JP6088657B2 - Agar-derived neoagalo-oligosaccharide composite composition having anti-obesity and anti-diabetic effects prepared by DagA enzyme reaction - Google Patents

Description

  The present invention relates to an anti-obesity and anti-diabetic-derived agaro-oligosaccharide complex composition prepared by DagA enzyme reaction, specifically, Streptomyces coelicolor DagA and agar or agarose. The present invention relates to a composition exhibiting an effect of preventing, treating or improving obesity or diabetes by containing a reaction product obtained by an enzymatic reaction as an active ingredient.

  Agar (Agar) is a typical seaweed-derived polysaccharide that has been widely used for food additives, pharmaceuticals, cosmetics, livestock feed, and industrial raw materials. It is one of the relatively abundant marine resources that reaches about 3,600 tons. However, in terms of actual use, only a part of the total production is simply processed and used as cheap raw materials, and the rest is largely left untouched and added value compared to the existing resources. The position is very low. Therefore, there is a great demand for research on development of new uses for rich domestic agar and improvement of added value.

  Agar is composed of agarose and agaropectin. Agarose is a unit consisting of D-galactose and 3,6-anhydro-L-galactose bound in the form of β-1,4. While agarobiose is a linear structure in which agarobiose is repeated and linked by α-1,3 bonds, and has a strong gelling power, agaropectin is composed of agarobiose units like agarose. Contains acidic groups such as sulfate groups and has low gelling power.

  In this, agarose is decomposed by neo-agarotetraose via neo-agarotetraose by β-agarase acting on β-1,4 bonds, and subsequently α- It is finally decomposed into galactose (D-galactose) and 3,6-anhydro-L-galactose by alpha-agarase acting on 1,3-linkage.

  On the other hand, Streptomyces coelicolor A3 (2), an actinomycete, is known to produce extracellular (secreted extracellularly) agarase that degrades agar (Stanier et al., 1942, J. et al. Bacteriol .; Hodgson and Chater, 1981, J. Gen. Microbiol.), The agarase is encoded by the dagA gene. In actinomycetes, the dagA gene is a beta-agarase gene whose function is only known, and is in an important position in the production research of agarase in actinomycetes. In particular, Streptomyces sericolor is the most widely used strain for molecular biology of actinomycetes and was published in 2002 by analyzing the sequence of chromosomal DNA at the Sanger center in the UK (Bentley et al. 2002, Nature).

  Since the agar is a polysaccharide in order to develop a method that can more effectively utilize agar, which is an abundant marine resource, the present inventor has a physiological process in the agar degradation process by appropriately utilizing a glycolytic enzyme. We focused on the possibility that various sugar-derived compounds can be produced that exhibit an active effect. Actinomycetes are microorganisms that produce useful physiologically active substances such as antibiotics. Since these physiologically active substances contain a variety of compounds related to sugar, agar breaks down in the actinomycetes. Or a strain possessing an enzyme that can be transformed into a useful compound by transformation. Therefore, a useful enzyme was searched from such actinomycetes and applied to the enzyme reaction of agar to produce a useful physiologically active substance.

High molecular polysaccharides such as agar produce oligosaccharides of relatively small size through enzymatic reactions. Some of these oligosaccharides are similar in molecular form to the oligosaccharides used for metabolism in the body, but the related enzymes cannot be degraded, resulting in inhibition of the activity of glycolytic enzymes. To do. The present inventor has focused on this point, and has attempted to develop a substance effective for obesity and diabetes by confirming the ability to inhibit the glycolytic enzyme activity against the enzyme reaction product of agar.
As a result, it was confirmed that the reaction product obtained through the enzymatic reaction of Streptomyces sericolor DagA, which is an actinomycete, and agar or agarose has an excellent effect in the prevention, treatment and improvement of obesity or diabetes. It came to.

  This patent application is the next generation BioGreen 21 project (Fund specific number: PJ009536, Research project name: Examination of anti-obesity efficacy of agar oligosaccharide) This is based on research results conducted with the support of development.

  Therefore, the main object of the present invention is to improve the usefulness of obesity or diabetes by regulating the differentiation and sugar metabolism of adipocytes, among other useful physiologically active substances produced through enzymatic reactions using agar as a substrate. It is to provide a physiologically active substance exhibiting an effect.

  According to one aspect of the present invention, the present invention relates to the prevention and prevention of obesity or diabetes comprising, as an active ingredient, a reaction product obtained by enzymatic reaction of Streptomyces coelicolor DagA and agar or agarose. A therapeutic pharmaceutical composition is provided.

  According to another aspect of the present invention, the present invention provides a functional food for preventing and improving obesity or diabetes, comprising the reaction product as an active ingredient.

According to still another aspect of the present invention, the present invention provides a feed for preventing and improving obesity or diabetes, comprising the reaction product as an active ingredient.
In the present invention, the enzyme reaction is preferably performed at a temperature of 35 to 45 ° C. and a pH of 6 to 8.

  In the present invention, the enzyme reaction is preferably performed by adding Dag A at a concentration of 20 to 100 units / ml to a 0.5 to 5% (w / v) agar or agarose solution. At this time, 1 unit was reacted with 50 mM phosphoric acid solution (pH 7) in which agarose was dissolved at 0.2% (w / v) for 5 minutes at 40 ° C. (4 ml of the reaction solution), and then the same amount as the reaction solution. Add DNS reagent (dinitrosalicylic acid 6.5g, 2M NaOH 325ml, glycerol 45ml / 1l distilled water) and boil for 10 minutes, then cool and measure absorbance (Å540nm) to produce absorbance (Å540nm) 0.001 Means quantity.

  In the present invention, the reaction product contains 45 to 85% by weight of a neo agarooligosaccharide mixture based on the total weight of the reaction product, and the neo agarooligosaccharide mixture is based on the total weight of the neo agarooligosaccharide mixture. It is preferable to contain 10% by weight or less of neoagarobiose, 50 to 70% by weight of neoagarotetraose and 30 to 50% by weight of neoagarohexaose.

  According to still another aspect of the present invention, the present invention contains a neo-agaro-oligosaccharide mixture as an active ingredient, and the neo-agaro-oligosaccharide mixture comprises neo-agarobiose, neo-agarotetraose or neo-agarotetraose. Disclosed is a pharmaceutical composition for preventing and treating obesity or diabetes, characterized by containing neoagarohexaose.

  According to still another aspect of the present invention, the present invention contains a neo-agaro-oligosaccharide mixture as an active ingredient, and the neo-agaro-oligosaccharide mixture comprises neo-agarobiose, neo-agarotetraose or neo-agarotetraose. Provided is a functional food for preventing and ameliorating obesity or diabetes, characterized by containing neoagarohexaose.

  According to still another aspect of the present invention, the present invention contains a neo-agaro-oligosaccharide mixture as an active ingredient, and the neo-agaro-oligosaccharide mixture is neo-agarobiose, neo-agarotetraose or neo-agarotetraose. Provided is a feed for preventing and improving obesity or diabetes, characterized by containing gallohexaose.

  In the present invention, the neo-agaro-oligosaccharide mixture is 10 wt% or less neooagarobiose, 50-70 wt-% neoagarotetraose and 30 wt% based on the total weight of the neoagaro-oligosaccharide mixture. Preferably it contains ˜50% by weight of neoagarohexaose.

  In the present invention, Streptomyces sericolor DagA means a protein having an amino acid sequence between 31 and 309 of SEQ ID NO: 2, and includes proteins produced from Streptomyces sericolor or from heterologous strains. In addition, as long as the function is changed to a completely different one or the agarase activity is not lost, a conventional gene recombination method, that is, a labeled amino acid is included to favor purification, or for heterologous expression. It includes proteins that have been recombined by methods such as changing the amino acid sequence.

  When originally translated from a gene into beta-agarase produced by Streptomyces sericolor, DagA has 309 amino acids of SEQ ID NO: 2, is produced with a molecular weight of about 35 kDa, and has an N-terminus of 30. One amino acid signal peptide is cut and secreted in the state of a finished cell outline protein (about 32 kDa).

  The DagA gene of Streptomyces sericolor encodes the DagA, and the dagA gene is represented by the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the base sequence of the gene present in the genome of Streptomyces sericolor A3 (2), and is named “SCO3471” on the NCBI (National Center for Biological Information) database. As confirmed through in vitro experiments, transcription of the dagA gene is regulated by 4 or 5 different promoters that are recognized by at least three different holoenzymes of RNA polymerase. Through analysis of the transcription phase of the dagA gene, it appeared that transcription was initiated at the upper 32, 77, 125 and 220th bases of the coding sequence.

  Originally, DagA of the present invention can be produced through the culture of Streptomyces sericolor, which is a DagA-producing strain. In order to increase production efficiency, an expression system for Streptomyces lividans, a heterologous strain, is used. It is preferable to use it. A method is used in which the dagA gene is inserted into a vector for actinomycetes to produce a recombinant vector, Streptomyces lividans is transformed with the recombinant vector, and the transformant is cultured. In this case, the recombinant vector is preferably constructed so that transcription of the dagA gene is regulated by a promoter derived from actinomycetes.

  There are various promoters derived from actinomycetes such as sgtR promoter (sgtRp), ermE promoter (ermEp), tipA promoter (tipAp), and these are selected when producing recombinant vectors. Can be used. Various types of vectors constructed so that transcription is regulated by these promoters have been developed. If SCO3471 is cloned into the vector, a set of structures in which transcription is regulated by actinomycete-derived promoters. Replacement vectors can be produced.

A transformant can be produced by transforming a host strain with a recombinant vector. However, since there are various methods for transformation depending on the host strain, an appropriate method is selected and used. For example, when Streptomyces lividans is used as a host strain, a transformation method mediated by PEG (polyethylene glycol) is used.
If a DagA-producing strain such as a transformant is cultured in a liquid medium, DagA can be produced. If a normal protein purification method such as ultrafiltration is used after obtaining a culture solution, high-purity DagA Can be produced. At this time, if agar or agarose is contained in the liquid medium, DagA can be produced more efficiently.

  According to the present invention, it was shown that neoagarohexaose, neoagarotetraose, and neoagarobiose are produced through an enzyme reaction between agar or agarose and DagA. Therefore, in the present invention, the enzyme reaction product may be either of these or a mixture of these. However, in addition to these three reaction products identified in the present invention, other reaction products can be generated, and therefore, the present invention is not necessarily limited to only these three reaction products.

  According to the present invention, the reaction product obtained through ultrafiltration after the enzymatic reaction of agar or agarose and DagA (neoagarohexaose, neoagarotetraose and neoagarobiose are all included) It is confirmed that each of these products is also excellent in preventing and treating obesity and diabetes. It was.

The composition of this invention can be used for uses, such as a pharmaceutical and a foodstuff.
The dosage form can be formulated based on the standard formulation of the pharmacological formulation of the Ministry of Food and Drug Safety (MFDS) or the standard formula of the dietary supplement. Depending on the therapeutic purpose, the active ingredient can be mixed and diluted with a pharmaceutically acceptable carrier or encapsulated in a carrier in the form of a container.

  When the carrier is used as a diluent, powders, granules for oral administration using one or more carriers selected from the group consisting of saline, buffer, water, Ringer's solution and ethanol, and parenteral administration It can be produced in dosage forms such as injections, syrups, solutions, tablets and the like. At this time, parenteral administration means administration of an active ingredient through vein, peritoneum, muscle, artery, transdermal route, inhalation, etc. in addition to oral administration.

  The dosage form further includes fillers, anti-agglomerating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc., and administered to a mammal to provide rapid, sustained or delayed release of the active ingredient. It can be formulated as provided. The dosage of the present invention is adjusted according to the patient's condition, administration route and dosage form, and is not limited. It is obvious that a person having ordinary knowledge in the field of the present invention can use it within various ranges according to symptoms. In general, however, in the present invention, it is determined that an experimentally effective amount of 10 to 200 mg / kg body weight can be administered continuously or intermittently per day.

  Based on the effective amount, the present invention provides a food product containing agar or agarose and DagA enzyme reaction product itself, or a composition in which a pharmaceutically acceptable carrier is mixed. Processed meat products, fish products, tofu, agar, porridge, ramen and udon noodles, soy sauce, miso, chili miso, mixed miso and other seasoned foods, sauces, sweets, processed milk products such as fermented milk and cheese, kimchi It can be used by including it in pickled foods such as fruit and vegetables, drink foods such as fruits, vegetables, soy milk and fermented drinks. The cooking method and the production method for each food are obvious to those who have ordinary knowledge in the field of the present invention, and a detailed description thereof will be omitted. Moreover, the pharmacologically acceptable carrier described above can also be used as the pharmaceutically acceptable carrier.

  On the basis of the effective amount, the present invention provides a feed containing a composition in which agar or agarose and DagA enzyme reaction product itself, or a feed acceptable carrier is mixed. The basic dietary material to be fed can be used by including the composition of the present invention. In particular, it is effective when used in pets such as dogs and cats and racehorses that may be obese due to lack of exercise.

  According to the present invention, since the enzyme reaction product of Streptomyces sericolor DagA and agar or agarose exhibits an excellent anti-obesity and anti-diabetic effect, if the composition of the present invention is administered or ingested, the obesity and diabetes effectively Can be prevented, treated and improved.

1 is a drawing of the pUWL201pw vector. It is a figure of the recombinant vector which concerns on one Example of this invention. It is the graph which showed the result of having analyzed the enzyme reaction product of this invention by HPLC-ELSD. Enzyme reaction product of the present invention (hereinafter referred to as “NAO”), or neo-agarohexaose (hereinafter referred to as “DP6”) or neo-agarotetraose (hereinafter referred to as “DP4”) obtained by purifying the enzyme reaction product. 1 is a graph showing the results of alpha-amylase inhibitory activity of neoagarobiose (hereinafter referred to as “DP2”). It is the graph which showed the alpha-glucosidase inhibitory activity result of NAO or DP6, DP4, DP2. Normal diet (hereinafter referred to as “ND”), high fat diet (hereinafter referred to as “HFD”), and high fat diet (hereinafter referred to as “NFD”) containing 0.25% (w / w) of the enzyme reaction product of the present invention. High fat diet (hereinafter referred to as “HFD-NAO 0.5%”) containing 0.5% (w / w) of the enzyme reaction product of the present invention (hereinafter referred to as “HFD-NAO 0.25%”). It is the graph which showed the weight gain according to the date of an experimental animal. It is the photograph which image | photographed the microscope of the liver tissue of the experimental animal which ingested ND, HFD, HFD-NAO0.25%, or HFD-NAO0.5% by H & E dyeing | staining. A shows ND, B shows HFD, C shows HFD-NAO 0.25%, and D shows HFD-NAO 0.5%. It is the graph which showed the hepatic steatosis degree of the experimental animal which ingested ND, HFD, HFD-NAO0.25%, or HFD-NAO0.5%. It is the graph which showed the magnitude | size of the testicular adipose tissue cell of the experimental animal which ingested ND, HFD, HFD-NAO0.25%, or HFD-NAO0.5%. Photographs (AD) of the testicular adipose tissue of laboratory animals fed with ND, HFD, HFD-NAO 0.25% or HFD-NAO 0.5%, taken with a microscope after H & E staining, and the peroral load test results It is the graph (E) which showed. A shows ND, B shows HFD, C shows HFD-NAO 0.25%, and D shows HFD-NAO 0.5%.

  Hereinafter, the present invention will be described in more detail through examples. These examples are merely to illustrate the present invention, and the scope of the present invention should not be construed as being limited by these examples.

Example 1. Production of DagA Using the chromosomal DNA of Streptomyces sericolor A3 (2) as a template, and using the following Asm-F and Asm-R primers, the dagA gene fragment (DagA signal peptide and completed peptide was encoded) A 947 bp fragment, in which the partial sequence of the primer is included in SEQ ID NO: 1), and the tagA gene was cloned into the pUWL201pw vector (see FIG. 1) using the restriction enzyme sites (NdeI / BamHI) of the fragment A recombinant vector (see FIG. 2) was prepared so that the transcription of the dagA gene was regulated by the ermE promoter.

Asm-F primer: 5'-GACATATGGTGCATCGACGGTGATC-3 '(NdeI) (SEQ ID NO: 3)
Asm-R primer: 5'-GGTGGATCCCCTACACGGCCTGATACG-3 '(BamHI) (SEQ ID NO: 4)

  A transformed strain obtained by transforming Streptomyces lividans TK24 with the recombinant vector was used for the production of DagA.

  This strain was inoculated into R2YE liquid medium containing 0.3% (w / v) agar and cultured at 28 ° C. for 48 to 72 hours with shaking at 120 rpm. After the entire culture process, main culture was carried out, and each culture time was 2.5 days.

  After centrifuging the culture solution obtained through the main culture to remove the cells, the upper layer solution is filtered through an ultrafiltration membrane (5 kDa cut-off membrane) to separate and isolate the protein that could not pass through the filtration membrane. Purified. The obtained concentrated solution (enzyme solution) was lyophilized and stored for use.

Example 2 Measurement of DagA agarase activity The agarase activity against DagA of Example 1 was measured by the reduction equivalent assay method (DNS method). 100 μl of the enzyme solution obtained in Example 1 was mixed with 3.9 ml of a 50 mM phosphoric acid solution (pH 7) in which agarose was dissolved at 0.2% (w / v) and reacted at 40 ° C. for 5 minutes. 4 ml of a reagent (dinitrosalicylic acid: 6.5 g, 2M NaOH: 325 ml, glycerol: 45 ml / 1 l distilled water) was added and boiled for 10 minutes, and then cooled and the absorbance (Å540 nm) was measured. Enzyme 1U was defined as the activity that produces an absorbance (Å540 nm) 0.001 after a 5-minute reaction.

Example 3 Enzyme reaction product of DagA
3-1. Degradation of agar or agarose using Dag A 1 l of a 20 mM Tris-HCl solution in which 0.5 to 5% (w / v) agar or agarose is dissolved is prepared and heated at 100 ° C. for about 10 minutes to dissolve thoroughly. After that, the temperature was lowered to 40 ° C., and the DagA enzyme 2,000 to 50,000 U was treated to carry out the enzyme reaction for 24 hours.

3-2. Pretreatment of DagA enzyme reaction product After removing the undegraded agarose by the enzyme reaction product, the supernatant was collected by centrifugation, and the enzyme reaction product was confirmed using TLC (thin layer chromatography). . The collected supernatant was partially purified through an ultrafiltration method using a 5 KDa cut-off membrane.

Example 4 Composition confirmation of DagA enzyme reaction product through HPLC-ELSD analysis The composition of the enzyme reaction product obtained in Example 4 was confirmed through HPLC-ELSD analysis. NH ₂ P-50 4E multimode column (250 × 4.6 mm) was used, and a mixed solution of acetonitrile and water (acetonitrile: water = 65: 35) was used as a mobile phase.

  As a result, as shown in FIG. 3, among the reaction products, neoagarobiose (hereinafter referred to as “DP2”), neoagarotetraose (hereinafter referred to as “DP4”), and neoagarohexaose (neoagarohexaose). , Hereinafter referred to as “DP6”), the total amount of neoagarooligosaccharides occupies 65 ± 20% (weight), and DP2, DP4, and DP6 are each 0 based on the total amount of neoagagarooligosaccharide. 10 to 50%, 50 to 70%, and 30 to 50% (weight).

Example 5 FIG. Purification of DagA enzyme reaction products using gel filtration chromatography
A product obtained by purely purifying DP6, DP4 and DP2 through gel permeation chromatography (GPC) using BioGel P-2 gel (Biorad, Cat. No .: 150-4115) is obtained through TLC and HPLC. The purity was confirmed.

Example 6 Confirmation test of anti-obesity and anti-diabetic efficacy of DagA enzyme reaction product
6-1. Inhibitory Activity of Alpha-Amylase (α-Amylase) DP2, DP4, DP6 obtained through purification in Example 5 and the enzyme reaction product obtained in Example 3 (hereinafter referred to as “NAO”) were used as samples.
Acarbose, which is widely known as a therapeutic agent for type 2 diabetes, is known to exhibit an antidiabetic effect by a mechanism that delays sugar absorption by suppressing the activities of alpha-amylase and alpha-glucosidase. Therefore, in order to confirm the diabetic relevance of neoagaro-oligosaccharide, it was investigated whether there was an enzyme activity suppression function similar to acarbose.
After mixing each sample with alpha-amylase solution and adding starch azure solution as a substrate and reacting for 10 minutes, the blue concentration that appears after degradation is measured by absorbance (Å595nm) to analyze enzyme activity did.
As a result, as shown in FIG. 4, each sample was shown to have an alpha-amylase inhibitory activity, but the inhibitory activity appeared to be lower than that of acarbose (positive control group).

6-2. Alpha-glucosidase inhibitory activity DP2, DP4, DP6 and NAO were used as samples.
Each sample is mixed with an alpha-glucosidase solution, p-NPG (p-nitrophenyl-α-D-glucopyranoside) solution is added as a substrate, reacted for 10 minutes, and then decomposed to produce p-nitrophenol (p- The enzyme activity was analyzed by measuring the concentration of nitrophenol by absorbance (酵素 405 nm).
As a result, as shown in FIG. 5, it was confirmed that all of DP2, DP4, DP6 and NAO had inhibitory activity, although it was not as high as that of acarbose (positive control group) which is a therapeutic agent for type 2 diabetes.

6-3. Confirmation of anti-obesity and anti-diabetic effects in obesity-induced mice When high-fat diet is fed to experimental animals (mice), excessive visceral fat increases, hyperglycemia, dyslipidemia, hyperinsulinemia and steatosis in liver tissue An obese animal model that develops and well reflects the characteristics that appear in obese patients can be established.
As a result, C57bl / 6 mice of 4 weeks old after the acclimatization period were divided into normal group (Normal diet, ND), high fat control group (High fat diet, HFD), high fat-0 by the randomized mass method according to body weight. After separation into 4 groups of 25% NAO mixed group (HFD-NAO 0.25%) and high fat-0.5% NAO mixed group (HFD-NAO 0.5%), samples for each were divided into high fat It was supplied with food for 9 weeks, and the anti-obesity and anti-diabetic effects of NAO were confirmed through blood biochemical examination, histological examination, and glucose tolerance examination of experimental animals.

  FIG. The body weight shows a statistically significant difference from one week after the high-fat diet is fed, and in the ninth week, which is the end of the experiment, the high-fat diet group (HFD) as the control group is compared with the normal diet group (ND). With an increase of 29.5%, it was possible to observe the induction of obesity in mice with a high fat diet. The body weight of the diet group to which NAO was added at 0.5% was significantly lower than that of the high fat diet group, and the body weight gain was smaller than that of the normal group. On the other hand, the diet group to which NAO was added at 0.25% did not show a significant difference from the high fat diet group.

Liver tissue and accessory testicular adipose tissue were collected from experimental animals fed the experimental diet for 9 weeks and observed with a microscope. The results of H & E staining (staining) of the liver tissue are shown in FIG.
In the case of liver tissue, the degree of steatosis was classified according to the method for comparing the degree of steatosis in Kleiner DE et.al. (2005). The results are shown in FIG. Fat accumulation in the liver tissue due to high-fat diet intake was significantly higher than in the normal diet group, indicating a form of micro-vescular steatosis and macro-vescular steotosis (Grade 1.75 ± 0.41). In contrast, in the NAO-added diet group, the number of fat vacuoles in both the 0.25% group (grade 0.75 ± 0.16) and the 0.5% (grade 0.37 ± 0.18) group It was confirmed that the size was reduced to a normal level, and it was confirmed that the deposition of fat in the liver tissue was suppressed by NAO.

  As a result of H & E staining of the adipose tissue around the accessory testicle, as shown in FIGS. 9 and 10, HFD (10.74 ± 1.44) is more abundant in the testicular adipose tissue than ND (4.65 ± 0.54). The size of adipocytes increased significantly, but in the diet group added with NAO, the 0.25% group (6.74 ± 0.38) and 0.5% (6.95 ± 0.82) groups In both cases, the size of adipocytes was reduced compared to the high fat diet group. Thus, NAO is thought to suppress fat accumulation in the liver and adipose tissue.

  In order to confirm the effect of high fat diet and NAO on lipid metabolism, the lipid content in serum was investigated and shown in Table 2. The serum total cholesterol content was 140.1 ± 5.2 mg / dL in the ND group, and increased to 182.0 ± 8.2 mg / dL in the HFD group. On the other hand, the diet group to which NAO was added showed a tendency to decrease to 173.0 ± 4.3 mg / dL in the 0.25% group, and significant in 159.6 ± 5.6 mg / dL in the 0.5% group. Decreased. In the case of serum neutral fat, the value was 48.7 ± 3.2 mg / dL in the ND group, whereas it increased to 64.8 ± 4.0 mg / dL in the HFD group. The diet group to which NAO was added showed a tendency to decrease to 56.7 ± 4.4 mg / dL in the 0.25% group, and significantly decreased to 47.3 ± 2.9 mg / dL in the 0.5% group. . Further, blood FFA (Free fatty acid) also showed a higher value than normal in the high fat diet group, but in the diet group added with NAO, a value similar to normal was shown. It is known that the blood FFA concentration and the TG concentration are closely related to fat deposition and insulin resistance in liver tissue. Therefore, it is judged that NAO has an effect of improving insulin resistance, which is one of the main causes of type 2 diabetes, by regulating blood FFA and TG.

  In order to confirm the effect of NAO in improving diabetes and improving insulin resistance, biochemical analysis related to oral GTT and insulin resistance was performed, and the results are shown in FIG. After ingesting NAO for 9 weeks, glucose was orally administered and blood glucose was measured. As a result, the high-fat diet group showed higher blood glucose levels than the normal group at 30 and 60 minutes after blood glucose administration, It was confirmed that insulin resistance was induced. On the other hand, in the diet group added with NAO, the blood glucose value was significantly lower than that in the high fat diet group at 30 and 60 minutes, and NAO was added even in the result of calculating and comparing AUC (Area under curve). Showed significantly lower in the diet group. This suggests that insulin resistance induced through a high-fat diet is restored to a level similar to normal by taking NAO together. Moreover, as shown in Table 3, as a result of blood insulin and blood glucose measurement, a numerical value lower than HFD was shown in the diet group to which NAO was added, and blood adiponectin was higher than HFD in the diet group to which NAO was added. did. Adiponectin numbers are known to be closely related to insulin resistance, but the adiponectin numbers of people with insulin resistance drop, and if these insulin resistances improve, the adiponectin number also rises. As shown in Table 3, the adiponectin concentration in the HFD group did not show a large difference from the normal group, but it was found that insulin resistance and diabetes due to obesity were not sufficiently developed in the HFD group. However, the value of adiponectin increased in the dietary group to which NAO was added compared to the normal group, suggesting that NAO can play an important function in improving insulin resistance. In this example, the progress from HFD to diabetes was not made, but the diabetes-related index tended to deteriorate, and it can be confirmed that a considerable part is normalized by taking NAO. It was. In addition, the OGTT test results showed a certain diabetes improvement effect in the diet group ingested NAO, and proved the possibility of NAO as an anti-diabetic functional material.

  According to the present invention, since the enzyme reaction product of Streptomyces sericolor DagA and agar or agarose exhibits an excellent anti-obesity and anti-diabetic effect, if the composition of the present invention is administered or ingested, the obesity and diabetes effectively Can be prevented, treated and improved. Thereby, the composition of this invention can be used for uses, such as a pharmaceutical, a foodstuff, and a feed for preventing, treating, and improving obesity and diabetes.

Claims (9)

A pharmaceutical composition for preventing and treating obesity or diabetes, comprising as an active ingredient a reaction product obtained by enzymatic reaction of Streptomyces coelicolor DagA and agar or agarose,
The reaction product contains 45-85% by weight of a neoagarooligosaccharide mixture, based on the total weight of the reaction product,
The neoagaro-oligosaccharide mixture is 10 wt% or less neooagarobiose, 50-70 wt% neoagarotetraose and 30-50 wt% based on the total weight of the neoagarooligosaccharide mixture. The pharmaceutical composition for the prevention and treatment of obesity or diabetes characterized by containing neoagarohexaose.   The pharmaceutical composition for prevention and treatment of obesity or diabetes according to claim 1, wherein the enzymatic reaction comprises a temperature of 35 to 45 ° C and a pH of 6 to 8.   2. The obesity according to claim 1, wherein the enzyme reaction is performed by adding DagA at a concentration of 20 to 100 units / ml to a 0.5 to 5% (w / v) agar or agarose solution. A pharmaceutical composition for the prevention and treatment of diabetes. A functional food for preventing and ameliorating obesity or diabetes, comprising as an active ingredient a reaction product obtained by enzymatic reaction of Streptomyces coelicolor DagA and agar or agarose,
The reaction product contains 45-85% by weight of a neoagarooligosaccharide mixture, based on the total weight of the reaction product,
The neoagaro-oligosaccharide mixture is 10 wt% or less neooagarobiose, 50-70 wt% neoagarotetraose and 30-50 wt% based on the total weight of the neoagarooligosaccharide mixture. The functional food for prevention and amelioration of obesity or diabetes characterized by containing neoagarohexaose.   The functional food for prevention and amelioration of obesity or diabetes according to claim 4, wherein the enzymatic reaction comprises a temperature of 35 to 45 ° C and a pH of 6 to 8.   The obesity according to claim 4, wherein the enzyme reaction is performed by adding DagA at a concentration of 20 to 100 units / ml to 0.5 to 5% (w / v) agar or agarose solution. Functional food for diabetes prevention and improvement. A feed for preventing and ameliorating obesity or diabetes comprising, as an active ingredient, a reaction product obtained by enzymatic reaction of Streptomyces coelicolor DagA and agar or agarose,
The reaction product contains 45-85% by weight of a neoagarooligosaccharide mixture, based on the total weight of the reaction product,
The neoagaro-oligosaccharide mixture is 10 wt% or less neooagarobiose, 50-70 wt% neoagarotetraose and 30-50 wt% based on the total weight of the neoagarooligosaccharide mixture. The above-mentioned feed for preventing and improving obesity or diabetes, characterized by containing neoagarohexaose.   The feed for preventing and improving obesity or diabetes according to claim 7, wherein the enzymatic reaction comprises a temperature of 35 to 45 ° C and a pH of 6 to 8.   The obesity according to claim 7, wherein the enzyme reaction is performed by adding DagA at a concentration of 20 to 100 units / ml to 0.5 to 5% (w / v) agar or agarose solution. Feed for prevention and improvement of diabetes.