KR20140061272A - Agar-derived neoagarooligosaccharide complex for the prevention and treatment of obesity and/or diabetes comprising enzyme reaction products of beta agarase daga and agar - Google Patents

Abstract

The present invention relates to an agar-derived neoagarooligosaccharide composite composition prepared by a dagA enzyme reaction and having anti-obesity and anti-diabetes effects and, more specifically, to a composition exhibiting the effects of preventing, treating, and remedying obesity or diabetes by containing, as an active ingredient, a reaction product obtained from the enzyme reaction of Streptomyces coelicolor dagA and agar or agarose. According to the present invention, the product from the enzyme reaction of the Streptomyces coelicolor dagA and the agar or agarose exhibits excellent anti-obesity and anti-diabetes effects, and thus the administration or intake of the composition can effectively prevent, treat, and remedy obesity and diabetes.

Description

[0001] The present invention relates to an agar-derived neoagarooligosaccharide complex for the prevention and / or treatment of obesity and / or diabetes-comprising enzyme reaction products of beta agarase DagA and agar.

The present invention relates to an agar-derived neoagarooligosaccharide composite composition having an anti-obesity and antidiabetic effect prepared by a DagA enzyme reaction. More specifically, the present invention relates to a neoagarooligosaccharide composition comprising Streptomyces coelicolor DagA and an agarose or agarose enzyme The present invention relates to a composition for preventing, treating and improving obesity or diabetes by containing a reaction product obtained as a reaction as an active ingredient.

Agar is a representative seaweed-derived polysaccharide that has been widely used in food additives, pharmaceuticals, cosmetics, livestock feed, and industrial materials for a long time and is one of the relatively abundant fisheries resources that produce about 3,600 tons per year in Korea . However, in actual use, only a part of the total production is simply processed and used as cheap raw materials, and the rest of the production is largely neglected. Therefore, there is a great demand for research on the development of new applications of agar and the improvement of added value.

Agar is composed of agarose and agaropectin. The agarose is composed of galactose (D-galactose) and agar (3-anhydro-L-galactose) Agarobiose is a straight chain structure in which agarobiose is repeated and connected by α-1 and 3 bonds, and agaropectin has a strong gelation power. Agaropectin, like agarose, is an agarobiose unit, but an acid group such as a sulfate group And has a weak gelling power.

The double agarose is degraded into neoagarobiose via neoagarotetraose by β-agarase acting on β-1, 4 bonds, and then α-1, 3-anhydro-L-galactose by galactose (D-galactose) and 3,6-anhydro-L-galactose by alpha-agarase acting on the 3-bond.

Streptomyces coelicolor A3 (2), which is actinomycetes, is known to produce extracellular (out-of-cell) agarases that degrade agar (Stanier et al., 1942, J. 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 the only known, and has an important role in study of agarase production in actinomycetes. In particular, Streptomyces sericola is the most widely used strain for the molecular biology of actinomycetes. In 2002, the sequence of chromosomal DNA was analyzed in the Sanger center in England (Bentley et al., 2002, Nature).

The present inventor intends to develop a method that can more effectively utilize agar, which is an abundant fishery resource. Since agar is a polysaccharide, various sugar-derived compounds that produce physiological activity during agar degradation can be produced by appropriately using sugar- The possibility of Actinomycetes are microorganisms which produce useful physiologically active substances such as antibiotics. Since these physiologically active substances contain various compounds related to sugars, actinomycetes include strains having enzymes capable of decomposing or transforming agar and converting them into useful sugar compounds . Therefore, we searched useful enzymes from these actinomycetes and applied them to the enzymatic reaction of agar to try to produce useful physiologically active substances.

Polymer polysaccharides such as agar can produce relatively small oligosaccharides through enzyme reaction. Such oligosaccharides are similar in molecular shape to oligosaccharides used for metabolism in living bodies, but related enzymes can not be degraded, and as a result, there are those that can inhibit the activity of sugar enzymes as a result. The present inventors paid attention to this point and attempted to develop a substance effective for obesity or diabetes by confirming the inferiority of sugar-enzyme activity to the enzyme reaction product of agar.

As a result, it was confirmed that DagA of actinomycetes Streptomyces sericola and the reaction product obtained through the enzymatic reaction of agar or agarose have an excellent effect on the prevention, treatment and improvement of obesity or diabetes mellitus, thereby completing the present invention .

Korean Patent Publication No. 10-2011-0093120

Stanier RY. 1942. Agar decomposing strains of the Actinomyces coelicolor species group. J. Bacteriol. 44: 555-570. Hodgson, D. A., and K. F. Chater. 1981. A chromosomal locus controlling extracellular agarase production by Streptomyces coelicolor A3 (2) and its inactivation by chromosomal integration of plasmid SCP1. J. Gen. Microbiol. 124: 339-348. Bentley SD, et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3 (2). Nature 417: 141-147.

Accordingly, it is a main object of the present invention to provide a physiologically active substance which is produced through an enzyme reaction using an agar as a substrate, particularly a physiologically active substance exhibiting excellent effects on obesity or diabetes by controlling adipocyte differentiation and glucose metabolism .

According to one aspect of the present invention, there is provided a method for preventing or treating obesity or diabetes comprising, as an active ingredient, a reaction product obtained by an enzyme reaction between Streptomyces coelicolor DagA and agarose or agarose ≪ / RTI >

According to another aspect of the present invention, there is provided a functional food for prevention and improvement of obesity or diabetes comprising the above-mentioned reaction product as an active ingredient.

According to still another aspect of the present invention, there is provided a feed for preventing or ameliorating obesity or diabetes comprising the above-mentioned reaction product as an active ingredient.

In the present invention, the enzyme reaction is preferably carried out 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 DagA to the agarose solution or agarose solution at a concentration of 20 to 100 unit / ml at 0.5 to 5% (w / v). 1unit was reacted with 50mM phosphoric acid solution (pH 7) dissolved in 0.2% (w / v) agarose at 40 ℃ for 5 minutes (reaction solution 4ml) and then equilibrated with the same amount of DNS reagent (dinitrosalicylic acid 6.5g, 325 ml of 2M NaOH, 45 ml of distilled water of glycerol, 1 liter of distilled water), boiled for 10 minutes, and cooled to measure an absorbance (Å 540 nm) to produce an absorbance (Å 540 nm) of 0.001.

In the present invention, the reaction product comprises a mixture of neoagarooligosaccharides in an amount of 45 to 85% by weight, based on the total weight of the reaction product, and the neoagarooligosaccharide mixture comprises 10% Of neoagarobiose, 50 to 70 wt.% Of neoagarotetraose and 30 to 50 wt.% Of neoagarohexaose.

According to another aspect of the present invention, there is provided a neoagarooligosaccharide mixture comprising as an active ingredient a neoagarooligosaccharide mixture, wherein the neoagaroligosaccharide mixture is selected from the group consisting of neoagarobiose, neoagarotetraose or neoagarotetraose The present invention provides a pharmaceutical composition for the prevention and treatment of obesity or diabetes, which comprises neoagarohexaose.

According to another aspect of the present invention, the present invention provides a neoagarooligosaccharide mixture as an active ingredient, wherein the neoagarooligosaccharide mixture is selected from neoagarobiose, neoagarotetraose or neoagarotetraose, The present invention provides a functional food for prevention and improvement of obesity or diabetes characterized by containing neoagarohexaose.

According to another aspect of the present invention, there is provided a neoagarooligosaccharide mixture comprising as an active ingredient a neoagarooligosaccharide mixture, wherein the neoagaroligosaccharide mixture is selected from the group consisting of neoagarobiose, neoagarotetraose or neoagarotetraose The present invention provides a feed for preventing or ameliorating obesity or diabetes, which comprises neoagarohexaose.

In the present invention, the neoagarooligosaccharide mixture may contain less than 10% neoagarobiose, 50-70% neoagarotetraose and 30-50% neoagarotrophin, based on the total weight of the neoagarooligosaccharide mixture. It is preferred that the composition contains neoagarohexaose by weight.

In the present invention, Streptomyces sericola DagA means a protein having an amino acid sequence of between 31 and 309 of SEQ ID NO: 2, and includes proteins produced from Streptomyces sericola or produced from bacillus. It is also possible to use a conventional method of gene recombination, that is, to include the labeled amino acid in order to make the purification advantageous or to change the amino acid sequence for heterologous expression within the range that the function is changed to completely different or does not lose the agarase activity Lt; RTI ID = 0.0 > recombinant < / RTI >

When DagA is originally translated from the gene into beta-agarase produced by Streptomyces sericola, it has 309 amino acids of SEQ ID NO: 2 and is produced with a molecular weight of about 35 kDa. N-terminal 30 amino acid signal peptides are cut And is released into the state of the completed extracellular protein (about 32 kDa).

The dagA gene of Streptomyces sericola encodes this DagA, and the dagA gene can be represented by the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the nucleotide sequence of the gene present in the genome of Streptomyces sericola A3 (2) and is named "SCO3471" on NCBI (US National Bioinformation Center) database. As confirmed by in vitro experiments, the transcription of the dagA gene is regulated by four or five other promoters that are recognized by at least the other three holoenzymes of the RNA polymerase. Through a transcriptional step analysis of the dagA gene, it was shown that the transcription was initiated at the 32nd, 77th, 125th and 220th bases of the coding sequence.

Although DagA of the present invention can be produced through the culture of Streptomyces lividans, which is a production strain of DagA, it is preferable to use an expression system of Streptomyces lividans, which is a host of Streptomyces lividans, in order to increase production efficiency. A method of inserting the dagA gene into a stactobacterium vector to prepare a recombinant vector, transforming the streptomyces ribidus with the recombinant vector, and culturing the transformant. In this case, the recombinant vector is preferably constructed such that the dagA gene can be transcribed by a promoter derived from actinomycetes.

Actinomycetes-derived promoter is because a variety of promoters such as a promoter sgtR (sgtRp), ermE promoter (ermEp), tipA promoter (tipAp) there can be selected and used them in the context of producing a recombinant vector. Several kinds of vectors constructed so that transcription can be regulated by these promoters have been developed. When SCO3471 is cloned into this vector, a recombinant vector having a structure in which transcription is regulated by actinomycetes-derived promoter can be produced.

The transformant can be prepared by transforming a host strain with a recombinant vector. Since there are various methods for transformation according to a host strain, an appropriate method can be selected and used. For example, when a Streptomyces ribidus is used as a host strain, a transformation method based on PEG (polyethylene glycol) can be used.

A DagA producing strain such as a transformant can be produced in a liquid medium to produce DagA, and a culture solution can be obtained, and a high purity DagA can be produced using a conventional protein purification method such as an ultrafiltration method. At this time, if agar or agarose is included in the liquid medium, DagA can be produced more efficiently.

According to the present invention, it has been shown that neoagarohexaose, neoagarotetraose, and neoagarobiose are produced through the enzymatic reaction of agar or agarose with DagA. Therefore, the enzyme reaction products in the present invention may be each of them, and they may be mixed. However, in addition to these three reaction products identified in the present invention, other reaction products may be produced, so that these reaction products are not necessarily limited to these three reaction products.

According to the present invention, the reaction products obtained by ultrafiltration after the enzymatic reaction of agar or agarose with DagA (forms in which neoagarohexaose, neoagarotetraose and neoagarobiose are all contained, and other In addition, each neoagarosehexaose, neoagarotetraose and neoagarobioxes were also found to be excellent in the prevention and treatment of obesity and diabetes.

The composition of the present invention can be used for medicines and foods.

It can be formulated based on the formulation standard of conventional pharmaceutical preparations of MFDS (Food and Drug Administration) or formulation standard of health supplement food, and can be formulated according to administration method, dosage form and therapeutic purpose in usual way May be mixed with a pharmaceutically acceptable carrier and diluted, or enclosed in a carrier in the form of a container.

When the carrier is used as a diluent, it may be formulated into powders, granules, injections, syrups, solutions, tablets and the like for oral administration and parenteral administration using at least one carrier selected from the group consisting of saline, buffer, water, Ringer's solution and ethanol And can be manufactured in the same formulation. In this case, parenteral administration means administration of the active ingredient through intravenous, peritoneal, muscular, arterial, transdermal, inhalation, etc. in addition to orally.

The formulations may further comprise a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, an antiseptic, etc. to formulate the composition so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The dosage of the present invention can be adjusted according to the patient's condition, route of administration, and dosage form, and is not limited, and any person skilled in the art will be able to use the dosage within a wide range, Generally, in the present invention, an experimentally effective amount of 10 to 200 mg / kg of body weight per day may be administered continuously or intermittently.

On the basis of the above effective amount, the present invention provides a food containing a composition comprising an agar or agarose and an enzyme reaction product itself of DagA or a pharmaceutically acceptable carrier. Sauces such as soy sauce, miso, kochujang, mixed potatoes, sauces, confectionery, milk products such as fermented milk and cheese, pickles such as kimchi and pickles, , Fruits, vegetables, soybean milk, fermented beverages, and the like. The cooking method and the production method of each food are obvious to those of ordinary skill in the art, and a detailed description thereof will be omitted. The pharmaceutically acceptable carrier may also be the above-mentioned pharmaceutically acceptable carrier.

On the basis of the above effective amount, the present invention provides a feed comprising a composition obtained by mixing an agar-agar or agarose with an enzyme reaction product itself of DagA or a carrier acceptable as a feed. The composition of the present invention can be used in the basic dietary material of the subject to be fed. Particularly, it may be effective to use as feed for pets such as dogs and cats and racehorses who are concerned about obesity because of lack of exercise.

According to the present invention, since the enzyme reaction product of Streptomyces sericola DagA and agar or agarose exhibits an excellent anti-obesity and antidiabetic effect, administration or ingestion of the composition of the present invention effectively prevents and treats obesity and diabetes And can be improved.

Figure 1 is a map of the pUWL201pw vector.
2 is a map of a recombination vector according to an embodiment of the present invention.
3 is a graph showing the results of HPLC-ELSD analysis of the enzyme reaction product of the present invention.
(Hereinafter, referred to as 'DP4') obtained by purifying the enzyme reaction product (hereinafter referred to as 'NAO') of the present invention or neoagarohexaose (hereinafter referred to as DP6), neoagarotetraose (Hereinafter, referred to as " DP2 ") and alpha-amylase inhibition activity of neoagarobiose (hereinafter referred to as DP2).
5 is a graph showing the results of alpha-glucosidase inhibition activity of NAO or DP6, DP4 and DP2.
FIG. 6 is a graph showing the results of a high-fat diet (hereinafter referred to as 'ND'), a high fat diet (hereinafter referred to as 'HFD') and 0.25% (w / (Hereinafter referred to as " HFD-NAO 0.5% ") or 0.5% (w / w) of the enzyme reaction product of the present invention A graph showing weight gain.
FIG. 7 is a microscopic photograph of H & E staining of hepatic tissues of experimental animals fed with ND, HFD, HFD-NAO 0.25% or HFD-NAO 0.5%. A represents ND, B represents HFD, C represents 0.25% of HFD-NAO, and D represents the tissue of 0.5% HFD-NAO.
FIG. 8 is a graph showing the degree of liver fat degeneration in experimental animals fed with ND, HFD, 0.25% HFD-NAO or 0.5% HFD-NAO.
FIG. 9 is a graph showing the sizes of epididymal adipose tissue cells of experimental animals fed with ND, HFD, 0.25% HFD-NAO, or 0.5% HFD-NAO.
FIG. 10 is a graph showing the results of photographs (A to D) of H & E staining of epididymal adipose tissue of experimental animals fed with ND, HFD, HFD-NAO 0.25% or HFD-NAO 0.5% (E). A represents ND, B represents HFD, C represents 0.25% of HFD-NAO, and D represents the tissue of 0.5% HFD-NAO.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Production of DagA

Using the chromosomal DNA of Streptomyces sericola A3 (2) as a template and using the following Asm-F and Asm-R primers, the dagA gene fragment (a 947 bp fragment encoding the signal peptide of DagA and the completed peptide, 1) was amplified. The dagA gene was cloned into the pUWL201pw vector (see FIG. 1) using the restriction enzyme site (NdeI / BamHI) of the fragment and the transcription of the dagA gene was amplified by the ermE promoter (See FIG. 2).

Asm-F primer: 5'-GACATATGGTGGTCAACCGACGTGATC-3 '(NdeI) (SEQ ID NO: 3)

Asm-R primer: 5'-GGTGGATCCCTACACGGCCTGATACG-3 '(BamHI) (SEQ ID NO: 4)

The transformant strain obtained by transforming Streptomyces lividans TK24 with this recombinant vector was used for the production of DagA.

This strain was inoculated into R2YE liquid medium containing 0.3% (w / v) of agar and incubated at 28 ° C for 48 to 72 hours with shaking at 120 rpm. After the pre-culture, the culture was performed and the incubation time was 2.5 days.

The culture solution obtained through the present culture was centrifuged to remove cells, and the supernatant was filtered through an ultrafiltration membrane (5 kDa cut-off membrane) to separate and purify proteins that did not pass through the filtration membrane. The obtained concentrate (enzyme solution) was used while being stored by freeze drying.

Example 2. Measurement of agarase activity of DagA

The agarase activity of DagA in Example 1 was measured by a reduction method (DNS method). 3.9 ml of a 50 mM phosphoric acid solution (pH 7) dissolved in 0.2% (w / v) of the enzyme solution obtained in Example 1 was added thereto and reacted at 40 ° C for 5 minutes. Then, the pH of the reaction solution was adjusted to 5 ml with a DNS reagent (dinitrosalicylic acid 6.5 g, 325 ml of NaOH, 45 ml of glycerol / 1 liter of distilled water), boiled for 10 minutes, and then cooled to measure the absorbance (540 nm). 1 U of enzyme was defined as the activity of generating 0.001 absorbance (540 nm) after 5 minutes of reaction.

Example 3. DagA enzyme reaction product

3-1. Degradation of agar or agarose using DagA

1 L of a 20 mM Tris-HCl solution in which 0.5 to 5% (w / v) agar or agarose was dissolved was prepared and heated at 100 ° C for about 10 minutes to sufficiently dissolve the solution. Then, the temperature was dropped to 40 ° C and 2,000 to 50,000 U of DagA enzyme And subjected to an enzymatic reaction for 24 hours.

3-2. Pretreatment of DagA enzyme reaction product

In order to remove undegraded agarose from the enzyme reaction product, the supernatant was recovered by centrifugation and the enzyme reaction product was confirmed by TLC (thin layer chromatography). The recovered supernatant was partially purified by ultrafiltration on a 5 kDa cut-off membrane.

Example 4. Confirmation of DagA Enzyme Reaction Product Composition by HPLC-ELSD Analysis

The composition of the enzyme reaction product obtained in Example 4 was confirmed by HPLC-ELSD analysis. NH ₂ P-50 4E multimode column (250 x 4.6 mm) was used as a mobile phase 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, neoagarobiose (hereinafter referred to as DP2), neoagarotetraose (hereinafter referred to as DP4) and neoagarohexaose DP2, DP4, and DP6 based on the total amount of the neoagarooligosaccharide. The amount of neoagarooligosaccharides in the total amount of the neoagarooligosaccharides was 65 ± 20% (weight) (0 ~ 10%, 50 ~ 70% and 30 ~ 50% respectively).

Example 5. Purification of DagA Enzyme Reaction Products Using Gel Filtration Chromatography

DP6, DP4 and DP2 were purified by gel permeation chromatography (GPC) using BioGel P-2 gel (Biorad, Cat. No. 150-4115) To confirm purity.

Example 6. Test for anti-obesity and antidiabetic efficacy of DagA enzyme reaction products

6-1. Alpha-amylase inhibitory activity

DP2, DP4 and DP6 obtained through purification in Example 5 and the enzyme reaction product (hereinafter referred to as 'NAO') obtained in Example 3 were used as samples.

It is known that acarbose, which is widely known as a treatment for type 2 diabetes, inhibits the activity of alpha-amylase and alpha-glucosidase and thus exhibits an anti-diabetic effect as a mechanism of delaying glucose uptake. Therefore, we investigated whether the neo - agarooligosaccharide has an enzyme - inhibitory activity similar to that of acarbose in order to confirm the diabetic relevance.

Amylase activity was analyzed by measuring the concentration of blue color in the solution by measuring the absorbance (Å 595 nm) after the 10 mM reaction of alpha-amylase solution and starch azure solution as a substrate.

As a result, as shown in FIG. 4, each sample showed alpha-amylase inhibitory activity, but the inhibitory activity was lower than that of acarbose (positive control).

6-2. Alpha-glucosidase inhibitory activity

DP2, DP4, DP6 and NAO were used as samples.

A solution of p-nitrophenol (p-nitrophenol) was prepared by dissolving α-glucosidase solution and each sample and reacting with p-NPG (p-nitrophenyl-α-D-glucopyranoside) The enzyme activity was measured by measuring the concentration by absorbance (405 nm).

As a result, as shown in FIG. 5, it was confirmed that DP2, DP4, DP6 and NAO inhibitory activity was not as high as that of acarbose (positive control), a type 2 diabetes treatment agent.

6-3. Identification of anti-obesity and antidiabetic effects in obese mice

Animal models of obesity can be established by feeding high fat diets to experimental animals (mice), which are characterized by excess visceral fat increase, hyperglycemia, dyslipidemia, hyperinsulinemia, and fatty degeneration in liver tissue have.

High fat diet (HFD), high fat-0.25% NAO mixed group (HFD), high fat diet (HFD) and high fat diet (NAO 0.25%) and high fat-0.5% NAO mixed group (HFD-NAO 0.5%). The samples were then fed with high fat diet for 9 weeks, and the blood biochemical test , Histological examination, and glucose tolerance test to confirm the anti - obesity and antidiabetic effect of NAO.

The weight gain and dietary intake of the experimental animals fed the 9-week experimental diet are shown in FIG. 6 and Table 1. (HFD) was 29.5% higher than that of the control group (ND), and at the end of the experiment (9 weeks after the end of the experiment), the control group, obesity induction . Body weight of dietary supplement with 0.5% NAO was significantly lower than that of high fat diet, and weight gain was lower than that of normal diet. On the other hand, there was no significant difference in the dietary group supplemented with 0.25% NAO from the high - fat diet group.

Salary before weight
(g) Weight after completion of pay
(g) Weight gain
(g / 10 wks) Dietary intake
(g / day) Feed efficiency ¹⁾ ND 21.13 ± 0.27 29.35 ± 0.27 8.22 ± 0.19 4.15 ± 0.09 2.02 + 0.07 HFD 21.30 ± 0.30 37.78 ± 1.04 ^† 16.48 ± 0.85 2.74 ± 0.05 ^† 6.40 ± 0.51 ^† HFD-NAO 0.25% 21.00 ± 0.25 37.59 + - 0.63 16.59 ± 0.58 ^† 2.56 + - 0.10 6.45 ± 0.36 HFD-NAO 0.5% 20.89 ± 0.27 34.37 ± 0.97 ^* 13.48 ± 0.94 ^* 2.77 ± 0.01 4.82 ± 0.36 ^*

¹⁾ Feed efficiency: Weight gain / Dietary intake

Results: Significant differences were found at p <0.05 by mean ± SD and T-test (n = 6 ~ 10).

P <0.05 for jeongsangsik That's ^{†, *} p <0.05 for the high fat That's

The liver tissue and epididymal adipose tissue were collected from an experimental animal fed a 9-week experimental diet and observed under a microscope. The H & E staining results of the liver tissues are shown in FIG.

For liver tissue, Kleiner DE et. The degree of fatty degeneration was classified according to the degree of steatosis of liver (2005). The results are shown in FIG. The lipid accumulation in the liver tissues was significantly higher than that of the normal diet, indicating micro-vescularsteotosis and macro-vescularsteotosis (grade 1.75 ± 0.41) . On the other hand, the number and size of fat follicles decreased to normal levels in both 0.25% (grade 0.75 ± 0.16) and 0.5% (grade 0.37 ± 0.18) in the NAO supplemented diet group, .

H & E staining of epididymal adipose tissue showed that adipose tissue was significantly increased in epididymal adipose tissue compared to ND (4.65 ± 0.54) in HFD (10.74 ± 1.44) as shown in Figures 9 and 10, In both 0.25% (6.74 ± 0.38) and 0.5% (6.95 ± 0.82) groups, the size of the adipocytes decreased compared to the high fat diet group. Therefore, NAO seems to inhibit lipid accumulation in liver and adipose tissue.

Table 2 shows the lipid content of serum in order to examine the effects of high fat diet and NAO on lipid metabolism. Serum total cholesterol content was 140.1 ± 5.2 mg / dL in the ND group and 182.0 ± 8.2 mg / dL in the HFD group. On the other hand, the NAO supplementation group showed a tendency to decrease to 173.0 ± 4.3 mg / dL in the 0.25% group and to 159.6 ± 5.6 mg / dL in the 0.5% group. Serum triglyceride levels were 48.7 ± 3.2 mg / dL in the ND group and 64.8 ± 4.0 mg / dL in the HFD group. The dietary group with NAO showed a tendency to decrease to 56.7 ± 4.4 mg / dL in the 0.25% group and to 47.3 ± 2.9 mg / dL in the 0.5% group. Serum free fatty acid (FFA) levels were also higher in the high - fat diet group than in the high fat diet group. Serum FFA and TG concentrations are known to be closely related to fat deposition and insulin resistance in liver tissue. Therefore, it is considered that NAO has an effect to improve insulin resistance, which is one of the main causes of type 2 diabetes, by regulating blood FFA and TG.

T-CHO
(mg / dL) TG
(mg / dL) FFA
(mEq / L) ND 140.1 ± 5.2 48.7 ± 3.2 1.28 + 0.07 HFD 182.0 ± 8.2 ^† 64.8 ± 4.0 ^† 1.52 ± 0.11 ^† HFD-NAO 0.25% 173.0 + - 4.3 56.7 ± 4.4 1.30 ± 0.05 ^* HFD-NAO 0.5% 159.6 ± 5.6 ^* 47.3 ± 2.9 ^* 1.22 ± 0.06 ^*

Results: Significant differences were found at p <0.05 by mean ± SD and T-test (n = 8).

P <0.05 for jeongsangsik That's ^{†, *} p <0.05 for the high fat That's

In order to confirm the diabetic improvement effect and the insulin resistance improving effect of NAO, biochemical analysis related to oral GTT and insulin resistance was performed, and the results are shown in FIG. 10 (E) and Table 3. After 9 weeks of NAO, glucose was orally administered and blood glucose levels were measured. As a result, the high-fat diet group showed higher blood glucose levels at 30 and 60 minutes after the administration of blood glucose, indicating insulin resistance was induced . On the other hand, in the dietary group supplemented with NAO, the blood glucose levels were significantly lower than those in the high-fat diet group at 30 minutes and 60 minutes, and the AUC (area under curve) Respectively. This suggests that insulin resistance induced by the high-fat diet may be restored to normal levels by ingesting NAO. In addition, as shown in Table 3, blood insulin and blood glucose levels were lower than those of HFD in NAO supplemented group, and adiponectin in blood was higher than HFD in NAO supplemented group. Adiponectin levels are also known to be closely related to insulin resistance. Adiponectin levels in people with insulin resistance are lower and their adiponectin levels increase when their insulin resistance improves. As shown in Table 3, the concentration of adiponectin in the HFD group did not show a significant difference from that in the normal group, suggesting that obesity-induced insulin resistance and diabetes did not sufficiently progress in the HFD group. However, in the NAO - supplemented group, the level of adiponectin was higher than that of the normal group, suggesting that NAO may play an important role in improving insulin resistance. In this example, the progression from HFD to diabetes did not progress, but the diabetic index tended to deteriorate, which was confirmed to be substantially normalized by ingestion of NAO. In addition, the OGTT test showed that the diabetic improvement effect in the dietary group fed with NAO proved the possibility of NAO as an anti-diabetic functional material.

Insulin
(ng / ml) Glucose
(Mg / dl) Adiponectin
(占 퐂 / ml) ND 0.72 + - 0.12 115.4 ± 7.3 47.29 + - 5.66 HFD 1.35 ± 0.14 ^† 153.3 ± 7.3 ^† 48.38 +/- 10.95 HFD-NAO 0.25% 0.92 + - 0.13 ^* 159.4 ± 4.0 55.35 ± 4.09 HFD-NAO 0.5% 0.94 + 0.14 156.8 ± 19.6 58.91 + - 11.45

Results: Significant differences were found at p <0.05 by mean ± SD and T-test (n = 8).

P <0.05 for jeongsangsik That's ^{†, *} p <0.05 for the high fat That's

&Lt; 110 > DyneBio Inc. <120> Agar-derived neoagarooligosaccharide complex for the prevention          and treatment of obesity and / or diabetes comprising enzyme          reaction products of beta agarase DagA and agar <130> PA131112-C01 <150> 10-2012-0128087 <151> 2012-11-13 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 930 <212> DNA <213> Streptomyces coelicolor A3 (2) <400> 1 gtggtcaacc gacgtgatct catcaagtgg agtgccgtcg cactcggagc gggtgcgggg 60 ctcgcgggtc ccgcacccgc cgctcatgcc gcagacctcg aatgggaaca gtaccccgtg 120 ccggccgccc ctggcggaaa caggtcctgg cagcttctcc ccagccattc ggacgacttc 180 aactacaccg gcaagcctca aaccttcagg ggcagatggc tggaccagca caaggatggc 240 tggtcgggcc cggccaacag cctctacagtg gcgcgccatt cctgggtggc tgacggaaat 300 ctcatcgtcg agggccgcag ggcgccggac gggagggtct actgcggcta cgtgacctcc 360 cgcaccccag tcgagtaccc tctctatacc gaagtactca tgcgtgtgag cgggctgaag 420 ctctcatcga atttctggct cctgagcaga gacgacgtca acgagattga cgtgatcgaa 480 tgctacggca acgagtcatt gcacggaaag cacatgaaca ccgcctacca catattccag 540 cggaacccct tcactgaact ggcgagaagc cagaaggggt atttcgcaga tgggagctac 600 gggtacaatg gtgagactgg gcaggtgttt ggggacggcg ccgggcaacc tcttcttcgg 660 aatggattcc accgctacgg cgtgcactgg ataagcgcca ccgaattcga tttctacttc 720 aacggcaggt tggtgcgccg gctgaaccgg tcgaacgacc tcagggaccc ccggagccgg 780 ttcttcgacc agccaatgca tctgatcctc aacaccgaga gtcatcagtg gcgcgtcgac 840 cgaggtatcg aacccacgga cgcggaactc gcagacccca gcatcaacaa catctactac 900 cgctgggtca ggacgtatca ggccgtgtag 930 <210> 2 <211> 309 <212> PRT <213> Streptomyces coelicolor A3 (2) <400> 2 Met Val Asn Arg Arg Asp Leu Ile Lys Trp Ser Ala Val Ala Leu Gly   1 5 10 15 Ala Gly Ala Gly Leu Ala Gly Ala Pro Ala Ala Ala His Ala Ala Asp              20 25 30 Leu Glu Trp Glu Gln Tyr Pro Val Pro Ala Ala Pro Gly Gly Asn Arg          35 40 45 Ser Trp Gln Leu Leu Pro Ser Ser Ser Asp Asp Phe Asn Tyr Thr Gly      50 55 60 Lys Pro Gln Thr Phe Arg Gly Arg Trp Leu Asp Gln His Lys Asp Gly  65 70 75 80 Trp Ser Gly Pro Ala Asn Ser Leu Tyr Ser Ala Arg His Ser Trp Val                  85 90 95 Ala Asp Gly Asn Leu Ile Val Glu Gly Arg Arg Ala Pro Asp Gly Arg             100 105 110 Val Tyr Cys Gly Tyr Val Thr Ser Arg Thr Pro Val Glu Tyr Pro Leu         115 120 125 Tyr Thr Glu Val Leu Met Arg Val Ser Gly Leu Lys Leu Ser Ser Asn     130 135 140 Phe Trp Leu Leu Ser Arg Asp Asp Val Asn Glu Ile Asp Val Ile Glu 145 150 155 160 Cys Tyr Gly Asn Glu Ser Leu His Gly Lys His Met Asn Thr Ala Tyr                 165 170 175 His Ile Phe Gln Arg Asn Pro Phe Thr Glu Leu Ala Arg Ser Gln Lys             180 185 190 Gly Tyr Phe Ala Asp Gly Ser Tyr Gly Tyr Asn Gly Glu Thr Gly Gln         195 200 205 Val Phe Gly Asp Gly Ala Gly Gln Pro Leu Leu Arg Asn Gly Phe His     210 215 220 Arg Tyr Gly Val His Trp Ile Ser Ala Thr Glu Phe Asp Phe Tyr Phe 225 230 235 240 Asn Gly Arg Leu Val Arg Arg Leu Asn Arg Ser Asn Asp Leu Arg Asp                 245 250 255 Pro Arg Ser Phe Phe Asp Gln Pro Met His Leu Ile Leu Asn Thr             260 265 270 Glu Ser His Gln Trp Arg Val Asp Arg Gly Ile Glu Pro Thr Asp Ala         275 280 285 Glu Leu Ala Asp Pro Ser Ile Asn Asn Ile Tyr Tyr Arg Trp Val Arg     290 295 300 Thr Tyr Gln Ala Val 305 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer (Asm-F) <400> 3 gacatatggt ggtcaaccga cgtgatc 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer (Asm-R) <400> 4 ggtggatccc tacacggcct gatacg 26

Claims (18)

Streptomyces coelicolor A pharmaceutical composition for the prevention and treatment of obesity or diabetes comprising, as an active ingredient, a reaction product obtained by an enzyme reaction of DagA with agar or agarose. The method according to claim 1,
Wherein the enzyme reaction is carried out at a temperature of 35 to 45 DEG C and at a pH of 6 to 8. The pharmaceutical composition for preventing and treating obesity or diabetes according to claim 1, The method according to claim 1,
Wherein the enzyme reaction is carried out by adding DagA to a 0.5 to 5% (w / v) agar or agar solution at a concentration of 20 to 100 unit / ml. The method according to claim 1,
Wherein the reaction product contains 45 to 85% neoagarooligosaccharide mixture based on the total weight of the reaction product and wherein the neoagarooligosaccharide mixture comprises less than 10% neoagarooligosaccharide based on the total weight of the neoagarooligosaccharide mixture, Characterized in that it contains neoagarobiose, 50 to 70% by weight of neoagarotetraose and 30 to 50% by weight of neoagarohexaose. &Lt; / RTI &gt; Streptomyces coelicolor Functional food for preventing or ameliorating obesity or diabetes, comprising as an active ingredient a reaction product obtained by an enzyme reaction of DagA with agar or agarose. 6. The method of claim 5,
Wherein the enzyme reaction is carried out at a temperature of 35 to 45 DEG C and a pH of 6 to 8. The functional food for prevention and improvement of obesity or diabetes. 6. The method of claim 5,
Wherein the enzyme reaction is carried out by adding DagA at a concentration of 20 to 100 unit / ml to an agar or agarose solution of 0.5 to 5% (w / v). 6. The method of claim 5,
Wherein the reaction product contains 45 to 85% neoagarooligosaccharide mixture based on the total weight of the reaction product and wherein the neoagarooligosaccharide mixture comprises less than 10% neoagarooligosaccharide based on the total weight of the neoagarooligosaccharide mixture, Characterized in that it contains neoagarobiose, 50 to 70% by weight of neoagarotetraose and 30 to 50% by weight of neoagarohexaose. Functional foods. Streptomyces coelicolor A feed for preventing or ameliorating obesity or diabetes comprising, as an active ingredient, a reaction product obtained by an enzyme reaction of DagA with agar or agarose. 10. The method of claim 9,
Wherein the enzyme reaction is carried out at a temperature of 35 to 45 DEG C and at a pH of 6 to 8. 10. The method of claim 9,
Wherein the enzyme reaction is carried out by adding DagA at a concentration of 20 to 100 unit / ml to an agar or agarose solution of 0.5 to 5% (w / v). 10. The method of claim 9,
Wherein the reaction product contains 45 to 85% neoagarooligosaccharide mixture based on the total weight of the reaction product and wherein the neoagarooligosaccharide mixture comprises less than 10% neoagarooligosaccharide based on the total weight of the neoagarooligosaccharide mixture, Characterized in that it contains neoagarobiose, 50 to 70% by weight of neoagarotetraose and 30 to 50% by weight of neoagarohexaose. Forage feed. A neoagarol oligosaccharide mixture as an active ingredient,
Wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose or neoagarohexaose, wherein the composition comprises a neoagarooligosaccharide, a neoagarooligosaccharide, . 14. The method of claim 13,
The neoagarooligosaccharide mixture may contain up to 10% neoagarobiose, 50-70% neoagarotetraose and 30-50% by weight of neoagarooligosaccharide, based on the total weight of the neoagarooligosaccharide mixture A pharmaceutical composition for the prevention and treatment of obesity or diabetes, which comprises neoagarohexaose. A neoagarol oligosaccharide mixture as an active ingredient,
Wherein said neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose or neoagarohexaose. 2. The functional food for preventing and improving obesity or diabetes according to claim 1, wherein said neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose or neoagarohexaose. 16. The method of claim 15,
The neoagarooligosaccharide mixture may contain up to 10% neoagarobiose, 50-70% neoagarotetraose and 30-50% by weight of neoagarooligosaccharide, based on the total weight of the neoagarooligosaccharide mixture A functional food for prevention and improvement of obesity or diabetes characterized by containing neoagarohexaose. A neoagarol oligosaccharide mixture as an active ingredient,
Wherein the neoagarooligosaccharide mixture contains neoagarobiose, neoagarotetraose or neoagarohexaose. The diarrheicidal composition according to claim 1, wherein the neoagarooligosaccharide mixture contains neoagarobiose, neoagarotetraose or neoagarohexaose. 18. The method of claim 17,
The neoagarooligosaccharide mixture may contain up to 10% neoagarobiose, 50-70% neoagarotetraose and 30-50% by weight of neoagarooligosaccharide, based on the total weight of the neoagarooligosaccharide mixture A functional food for prevention and improvement of obesity or diabetes characterized by containing neoagarohexaose.

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