative lactic acid bacteria, significantly increased compared to the non-feeding group, and included a number of short-chain fatty acid-producing bacteria. The present invention was completed by confirming that the bacteria counts of the Clostridiaceae family and the Erysipelotrichaceae family were significantly increased compared to the non-supplied group.

In order to solve the above problems, the present invention provides a prebiotic containing a seaweed extract as an active ingredient.

In addition, the present invention provides a livestock feed additive containing the prebiotics as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving bowel disease comprising the prebiotics as an active ingredient.

The seaweed extract according to the present invention can be used not only as a prebiotics material, but also can be used as a food additive, a health functional food or a livestock feed additive because it has an excellent intestinal function improvement effect, thereby improving the utilization of seaweed. It seems to be.

1 shows an animal test plan for the functional evaluation of the present invention and the comparative control of the positive control.
2 is an overall experimental schematic diagram of the present invention.
3 is an intestinal microbial state of all populations shown using a heatmap on the start date of an animal experiment for investigating the effect of intestinal health improvement by extracting hot water of Kim.
4 is a result of comparative analysis of intestinal microbial ecology using NMDS (Non-metric multidimensional scaling), (A) 14 days after sample administration, and (B) 28 days after sample administration. BLK: Normal Control, WTD: Negative Control, FOS: Positive Control, EPT100: 100 mg/kg bw, EPT300: 300 mg/kg bw, EPT500: 500 mg/kg bw Arrow: Microorganisms that significantly affect NMDS Indicated (p<0.05).
Figure 5 shows the microorganisms showing significant difference (p<0.05) by comparing the control group BLK and the intestinal microbial ecology at the genus level after 14 days of animal testing. (A): BLK-WTD, (B): BLK-FOS, (C): BLK-ETP100, (D): BLK-ETP300, (E): BLK-ETP500. BLK: normal control, WTD: negative control, FOS: positive control, EPT100: 100 mg/kg bw, EPT300: 300 mg/kg bw, EPT500: 500 mg/kg bw
FIG. 6 shows microorganisms showing significant difference (p<0.05) by comparing BLK, a normal control, and intestinal microbial ecology at the genus level after 28 days of animal testing. (A): BLK-WTD, (B): BLK-FOS, (C): BLK-ETP100, (D): BLK-ETP300, (E): BLK-ETP500. BLK: normal control, WTD: negative control, FOS: positive control, EPT100: 100 mg/kg bw, EPT300: 300 mg/kg bw, EPT500: 500 mg/kg bw
Figure 7 shows the immune system-related disease reduction effect by the hot water extract. (A): BLK-EPT100, (B): BLK-EPT300, (C): BLK-EPT500. BLK: normal control, WTD: negative control, FOS: positive control, EPT100: 100 mg/kg bw, EPT300: 300 mg/kg bw, EPT500: 500 mg/kg bw

In order to achieve the object of the present invention, the present invention provides a prebiotic containing a seaweed extract as an active ingredient.

The term'prebiotics' can be defined as a short-chain carbohydrate, oligosaccharide, which minimizes hydrolysis and absorption by animals and induces the advantage of beneficial bacteria in the gut microflora. Most prebiotics can be selectively used as a substrate by beneficial bacteria in the gut, and typically include FOS (fructooligosaccharide), TOS (transgalactooligosaccharide), OF (oligofructose), or inulin. Probiotics, also called beneficial bacteria, provide beneficial effects on the host, such as maintaining normal host gastrointestinal microflora and increasing resistance to pathogenic bacteria, which support the growth or maintenance of these probiotics. This is prebiotics.

In the pre- biotics of the present invention, the seaweed extract is radiated seaweed ( Pyropia yezoensis ), but is not limited thereto.

The seaweed extract according to the present invention can induce an increase in useful microorganisms in the intestine having a probiotic function when fed to an animal model, so that it can function as a prebiotic.

In the prebiotic composition of the present invention, the seaweed extract is Lactobacillus, Bifidobacterium, Coprobacillus, Roseburia, Coprococcus genus (Coprococcus), Allobaculum, Peptococcaceae, Christensenellaceae, Clostridiaceae and Erysipelotrichaceae It may be to increase any one or more intestinal microbial flora selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a livestock feed additive containing the prebiotics as an active ingredient.

The livestock feed additive of the present invention can be used as it is, or additionally, a known carrier such as cereals and by-products allowed for livestock, stabilizers, etc. can be added, and if necessary citric acid, humic acid, adipic acid, lactic acid, malic acid, etc. Phosphates such as organic acids, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenols, catechins, alpha-tocopherols, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid Natural antioxidants such as antioxidants, antibiotics, antimicrobials, and other additives may be added, and the shape may be in a suitable state such as powder, granules, pellets, suspension, etc., and when the feed additive is supplied, it is solely used for livestock, etc. It can be supplied by mixing with or in feed.

In addition, the present invention provides a health functional food composition for preventing or improving bowel disease comprising the prebiotics as an active ingredient.

The prebiotics according to the present invention contain a seaweed extract, more preferably a hot water extract of P. yezoensis as an active ingredient.

In the health functional food composition according to an embodiment of the present invention, the intestinal disease is functional dyspepsia, ulcerative dyspepsia, non-ulcerative dyspepsia, gastro-esophageal reflux disease, reflux esophagitis, gastritis, false intestinal obstruction, gastrointestinal palsy , Constipation, irritable bowel syndrome, irritable colitis, diabetic gastrointestinal dyskinesia, gastrointestinal dyskinesia due to chemotherapy, gastrointestinal obstruction due to gastrointestinal dyskinesia, gastrointestinal dyskinesia caused by myotonic dystrophy and non-sympathetic chest pain It may be one, but is not limited thereto.

Health functional food composition of the present invention can be used in various ways, such as drugs, food and beverages for the prevention of oxidation. The functional food of the present invention includes, for example, various foods, candy, chocolate, beverage, gum, tea, vitamin complex, health supplements, and the like, and can be used in the form of powder, granule, tablet, capsule, or beverage.

Pre-biotics containing seaweed extract or seaweed extract of the present invention may be added to food or beverages for the purpose of propagating beneficial bacteria in the intestine. At this time, the amount of the prebiotic containing the seaweed extract or seaweed extract in a food or beverage is generally added to the health functional food composition of the present invention at 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight. Health drink composition may be added at a rate of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The healthy beverage composition of the present invention has no particular limitation on the liquid component except containing the seaweed extract as an essential component in the indicated proportions, and may contain various flavoring agents or natural carbohydrates and the like as additional components as in conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides, for example, disaccharides such as glucose and fructose, for example, polysaccharides such as maltose and sucrose, for example, conventional sugars such as dextrin and cyclodextrin. And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g per 100 ml of the composition of the present invention, preferably about 5 to 12 g.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Materials and methods

1. Experimental design

The animal experiment was conducted at the Wando Marine Bio Research Center of the Jeonnam Biological Industry Promotion Agency by an animal testing agency. In the present invention, 60 male 6-week-old Spraugue-Dawley rats were normal control (Blank, BLK, n=10), negative control (Water drink, WTD, n=10), positive control (Fructooligosaccharides, FOS, n=10). , Kim hot water extract 100 mg/kg bw (Extract of Porphyra yezoensis, EPT100, n=10), Kim hot water extract 300 mg/kg bw (Extract of Pophyra tenera, EPT300, n=10), Kim hot water extract 500 mg/kg Divided into a total of 6 groups including bw (Extract of Pophyra tenera, EPT500, n=10), samples were administered orally for 28 days, and animal experiments were conducted to monitor changes in intestinal microbial ecology.

In order to extract the polysaccharide derived from seaweed, hot water extraction was selected as the extraction method, and 300 mL of distilled water was added to 15 g of dried seaweed, and the mixture was repeatedly extracted three times at 100°C for 3 hours. After filtering the extract with filter paper (25㎛) and concentrated using a rotary concentrator to 50℃, 50psi condition, the freeze dryer was dried and the yield of solids was measured. The extract extracted three times was found to have a solid content of 6.48 g and a yield of 43.2%.

2. Fecal sampling and DNA extraction

During this animal experiment, for analysis of intestinal microbial ecology, feces were collected weekly for each individual and stored at -20°C until DNA extraction, and after completion of the experiment, a fecal sample was obtained from an animal testing agency and used in the present invention. To extract DNA from feces, a QIAamp Powerfecal DNA kit (QIAGEN, USA) was used, and DNA was extracted according to the protocol provided by the manufacturer. The extracted DNA was stored at -20°C until the preparation of a library for analyzing intestinal microbial ecology using Illumina's MiSeq platform.

3. Preparation of MiSeq

Prepared for microbial ecological analysis using MiSeq according to the '16S Metagenomic Sequencing Library Preparation' guidelines provided by Illumina Corporation (USA). Among the 9 variable regions present in the 16S rRNA, V4 was amplified through PCR using the KAPA HiFi HotStart ReadyMix PCR kit (Kapabiosystems, USA), and the PCR product was purified using HiAccuBead (Accugene, Korea). The purified PCR product was attached with a barcode sequence for MiSeq analysis using another PCR. The primer was removed in the same manner as the method for purification of PCR products before, and the concentration of the final PCR products of each sample was measured by a Qubit assay (Invitrogen, USA). The final PCR products of all samples were collected in an Eppendorf tube to have the same concentration, and sequencing was requested (Macrogen, Korea).

4. MiSeq result analysis

The sequence data obtained from MiSeq was performed based on Linux on the server (Dell PowerEdge R920, Memory 1.5TB, Hard 900Gb) possessed by this lab, and analyzed using software called Mothur. From the DNA extraction as a whole, microbial genomic analysis is performed as disclosed in FIG. 2, and the result data obtained through the analysis process shown in FIG. 2 are software called R (https://www.r-project.org/) and STEMP (http Statistical analysis and visualization through ://kiwi.cs.dal.ca/Software/STAMP) showed the results of comparative analysis on microbial ecology with Heatmap, Biplot, etc.

Example 1. Sample information and sequence number

In order to investigate intestinal microbial changes caused by the extract of polysaccharide derived from seaweed in the present invention, animal experiments were performed using rats, one of the experimental animals. After 14 and 28 days, DNA was extracted from the sample, and V4 in the 16S rRNA gene of the microorganism was amplified through PCR (Table 1). This PCR product was subjected to sequencing using MiSeq, one of Illumina's platforms, and a total of 9,754,223 reads were obtained from 150 fecal samples, and the error sequence was removed according to Mothur SOP. As a result, a total of 6,263,648 reads were used for ecological analysis of intestinal microorganisms (Tables 2 to 5). Sequence data analysis was performed using Mothur (https://www.mothur.org/wiki/MiSeq_SOP) by standardizing the number of sequences per sample to the lowest sequence number of 18,998 reads, and the result obtained from this is R software, STEMP Statistical analysis was performed through and visualized in a table or picture.

Example 2. Analysis of ecological changes in intestinal microflora

Prior to performing this experiment using DNA extracted from feces collected immediately before sample administration, the intestinal microbial ecology of all populations was analyzed through heatmap (FIG. 3). It was confirmed that the populations belonging to each group are not grouped in a specific form of intestinal microbial ecology, but are randomly mixed, and thus there is no problem in conducting an investigation of intestinal microbial ecological change by Kim Yeol-su extract.

The intestinal microbial ecology of all groups on the 14th and 28th day after sample administration was analyzed through NMDS (Non-metric multidimensional scaling), which can show the difference in the intestinal microbial ecology between samples in two dimensions (FIG. 4). In the comparative analysis of intestinal microbial ecology using AMOVA (Analysis of molecular variance), it was confirmed that intestinal microbial ecology was significantly different between all groups after 14 days of sample administration (p<0.05) (Table 6), but normal control was found in NMDS results. It was confirmed that only the BLK group of phosphorus showed a significantly different intestinal microbial ecology, and there was no significant difference in the intestinal microbial ecology between different groups (FIG. 4A). Through this, the intestinal microbial ecology was not significantly different between the groups that received the sample for 14 days, but it was confirmed that the intestinal microbial ecology was changed by the sample administration. In addition, in the AMOVA results after 28 days of sample administration, it was confirmed that intestinal microbial ecology was significantly different between all groups (p<0.01) (Table 3), but in the NMDS results, among the groups that administered the normal control BLK and Kim Yeolsu extract EPT300 showed similar intestinal microbial ecology, and EPT100 and EPT500 showed similar intestinal microbial ecology among the groups to which the Kim Yeolsu extract was administered. The positive control FOS administered with fructooligosaccharide was found to have similar intestinal microbial ecology of WTD administered with the excipient water (FIG. 4B). The arrows shown in FIG. 4 indicate microorganisms that significantly affect the coordinates representing the relative intestinal microbial ecology of all samples, among which, after 28 days of sample administration, fructooligosaccharide was administered compared to the relative control BLK. In FOS, it was confirmed that the genus Bifidobacterium, which is widely known as a lactic acid bacterium and beneficial to the body, appears (p<0.05).

Example 3. Analysis of intestinal microbial ecological changes at genus level

By comparing and analyzing the intestinal microbial ecology between the normal control group BLK and all groups administered the sample at the genus level, it was confirmed that there were significantly different microorganisms (p<0.05). Microorganisms that showed differences were shown as heatmaps (FIGS. 5 and 6). After 14 days of sample administration, it was confirmed that Lactobacillus (Lactobacillus), known as beneficial bacteria, increased in all groups except EPT300, which was treated with the Kim Yeolsu extract, compared to the normal control BLK, and treated with the positive control FOS and the Kim Yeolsu extract. In EPT100 and EPT500, it was confirmed that Bifidobacterium genus increased significantly. In addition, compared to IBS (Irritable bowel syndrom) patients, coprobacillus, which has been reported to be present in the intestines of healthy people, is treated with WTD and Kim hot water extract with water as an excipient compared to the normal control BLK. It was found to increase significantly in one EPT300 and EPT500. Among the groups treated with the extract of Kim Yeol-su, only EPT100 is involved in various metabolic regulation in the body, and it is reported to have beneficial effects on short-chain fatty acids (SCFAs). Coprococcus) was found to increase significantly.

After 28 days of sample administration, the allobaculum known as butyrate-producing bacteria, one of the SCFAs, was significantly increased in all groups except EPT500 treated with Kim Yeol-soo extract, compared to BLK, the normal control group. It was confirmed that the coprococcus genus reported to generate butyrate including butyrate kinase or CoA transferase in EPT300 only was significantly increased in the group treated with the Kim Yeolsu extract.

The Peptococcaceae family and the Christensenellaceae family were found to increase significantly only in EPT100 and EPT500. The Peptococcaceae family belongs to the phylum Firmicutes, It has been reported to inhabit the gut and produce representative SCFAs, acetate, propionate, and butyrate, from carbohydrates, and has been reported to have high levels in intestinal microflora in dry or healthy humans in Christensenella. In addition, it was confirmed that the Clostridiaceae family and the Erysipelotrichaceae family were significantly increased in all the groups treated with the extract of Kim Yeonsu. Clostridiaae is a member of the Pyrmicus family, similar to peptocococcus, and has been reported to inhabit the human gut and produce representative SCFAs, acetate, propionate, and butyrate, from carbohydrates. It has been reported to play an important role in bile acid metabolism and enhance anti-inflammatory responses. As described above, the metabolic process can be predicted through the intestinal microbial ecology, and it was also confirmed that the difference in the intestinal microbial ecology by the extract of Kim Yeol-su can reduce the disease related to the immune system (FIG. 7 ).

Claims (5)

Prebiotics containing seaweed extract as an active ingredient. The prebiotics according to claim 1, wherein the seaweed extract is a hot water extract of Pyropia yezoensis . The method according to claim 1, wherein the extract of laver is Lactobacillus, Bifidobacterium, Coprobacillus, Roseburia, Coprococcus, From the group consisting of Allobaculum, Peptococcaceae, Christensenellaceae, Clostridiaceae and Erysipelotrichaceae Prebiotics, characterized in that to increase any one or more intestinal microbes selected. A livestock feed additive comprising the prebiotics of any one of claims 1 to 3 as an active ingredient. A health functional food composition for preventing or improving bowel disease, comprising the prebiotic of any one of claims 1 to 3 as an active ingredient.

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