KR20190141349A - COMPOSITION FOR IMPROVING INTESTINAL MICROFLORA AND PREVENTING, TREATING OBESITY COMPRISING POLYSACCHARIDES FROM FERMENTED Aureobasidium pullulans AS AN EFFECTIVE INGREDIENT - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for alleviating intestinal bacterial flora and treating obesity, and a food composition for alleviating intestinal bacterial flora and treating obesity containing fermented Aureobasidium pullulans polysaccharide as an active component, which is obtained by inoculating Aureobasidium pullulans in a medium composition comprising a carbon source, a nitrogen source, and minerals, followed by culturing the same. By the same, it is easy to manufacture a composition by using components derived from nature, a manufacturing cost can be reduced, intestinal bacterial flora can be alleviated safely to the human body, and obesity can be suppressed.

Description

COMPOSITION FOR IMPROVING INTESTINAL MICROFLORA AND PREVENTING, TREATING OBESITY COMPRISING POLYSACCHARIDES FROM FERMENTED Aureobasidium pullulans AS AN EFFECTIVE INGREDIENT}

The present invention relates to a composition for improving intestinal flora and preventing and treating obesity, and more particularly, for improving the intestinal flora and comprising a fermented polysaccharide derived from black yeast (Aureobasidium pullulans) as an active ingredient and improving the intestinal flora. And it relates to a pharmaceutical composition for the treatment of obesity.

The human intestinal microflora is divided into four doors: Gram-negative bacteria, Bacteroidetes and Proteobacteria , and Gram-positive bacteria, Firmicutes and Actinobacteria . The Bacteroidetes Clostridia and Bacteroides species River consisting mostly accounted for most of the Firmicutes, less than 10% of the remaining doors. The first evidence that obesity is associated with changes in the gut microbiota is a study of increased Firmicutes and decreased Bacteroidetes in the intestinal microbiota of Leptin deficient ob / ob mice. Providing high-fat, high-protein meals to wild-type rats results in an increase in the Firmicutes statement and a decrease in the Bacteroidetes statement. Even when the same calories are consumed, sterile mice receiving intestinal microorganisms from obese mice gain more weight than mice receiving intestinal microorganisms from dry mice.

These results indicate that intestinal microbes play an important role in the accumulation of energy and the development of obesity. An increase in the ratio of Firmicutes / Bacteroidetes was also observed in the intestinal microbiota of obese individuals. The fraction of Bacteroidetes increases when a low-carb or low-fat diet is supplied. Twin studies have shown that obesity has reduced intestinal microbial diversity, reduced Bacteroidetes , and is rich in microorganisms with genes involved in carbohydrates and fat metabolism. However, there are also reports of contradictory findings that the ratio of Firmicutes / Bacteroidetes is reduced in obese people and that there is no difference in phylum levels between obese and skinny people.

Gastric bypass surgery reduces Firmicutes and increases the percentage of Gammaproteobacteria compared to normal weight and obese people, which may be due to changes in intestinal environment and dietary intake due to a decrease in stomach and short intestine after surgery. . In obese people, there are increasing numbers of Prevotellaceae families that produce hydrogen and archaea that use hydrogen, which seems to increase energy accumulation by promoting polysaccharide fermentation of plants to produce short-chain fatty acids.

A study of enterotypes correlated the body mass index of humans with three marker molecules, such as the ATPase complex, indicating that functional biomarkers analyzed by metagenomics are more important than differences in sentence composition. Suggest.

When mice are fed a high-fat diet, the intestinal permeability increases, and the microporous lipopolysaccharide (LPS) increases in the blood, and the increase of LPS causes obesity, low inflammation, and insulin resistance. Eliminating toll-like receptor (TLR) -4 that recognizes LPS can prevent food-induced obesity and insulin resistance. Mice lacking TLR-5 that recognize flagellin develop metabolic syndrome with changes in the intestinal microflora. These results suggest that intestinal microorganisms interact with the innate immune system through the host's TLRs and are involved in metabolic regulation. Increasing Bifidobacteria by the administration of prebiotics to obese mice increases the production of glucagon-like peptide 2, reducing intestinal permeability and decreasing blood LPS levels, improving inflammation and glucose tolerance. Transplantation of dry gut microbiota into patients with metabolic syndrome increases insulin sensitivity along with changes in the intestinal microflora producing butyrate.

Obesity has been found to alter the environment of the intestinal microflora, and conversely, studies have been made on whether the composition of the microbial flora can cause obesity. Enteric microbial flora breaks down fluorinated foods and is involved in energy metabolism.In the study, sterile rats gained 60% more body fat and increased insulin resistance despite no increase in food intake from normal rat enteric microorganisms. It is thought that the components of the intestinal microflora influenced the efficiency of getting energy from food.

In other words, the intestinal microbial flora of obese rats can be seen to be more efficient in obtaining and storing nutrition from food than in lean controls. In another study, when leptin-deficient obese rats were transplanted into the intestinal lean mice, fat increased after 2 weeks, which was significantly different from that of control mice transplanted with lean mice. Taken together, the difference in the efficiency of extracting calories from food can be seen to be due to the composition ratio of the microbial flora, resulting in a difference in weight.

The mechanism by which microorganisms cause weight gain is: 1) increased intestinal sugar absorption, 2) increased caloric absorption from fluorinated foods (forming short fatty acid chains through fermentation), and 3) hyperinsulinemia caused by high blood sugar, resulting in fat production ( This can be explained by promoting lipogenesis.

Lipoprotein lipase (LPL), which is involved in fat storage, promotes the formation of triglycerides in the liver by fermenting and absorbing polysaccharides into monosaccharides or short-chain fatty acids, which are fasting induced adipose factors (FIAF) in the intestinal epithelial cells. ) Inhibits the activity of LPL. Inhibition of FIAF due to changes in the intestinal microflora results in LPL activity in adipocytes, increasing the accumulation of triglycerides produced in the liver. Weight gain after transplantation of intestinal microorganisms from normal rats into the intestine of sterile rats may be attributed to LPL activity due to inhibition of FIAF. These results suggest that the intestinal microflora affects body weight by regulating energy storage.

Acquisition of early intestinal microflora can be seen as protection against pathogens and the development of natural immune processes. Genome and environment are important factors in determining the balance of these intestinal environments. A study was conducted on the effects of early acquired enteric microbial flora on obesity in the future. There were many bifidobacteria, and Staphylococcus in obese children Aureus was found to be many. In the first months of life, most infants are transfused with S. aureus through the mother's skin, which later produces toxins such as superantigens that carry inflammatory precursors. S. aureus of Intestinal Microorganisms in Early Childhood Translocation of the same organisms seems to be associated with obesity by increasing the systemic marker of inflammation, CD14, causing a persistent inflammatory state.

As the composition of the intestinal microorganisms has been found to have a close effect on obesity, studies of microorganisms that are helpful for the treatment of obesity are also being actively conducted. Prebiotic oligofructose intake in obese rats induced by high fat diet restored the number of bifidobacteria in the intestine, normalized serum LPS levels, reduced inflammatory conditions, and improved glucose tolerance and insulin secretion. Other studies have also reported that prebiotic intake was effective in increasing bifidobacteria, such as body weight, fatty liver, and metabolic disorders.

Conjugated linoleic acid (CLA), which is known to help obesity by reducing body fat, is usually supplied through food intake. The intestinal probiotics of lactic acid-producing bacterium (LABs) are trans-10, Cis-12-CLA is expected to be effective in treating obesity. In particular, probiotics can supply CLA continuously in the intestine, which has the advantage of being longer lasting than eating pure CLA. Probiotics lower LDL cholesterol levels. Enterococcus Yogurt containing probiotics such as faecium and Streptococcus thermophilus reduced LDL cholesterol levels by 8.4%. However, different probiotics have different effects, so Lactobacillus acidophilus , Lactobacillus Casei and Bifidobacterium bifidum have no effect on body weight, fat and cholesterol, and further studies are needed.

Korea Patent Registration No. 10-1843976 Korea Patent Registration No. 10-1724529

An object of the present invention is to provide a pharmaceutical composition for the improvement of intestinal flora and easy to manufacture by using the black yeast fermented polysaccharide which is a polysaccharide obtained by culturing black yeast, can reduce the manufacturing cost, easy to oral administration There is.

Another object of the present invention is easy to manufacture by using a fermented polysaccharide derived from black yeast, it is possible to reduce the manufacturing cost, food composition for improving intestinal flora and obesity prevention can be easily added to various foods To provide.

According to one aspect of the invention,

Provided is a pharmaceutical composition for improving intestinal flora and treating obesity, comprising black yeast fermented polysaccharide prepared by inoculating black yeast (Aureobasidium pullulans) in a medium composition comprising a carbon source, a nitrogen source and a mineral as an active ingredient.

In the medium composition, the carbon source is a liquid fructose, the nitrogen source is an ammonium salt, and the mineral may include vitamin C, potassium hydrogen phosphate (K ₂ HPO ₄ ), and magnesium sulfate (MgSO ₄ ).

Preferably, the beta glucan is β-1,4 glucan; And a glucose main chain linked by β-1,3 bonds, wherein at least one of glucose in the main chain is β-1,3 / 1,6-glucan linked to other glucose by β-1,6 bonds; It may include one or more selected from.

The black yeast fermentation polysaccharide is 40 to 50 parts by weight of α-1,4 / 1,6-glucan based on 100 parts by weight of β-1,4 glucan; And 20 to 30 parts by weight of β-1,3 / 1,6-glucan.

The black yeast fermentation polysaccharide may be to inhibit Permicutes (Firmicutes) of the intestinal microorganisms and increase Bacteroidetes (Bacteroidetes).

According to another aspect of the invention,

Provided is a food composition for improving intestinal flora and preventing obesity, which comprises black yeast fermented polysaccharide prepared by fermenting black yeast (Aureobasidium pullulans) as an active ingredient.

The pharmaceutical composition for improving intestinal flora and obesity of the present invention is easy to manufacture by using black yeast fermented polysaccharide derived from black yeast, can reduce manufacturing cost, and is easy to oral administration.

In addition, the food composition for improving intestinal flora and obesity of the present invention is easy to manufacture by using the black yeast fermented polysaccharide derived from black yeast, can reduce the manufacturing cost, can be easily added to various foods .

Figure 1 shows the results of classification of the phylum level (phylum) of the intestinal microorganism according to Test Example 2.
Figure 2 shows the similarity of intestinal microflora between treatments at the Phylum level according to Test Example 2.
Figure 3 shows the result of classifying the level of the class (Class) of intestinal microorganisms according to Test Example 2.
Figure 4 shows the results of intestinal lactic acid bacteria and enteric microbial analysis using qPCR according to Test Example 4.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, to describe the pharmaceutical composition for improving the intestinal flora and obesity of the present invention.

The pharmaceutical composition for improving intestinal flora and obesity of the present invention comprises black yeast fermented polysaccharide prepared by inoculating black yeast (Aureobasidium pullulans) in a medium composition comprising a carbon source, a nitrogen source, and a mineral as an active ingredient. do. The black yeast fermentation polysaccharide may be obtained by concentrating and drying the filtrate after filtering the culture solution.

Preferably, in the medium composition, the carbon source is liquid fructose, the nitrogen source is an ammonium salt, and the mineral may include vitamin C, potassium hydrogen phosphate (K ₂ HPO ₄ ), and magnesium sulfate (MgSO ₄ ).

More preferably, 3 to 10 parts by weight of ammonium salt, 1 to 10 parts by weight of vitamin C, 1 to 8 parts by weight of potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (100 parts by weight of liquid fructose in the medium composition) MgSO ₄ ) may include 1 to 8 parts by weight.

The ammonium salt is ammonium chloride (NH ₄ Cl), ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), ammonium nitrate ((NH ₄ ) NO ₃ ), ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and ammonium acetate (CH ₃ COONH ₄ ) It may be one or more selected from.

The black yeast fermentation polysaccharide includes alpha glucan and beta glucan.

The alpha glucan is preferably α-1,4 / 1,6-glucan.

The α-1,4 / 1,6-glucan includes a glucose main chain connected by α-1,4 bonds, and at least one of glucose in the main chain is connected to other glucose by α-1,6 bonds. .

It is preferable that the said beta glucan is 1 or more types chosen from (beta) -1,4 glucan and (beta) -1,3 / 1,6-glucan.

The β-1,3 / 1,6-glucan includes a glucose main chain connected by β-1,3 bonds, and at least one of glucose in the main chain is connected to other glucose by β-1,6 bonds. .

More preferably, the black yeast fermentation polysaccharide is 40 to 50 parts by weight of α-1,4 / 1,6-glucan based on 100 parts by weight of β-1,4 glucan; And 20 to 30 parts by weight of β-1,3 / 1,6-glucan.

The black yeast fermentation polysaccharide is characterized in that it inhibits Permicutes (Firmicutes) in the intestinal microorganisms and increases Bacteroidetes (Bacteroidetes).

Hereinafter, a method for preparing a pharmaceutical composition for improving intestinal flora and obesity treatment comprising the black yeast fermentation polysaccharide of the present invention as an active ingredient will be described. Specifically, the method for producing the black yeast fermentation polysaccharide is as follows.

First, inoculate the black yeast (Aureobasidium pullulans) in the above-mentioned medium composition, and then culture to prepare a spawn culture (step a).

The culture of this step is preferably carried out for 1 to 2 days at 25 to 35 ℃ at 150 to 200 rpm to prepare a seed culture.

Next, the seed culture is inoculated at 1 to 2% by weight in the above-described medium composition to incubate for 2-3 days under aerobic conditions to prepare a culture (step b).

Cultivation of this step is preferably carried out at aerobic conditions of 150 to 200 rpm at 20 to 30 ℃.

When the culture conditions are out of the above, the yield of the finally obtained black yeast fermentation polysaccharide may be lowered. In particular, when the culture of this step is not performed, almost no black yeast fermentation polysaccharide is produced.

Thereafter, the culture solution is centrifuged to obtain a supernatant (step c).

The centrifugation is preferably centrifuged at 5000 to 7000 xg for 10 to 30 minutes to obtain a supernatant from which the cells are removed.

Next, the obtained supernatant is filtered through an ultrafiltration membrane (UF) (step d).

Such filtration can remove the low molecular weight material containing the salt component in the supernatant.

Finally, the filtered filtrate is lyophilized to yield black yeast fermentation polysaccharide (step e).

On the other hand, the term 'comprising as an active ingredient' herein means containing an amount sufficient to achieve the efficacy or activity of the black yeast fermentation polysaccharide. In one embodiment of the invention, the black yeast fermentation polysaccharide in the composition of the invention is for example at least 0.001 mg / kg, preferably at least 0.1 mg / kg, more preferably at least 10 mg / kg, even more Preferably at least 100 mg / kg, even more preferably at least 250 mg / kg, most preferably at least 1 g / kg.

Since the black yeast fermented polysaccharide has no adverse effects on the human body even when excessively administered as a natural product, the upper limit of the quantity of the black yeast fermented polysaccharide included in the composition of the present invention can be selected and performed by those skilled in the art within an appropriate range.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, wherein the adjuvant includes excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants Or a flavoring agent can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible with saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and these One or more components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like, and preferably, oral administration.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

In addition, the present invention provides a food composition for improving the intestinal flora and obesity prevention comprising the black yeast fermentation polysaccharide as an active ingredient.

Since the description of the black yeast fermentation polysaccharide is the same as described in the pharmaceutical composition, the details will be referred to the above description.

The food composition according to the present invention can be used as a functional food or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meats, chocolates, pizzas, bread ramen noodles, other noodles, gums, ice creams, vitamin complexes, and health supplements. Foods;

The food composition of the present invention may include not only the black yeast fermentation polysaccharide as an active ingredient, but also components commonly added in preparing foods, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. do. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared with drink and beverages, in addition to the black yeast fermentation polysaccharide of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included. have.

In addition, the present invention provides a health functional food comprising a food composition for improving the intestinal flora and obesity prevention containing black yeast fermented polysaccharide as an active ingredient. Health functional food is a food made by adding black yeast fermented polysaccharide to food materials such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, suspensions, etc. It means coming, but unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. The amount of the black yeast fermented polysaccharide added to the health functional food can not be defined uniformly depending on the type of the health functional food to be treated, but may be added within a range that does not impair the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the dietary supplement of the present invention may be in the form of pills, tablets, capsules or beverages.

EXAMPLE

Example : Black yeast  Fermentation Polysaccharide Preparation

The medium composition used was 0.2 parts by weight of NH ₄ Cl, 0.2 parts by weight of vitamin C, 0.05 parts by weight of potassium hydrogen phosphate (K ₂ HPO ₄ ) and 0.05 parts by weight of magnesium sulfate (MgSO ₄ ), the liquid fructose as a carbon source 2.5 It was used after sterilization by mixing by weight. The whole culture was inoculated in 100 ml of the medium composition prepared by sterilization in a 250 ml flask by taking black yeast (Aureobasidium pullulans SM2001 (KCCM 10307)) incubated in a solid medium for a predetermined time, and then shaking speed of 200 rpm at 25 ℃. The culture was shaken for 36 hours to prepare a seed culture. The culture was inoculated at 2% by weight in 200 ml of the same medium composition prepared by sterilization of the cultured spawn culture solution in a 500 ml flask, and cultured for 3 days in the same manner as in the previous culture to obtain a culture solution. Then, the culture solution was centrifuged for 20 minutes in the range of 7000Xg and the supernatant from which the cells were removed was recovered. The recovered supernatant was filtered through an ultrafiltration membrane (UF) for 2 days and lyophilized to recover the black yeast fermentation polysaccharide in powder form.

The black yeast fermented polysaccharide prepared as described above was composed of 79.5% carbohydrate, 4.2% protein, 0.3% fat, 3.2% moisture, 12.8% ash and other components 12.8%. In addition, the carbohydrate component is a weight ratio of α-1,4 / 1,6-glucan 19.4%, β-1,4 glucan 44.0%, β-1,3 / 1,6-glucan 11.6%, other carbohydrates 25% Configured.

[Test Example]

To investigate the efficacy of black yeast fermented polysaccharides, 36 male 6-week-old male rats were used in the feeding experiment. Group separations were repeated three times, three per cage, using a total of nine animals per group. The control group fed the basic feed (C), the 0.1 wt% black yeast fermentation polysaccharide added to the basic feed (T1), the 0.2 wt% black yeast fermentation polysaccharide added to the basic feed (T2), the 0.4 wt% black yeast fermented to the basic feed The diet was divided into polysaccharide added groups (T3) for 28 days.

Test Example  1: Blood component analysis

To investigate the effect of black yeast fermentation polysaccharide on blood components, control (C), black yeast fermentation polysaccharide 0.1% treatment (T1), black yeast fermentation polysaccharide 0.2% treatment (T2), black yeast fermentation polysaccharide 0.4% treatment (T3 4 weeks after the experimental animal specification test, blood was collected, plasma and serum were separated, and the biochemical components of the blood were examined and the results are shown in Table 1 below.

Item C T1 T2 T3 Total Cholesterol (mg / dL) 81.25 ± 8.77 83.50 ± 11.33 74.75 ± 5.25 80.75 ± 10.78 Total triglycerides (mg / dL) 60.00 ± 9.83 50.50 ± 7.19 52.50 ± 17.82 40.00 ± 8.16 Low density cholesterol (mg / dL) 15.50 ± 4.51 15.75 ± 4.11 14.00 ± 1.83 15.75 ± 4.57 High Density Cholesterol (mg / dL) 62.00 ± 3.83 64.25 ± 9.22 53.25 ± 4.65 58.00 ± 6.48 Leptin (ng / mL) 0.83 ± 0.23 0.85 ± 0.34 0.93 ± 0.31 1.53 ± 0.30

According to the results, the concentration of triglyceride in blood was the highest in the control group 60.00 mg / dL, 0.4% treated with black yeast fermented polysaccharide 0.4% showed the lowest significantly (40.00 mg / dL).

Test Example  2: gut Flora  analysis

To examine the intestinal flora of 0.1% treatment (T1), 0.2% treatment (T2), and 0.4% treatment (T3) of control (C), black yeast fermentation polysaccharides, three repetitions for each treatment after 28 days of test for each treatment. Pyrosequencing was performed.

Genomic DNA from fecal samples from each group of animals is extracted with ZR Fecal DNA MiniPrep ™ (Zymo Research Corporation, USA), and next-generation sequencing is performed using Illumina HiSeq 2500 (Chunlab Inc., Seoul, Korea). Geneinration sequencing was performed and bioinformatics was analyzed by modifying Derakhshani et al.

The microbial flora analyzed is phylogenetic at the Phyla, Class, Order, Family, Genus, and Species levels. The read number of pyrosequencing is shown in Table 2 below.

Item C T1 T2 T3 Phyla 7 8 8 7 Class 11 15 13 11 Order 14 19 17 13 Family 41 42 47 38 Genus 188 186 179 142 Species 434 468 411 322

According to the results, the species (species) in the control group (C) was found 434 species, T1 468 species, T2 411 species, T3 322 species were identified. In particular, T1 increased more than 34 species compared to the control group (C) showed the largest increase in the intestinal microbial diversity.

1 is a result of classification at the phylum level of the phylotype of microorganisms present in the feces of rats. According to the results, Firmicutes and Bacteroidetes accounted for more than 90% of all treatments, indicating that they are dominant bacteria among intestinal microorganisms.

Figure 2 shows the similarity of intestinal microflora between treatments at the Phylum level. According to the results, the black yeast fermentation polysaccharide treatment groups T1, T2 and T3 showed decreased Firmicutes and increased Bacteroidetes compared to the control group fed the basic feed. Black yeast fermented polysaccharides can inhibit obesity by inhibiting Firmicutes among intestinal microorganisms and increasing Bacteroidetes . There is a correlation between the signaling system of fat accumulation of fat cells including inflammation induction and insulin action, so that inflammation and obesity are highly correlated.

Figure 3 shows the results of the classification of steel (Class) of intestinal microorganisms according to the feed of black yeast fermentation polysaccharide. According to this, the major classes that make up the intestinal microflora are Clostridia ( 44-66 %), Bacilli ( 13-45 %), and Erysipelotrichi. (4-10%), and Clostridia was the most dominant microorganism in all treatments. Clostridia containing a lot of harmful microorganisms accounted for 65% and 66% of the control (C) and T1 treatments, which were higher than the rest of the treatments, while T2 and T3 treatments showed 57% and 44%, respectively. Showed. In particular, black yeast fermented polysaccharide 0.4% treatment (T3) was the lowest. Bacilli containing lactic acid bacteria was the highest with 45% of T3 treatments and the lowest with 13% of T1 treatments. Mollicutes, known as pathogenic microorganisms, showed the highest value of 0.65% in the control and T3 treatment decreased to 0.1%. Feeding of black yeast fermented polysaccharide at the class level has been shown to reduce harmful bacteria and increase beneficial bacteria.

Test Example  3: Analysis of intestinal pathogenic microorganisms and changes in lactic acid bacteria

Table 3 shows the changes in intestinal pathogenic microorganisms according to the feed of black yeast fermented polysaccharide, and Table 4 shows the change of intestinal lactic acid bacteria according to the feed of black yeast fermented polysaccharide as the number of pyrosequecing reads.

Class Genus Species C T1 T2 T3 Clostridia Hungatella Clostridium asparagiforme One 0 0 0 Bacteroidia Bacteridees Bacteridees _ uc 2 3 0 0 Bacteroidia Bacteridees Bacteridees xylanisolvens One One 0 0 Mollicutes EU381820 _g 4 P003335 _s One 0 0 0 Mollicutes EF445272 _g HM124127 _s 36 10 9 5 Mollicutes AM275436 _f_ uc AM275436 _f_ uc _s 15 6 7 3 Mollicutes EF445272 _g EF445272 _g_ uc 8 10 5 3 Mollicutes EF445272 _f_ uc EF445272 _f_ uc _s 7 2 3 One Mollicutes EU844239 _g AB702926 _s 2 0 0 0 Mollicutes AM275436 _o_ uc _g AM275436 _o_ uc _s One 0 0 0

Class Genus Species C T1 T2 T3 Actinobacteria _c Bifidobacterium Bifidobacterium pseudolongum 0 6 19 24 Bacilli Lactobacillus Lactobacillus johnsonii 1240 686 1540 2897 Bacilli Lactobacillus Lactobacillus taiwanensis 91 48 124 319 Bacilli Lactobacillus Lactobacillus _ uc 22 14 33 52 Bacilli Pediococcus Pediococcus acidilactici group 0 0 2 8 Bacilli Lactobacillus Lactobacillus rodentium 0 0 0 3 Bacilli Lactobacillus Lactobacillus ruminis 0 0 0 One Bacilli Leuconostoc Leuconostoc lactis 0 0 0 84 Bacilli Leuconostoc Leuconostoc _ uc 0 0 0 One Bacilli Enterococcus Enterococcus  thailandicus group 0 0 0 3 Bacilli Enterococcus Enterococcus lactis 0 0 0 One Bacilli Streptococcus Streptococcus _ uc 0 0 0 One Bacilli Lactobacillus EU454989 _s One 0 0 0 Bacilli Enterococcus Enterococcus casseliflavus 4 0 0 0

According to this, the intestinal pathogenic microorganism is Clostridium Ten species, including asparagiforme , were reduced by fermentation of black yeast fermentation polysaccharide. Enteric lactic acid bacteria were reduced in two species including EU454989_s , but Bifidobacterium Five species including pseudolongum increased dependently on the fermentation of black yeast fermentation polysaccharide, and seven species of lactic acid bacteria appeared only in the black yeast fermentation polysaccharide enrichment tool (T3).

Test Example  4: Quantitative PCR ( qPCR Of Enteric Lactobacillus and Intestinal Microorganisms

In order to analyze the effect of black yeast fermented polysaccharides on the formation of intestinal flora, real-time PCR was performed after the end of the specification experiment using specific primers for intestinal lactic acid bacteria and 12 major microorganisms. Shown in

Species C T1 T2 T3 Bacteridees spp . 1.00 ^ab 1.26 ± 0.235 ^b 0.51 ± 0.080 ^a 0.049 ± 0.038 ^a Roseburia spp . 1.00 ^c 0.18 ± 0.081 ^b 0.08 ± 0.068 ^ab 0.03 ± 0.025 ^a Faecalibacterium prausnitzii 1.00 ^b 0.24 ± 0.035 ^a 0.09 ± 0.025 ^a 0.19 ± 0.197 ^a Ruminococcus spp . 1.00 ^ab 2.09 ± 0.356 ^c 1.24 ± 0.082 ^b 0.41 ± 0.165 ^a Bifidobacterium spp . 1.00 ^a 6.15 ± 3.746 ^a 22.22 ± 7.122 ^b 534.51 ± 158.462 ^c Methanogens 1.00 ^a 1.31 ± 0.458 ^ab 1.16 ± 0.292 ^ab 2.26 ± 0.316 ^b Oscillospira spp . 1.00 ^a 5.75 ± 1.246 ^b 0.46 ± 0.182 ^a 0.56 ± 0.258 ^a Leuconostoc mesenteroides 1.00 ^a 1.02 ± 0.159 ^a 0.83 ± 0.075 ^a 1.84 ± 0.443 ^b Leuconostoc citreum 1.00 ^b 0.84 ± 0.137 ^ab 0.61 ± 0.140 ^a 8.62 ± 0.162 ^c Weissella cibaria 1.00 ^c 0.37 ± 0.173 ^b 0.02 ± 0.015 ^a 0.28 ± 0.167 ^ab Weissella koreensis 1.00 0.87 ± 0.235 0.98 ± 0.251 0.67 ± 0.341 Lactobacillus sakei 1.00 ^ab 1.11 ± 0.029 ^b 0.82 ± 0.085 ^a 0.85 ± 0.201 ^a

As a result of qPCR of 12 representative enteric lactic acid bacteria and microorganisms, Bifidobacterium spp . , Leuconostoc mesenteroides , Leuconostoc citreum and Methanogens were increased with black yeast fermentation polysaccharide treatment compared with control group.

Test Example  5: Black yeast  Growth Efficiency Analysis According to Fermented Polysaccharide Supplement

To investigate the effect of feeding black yeast fermented polysaccharide on the growth efficiency of rats, the daily gain, total feed intake and feed efficiency during the total feeding period were calculated and shown in Table 6 below.

Item C T1 T2 T3 Initial weight (g) 243.2 ± 15.1 244.4 ± 13.1 245.0 ± 11.9 245.6 ± 11.2 Final weight (g) 365.4 ± 22.4 339.6 ± 18.7 324.8 ± 15.4 320.9 ± 12.7 Average total weight increase (g) 4.36 ± 1.35 3.40 ± 0.57 2.85 ± 0.51 2.69 ± 0.37 Average daily feed intake (g) 20.64 ± 1.79 17.30 ± 3.07 17.63 ± 2.89 17.67 ± 3.01 Feed conversion 21.14 19.64 16.16 15.28

According to the results, the daily weight gain was 3.40g, 2.85g, and 2.69g in the T1, T2, and T3 treatments, respectively, and decreased in the treatments. Indicated. Daily feed intake was 17.30g, 17.36g, 17.67g in T1, T2, and T3 treatments, respectively, while control showed 20.64g significantly higher daily feed intake.

The conversion rate was calculated according to the mean total weight increase and the average daily intake. The higher the amount of black yeast fermented polysaccharides, the lower the conversion rate was.

As described above, embodiments of the present invention have been described, but those skilled in the art may add, change, delete, or add elements within the scope not departing from the spirit of the present invention described in the claims. The present invention may be modified and changed in various ways, etc., which will also be included within the scope of the present invention.

Claims (8)

A pharmaceutical composition for improving intestinal flora and obesity treatment comprising black yeast fermented polysaccharide prepared by inoculating black yeast (Aureobasidium pullulans) in a medium composition comprising a carbon source, a nitrogen source and a mineral as an active ingredient. The method of claim 1,
In the medium composition, the carbon source is a liquid fructose, the nitrogen source is an ammonium salt, and the minerals include vitamin C, potassium hydrogen phosphate (K ₂ HPO ₄ ) and magnesium sulfate (MgSO ₄ ) improvement and Pharmaceutical composition for the treatment of obesity. The method of claim 1,
The black yeast fermentation polysaccharide is an intestinal flora improved and characterized in that it contains beta glucan and pharmaceutical composition for the treatment of obesity. The method of claim 3,
The alpha glucan includes a glucose main chain connected by α-1,4 bonds, and at least one of the glucose of the main chain is α-1,4 / 1,6-glucan linked with other glucose by α-1,6 bonds. Pharmaceutical composition for improving the intestinal flora and obesity, characterized in that. The method of claim 3,
The beta glucan,
β-1,4 glucan; And
a glucose main chain linked by β-1,3 bonds, wherein at least one of the glucose of the main chain is β-1,3 / 1,6-glucan linked to other glucose by β-1,6 bonds; Intestinal flora total improvement and obesity treatment pharmaceutical composition comprising at least one selected from. The method of claim 3,
The black yeast fermentation polysaccharide,
To 100 parts by weight of β-1,4 glucan,
40-1 to 50 parts by weight of α-1,4 / 1,6-glucan; And
β-1,3 / 1,6-glucan 20 to 30 parts by weight; intestinal flora improved and characterized in that it comprises a pharmaceutical composition for the treatment of obesity. The method of claim 1,
The black yeast fermentation polysaccharide inhibits intestinal microflora (Firmicutes) in the intestinal microorganisms, and improves the intestinal flora, characterized in that to increase Bacteroidetes (Bacteroidetes) pharmaceutical composition for obesity. A food composition for improving intestinal flora and preventing obesity, comprising black yeast fermented polysaccharide prepared by inoculating black yeast (Aureobasidium pullulans) in a medium composition comprising a carbon source, a nitrogen source, and a mineral.

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