KR101656479B1 - Composition for anti-obesity containing extract of pseudomonas culture - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating obesity or metabolic diseases caused by obesity, which comprises a surfactant isolated from a culture medium of Pseudomonas sp. The present invention also relates to a health functional food for preventing or ameliorating obesity or a metabolic disease caused by obesity, which contains a surfactant separated from a culture medium of a microorganism of the genus Pseudomonas as an active ingredient. The surfactant according to the present invention can be applied to anti-obesity applications or weight control applications by inhibiting lipid digestion.

Description

TECHNICAL FIELD [0001] The present invention relates to an anti-obesity composition comprising an extract of Pseudomonas sp.

The present invention relates to a pharmaceutical composition for preventing or treating obesity or a metabolic disease caused by obesity containing a surfactant isolated from a culture medium of Pseudomonas sp . The present invention also relates to a health functional food for preventing or ameliorating obesity or a metabolic disease caused by obesity, which contains a surfactant separated from a culture medium of a microorganism of the genus Pseudomonas as an active ingredient.

Obesity is a state of excess body fat accumulation which is caused by an increase in the number of adipocytes or an increase in the size of adipocytes because the amount of adipocyte synthesis in the adipose tissue is greater than that of the adipocyte. Obesity is the most directly related to the accumulation of excess energy, among which there are caloric intake, endocrine disruption, lack of exercise, genetic factors, and about 95% of obese patients have low energy consumption It belongs to the simplicity obesity brought about. Also, obesity can lead to complications such as diabetes, hypertension, cardiovascular related diseases, joint diseases caused by excessive weight, lung diseases and cancer. Obesity, which is regarded as a risk factor for lifestyle-related diseases, is now recognized as an independent disease. As interest in obesity around the world is increasing, competition for new drug development is accelerating. Obesity, which is caused by unbalance of energy consumption and consumption, is applied with exercise therapy, low calorie diet, etc. depending on the degree. Drug therapy is applied to decrease food intake, change nutrition absorption and metabolism, increase energy consumption .

Currently, the global anti-obesity material market is over $ 200 billion, and it is expected to grow rapidly every year due to the increase in the obesity population. However, various side effects such as orlistat and sibutramine, which are representative anti-obesity drugs currently being used, have been reported, and research and development of anti-obesity substances to replace them have been actively carried out. Representative natural materials include green tea catechin, grape seed extract, black bean, and red ginseng, and focus mainly on inhibiting the lipase activity, a lipase. Pancreatic lipase is an enzyme involved in the metabolism of fat, which promotes the digestion of fat and helps the intestinal epithelial cells to absorb degradation products. As the fat is decomposed and absorbed by the pancreatic lipase, inhibition of the activity of the lipase enzyme may lower the fat absorption in the blood.

Pseudomonas is truly a genus that contains as many as 150 species in the genus Pseudomonas. Cells are either non-exercise bacterium, which usually have fluorescence, or that emit green, blue, purple, yellow, or some other insoluble purple or yellow pigment . Gram-negative bacteria and widely distributed in soil, fresh water, and seawater. Aerobic, but denitrification or nitrate respiration also anaerobically grows, but does not fix nitrogen. Aliphatic hydrocarbons, phenols, terpenes, steroids, and the like. These include P. aeruginosa and P. fluorescens .

However, it is not known at present that extracts of Pseudomonas sp. Cultures can be used for anti-obesity because of their lipid digestion inhibitory effect.

It is an object of the present invention to provide a natural substance which can suppress lipid digestion in digestive organs such as duodenum, control bioavailability of lipid, and ultimately be used for anti-obesity use.

The inventors of the present invention have conducted intensive studies in order to achieve the above object, and as a result, discovered that the surfactant isolated from the culture medium of Pseudomonas sp. Cultures, particularly Pseudomonas sp. Cultures, has excellent lipid digestion inhibitory activity and completed the present invention .

The surfactant separated from the culture medium of Pseudomonas sp. Microorganism according to the present invention can be applied to anti-obesity use or weight control use by suppressing lipid digestion.

1 is a schematic diagram showing an example of a lipid digestion system.
FIG. 2 shows the Pseudomonas sp . Z1 and the surface tension of the culture medium.
Figure 3 is a graphical representation of Pseudomonas sp . Z1 < tb >< SEP >
Fig. 4 shows the Pseudomonas sp . Z1 Production It is a graph showing changes in emulsification index and surface tension according to the pH of a surfactant.
FIG. 5 shows the results of Pseudomonas sp . Z1 Production It is a graph showing changes in emulsification index and surface tension according to temperature change of a surfactant.
FIG. 6 is a graph showing Pseudomonas sp . Lt; RTI ID = 0.0 > Z1 < / RTI > culture on lipid digestion of free fatty acids.
FIG. 7 shows the Pseudomonas sp . Z1 < / RTI > culture on the surface tension of emulsions during lipid digestion.
Figure 8 shows that Pseudomonas sp . Lt; RTI ID = 0.0 > Z1 < / RTI > culture on the particle size of the emulsion during lipid digestion.

Hereinafter, the present invention will be described in detail. However, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases caused by obesity or obesity, which comprises a surfactant separated from a culture medium of Pseudomonas sp. Microorganism as an active ingredient. In particular, the present invention relates to the use of Pseudomonas sp . The present invention relates to a pharmaceutical composition for preventing or treating obesity or metabolic diseases caused by obesity containing a surfactant separated from a culture medium of Z1 (accession number KCTC 12364BP) as an active ingredient. The term " surfactant " used in the present invention means a substance having the hydrophilic and hydrophobic characteristics produced by Pseudomonas aeruginosa in vitro.

The surfactant according to the present invention is excellent in inhibiting lipid digestion in the human body and can be used for controlling the bioavailability of lipid.

Accordingly, the pharmaceutical composition of the present invention can be used for prevention or treatment of obesity. In addition, the pharmaceutical composition of the present invention can be used for prevention or treatment of obesity-related or metabolic diseases caused by obesity. The metabolic diseases may be, but are not limited to, one or more diseases selected from the group consisting of hyperlipidemia, fatty liver, arteriosclerosis, cardiovascular disease, or metabolic syndrome in which the diseases occur simultaneously.

In the present invention, a surfactant is separated from a microorganism of the genus Pseudomonas, and emulsified using the surfactant. The method for producing the emulsion is not particularly limited, and a conventional method using an emulsifier can be used. The emulsion may further contain a buffer and an oil, and may contain 30 to 50% (w / v) of the active ingredient.

As an example of a method for producing an emulsion, there are a step of removing microbial cells from a culture solution in which a microorganism of the genus Pseudomonas is cultured, And extracting the surfactant from the supernatant from which the cells have been removed.

In the present invention, Pseudomonas sp . Z1 culture extracts were separated and the lipid digestion inhibition effect was investigated by investigating the interfacial characteristics (free fatty acid, surface tension, lipid particle size, etc.) of the emulsions during lipid digestion under the environmental conditions of small intestine after lipid preparation Respectively.

The effect of the surfactant isolated from the culture medium of Pseudomonas sp. Microorganism on the interfacial characteristics of the emulsions during the digestion of lipids was investigated. As a result, the content of free fatty acid in the emulsion was higher than that of the surfactant (lecithin and tween 80) , Changes in surface tension and changes in emulsion particle size were significantly low. This is because the surface active substance isolated from the culture medium of Pseudomonas sp. Is a sugar-bound glycolipid, and the bound sugar imparts steric hindrance to adsorption and hydrolysis of lipid particles such as bile acid and lipase, The hydrolysis was suppressed.

In this regard, it has been previously reported that the interfacial properties of lipid emulsion droplets influence the rate and extent of lypolysis (Chu BS et al., Langmuir. 2009 Aug 18; 25 16): 9352-60).

Generally, microorganisms produce biosurfactant when the surface tension of cultured microorganism is 40 mN / m or less. If the surface tension is 35 mN / m or less, mass production of biosurfactant Pseudomonas spp. sp . The surface tension of the extract of Z1 culture was 26 mN / m, indicating excellent performance of the surfactant. In addition, Pseudomonas sp . The extract of Z1 culture is more soluble in mixed solvents such as chloroform / methanol than n-hexane, and thus it is presumed to be a biosurfactant of glycolipid showing the dissolution characteristics of glycolytic system.

The pharmaceutical composition according to the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppository syringes, pre-filled syringes ) Solution form, or a lyophilized form, but the present invention is not limited thereto.

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like are included in addition to water and liquid paraffin which are conventionally used simple diluents . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The preferred dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the condition and body weight of the patient, the degree of obesity, the type of disease, the drug form, the administration route and the period. It is preferable for the composition of the present invention to allow the active ingredient to be administered at 0.2 to 200 mg / kg per day for optimal efficacy. The administration may be carried out once a day or divided into several doses, but is not limited thereto.

In another aspect, the present invention relates to a health functional food for preventing or ameliorating obesity or a metabolic disease caused by obesity, which comprises a surfactant separated from a culture medium of a microorganism of the genus Pseudomonas as an active ingredient. In particular, the present invention relates to the use of Pseudomonas sp . Z1 (Accession No. KCTC 12364BP) as an active ingredient, or a metabolic disease caused by obesity.

The health functional food according to the present invention can be utilized not only as a patient for obesity but also as a functional food for weight control of a normal weight person.

The health functional food of the present invention may be manufactured in various forms including, but not limited to, various drinks, gum, tea, confectionery, vitamin complex, health supplement, and food for controlling body weight.

The preferred intake amount of the health functional food of the present invention varies depending on the condition and body weight of the recipient, the degree of obesity, the type of food, the period of consumption, and can be appropriately selected. It is preferable for the composition of the present invention to allow the active ingredient to be ingested at 0.2 to 200 mg / kg per day for optimal effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Materials and methods

(1) Culture of microorganisms

Pseudomonas sp . Z1 was cultured in Luria-Bertani medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of bacto agar, DW 1 L) and used in the experiment . Pseudomonas sp . The Z1 pre-culture solution was inoculated in a 1 L nutrient medium and cultured at 30 ° C with shaking.

(2) Growth of strain

1 mL of the culture solution of the strain was collected at predetermined time intervals, and the absorbance was measured at 600 nm using a UV-VIS spectrophotometer.

(3) Surface tension measurement

The surface tension was measured by centrifuging the culture at 8,000 rpm for 15 minutes, removing the cells, measuring the surface tension three times by the ring method using a surface tension meter (Sigma 702, Attention by KSV, Finland) .

(4) Investigation of lipid digestion and interfacial characteristics

The lipid digestion system of Fig. 1 was applied to investigate the lipid digestion characteristics of the emulsions. The emulsion was preheated at 37 ° C for 10 minutes and then added with 2 ml of bile salt (5 mg / ml) and lipase (8 mM) in a 2 mM Bis-tris buffer (0.15 M NaCl, 0.01 M CaCl ₂ , pH 7) mg / mL), and incubated for 2 hours at 200 rpm and 37 ℃ for 2 hrs. The samples were collected at regular intervals to determine the interfacial characteristics such as free fatty acid content, surface tension, and emulsion particle size. Respectively. The free fatty acid was titrated to the pink color with 0.05 N NaOH after dropping 3 drops of 1% phenolphthalein in the lipid digestion solution for each reaction time. The surface tension was determined by the ring method using the surface tension meter and the emulsion particle size Were measured using a particle size analyzer (Nanotrac 250, Microtrac, USA).

(5) Of the surfactant  detach

Pseudomonas sp . The culture broth of Z1 was centrifuged (8000 rpm, 15 min) to remove the microbial cells. The pH of the supernatant was adjusted to pH 2.0 by slowly adding 1N HCl solution to the supernatant. The same amount of chloroform: methanol: water : 1) was added and the mixture was allowed to stand overnight at 4 ° C with stirring. After that, only the organic solvent layer was recovered through a separatory funnel, concentrated under reduced pressure, dissolved in 0.2 M phosphate buffer (pH 7.0), and powdered using a freeze dryer.

(6) Critical micelle concentration ( Critical micelle concentration , CMC ) Measure

CMC was measured by repeating the surface tension three times at 25 ° C while diluting the separated surfactant in 0.2M phosphate buffer (pH 7.0) at a constant concentration. At this time, the concentration at the point where the surface tension suddenly increased according to the dilution ratio was determined as the CMC value.

(7) Measurement of substrate specificity

Substrate specificity was determined by the addition of 2 mL of a solution of the separated surfactant in 0.2 M phosphate buffer (pH 7.0) at a concentration of 1 g / L and a mixture of sunflower oil, hexadecane, kerosene, hexane, Benzene were mixed with each other, and the mixture was emulsified for 2 minutes with vigorous stirring. After standing for 24 hours at room temperature, the height of the emulsion was measured. Using the height of the measured emulsion, E ₂₄ (emulsification index,%) was obtained according to the following formula.

E ₂₄ (%) = (height of emulsion layer / height of total solution) * 100

(8) pH  Stability measurement

pH stability is respectively adjusted to pH 2~12 with surface-active substances removed with HCl and NaOH were dissolved in 1g / L concentration of a 2M phosphate buffer (pH 7.0), and then after which had been allowed to stand at room temperature for 24 hours and the surface E ₂₄ The tension was measured.

(9) Measurement of thermal stability

The thermal stability was determined by dissolving the separated surfactant in 0.2 M phosphate buffer (pH 7.0) at a concentration of 1 g / L, and then heat-treating the resultant at a temperature ranging from 4 to 121 ° C for 45 minutes, then allowing to stand at room temperature for 24 hours, ₂₄ and surface tension were measured.

Example  One. Pseudomonas sp . Z1 ≪ / RTI > Of the surfactant  Physicochemical properties

1-1. Growth of the strain and surface tension of the culture

Pseudomonas sp . The growth of Z1 and the surface tension of the culture broth were investigated . Pseudomonas sp . It was confirmed that the surface tension was greatly decreased from the initial 52 mN / m to 26 mN / m during 3 days culture of Z1, and it was confirmed that the strain was able to produce an excellent culture extract. Also, . However, after 3 days, the growth was no longer active and the surface tension was not substantially decreased, indicating that the optimum culture period was 3 days (see FIG. 2).

1-2. Isolate from the strain Of the surfactant CMC

The surface tension of pure water containing no surfactant was 69 mN / m, whereas when the initial concentration of surfactant was 200 mg / L, the surface tension of water was greatly reduced by the added surfactant, which was 29.3 mN / m Surface tension. As a result, the surface tension was not changed even when diluted to the concentration of 10 mg / L, but it was increased rapidly at the lower concentration. The concentration of 10 mg / L, which is the concentration at which the surface tension rapidly changes, is called Pseudomonas sp . And the CMC of the surfactant produced by Z1 (see Fig. 3).

1-3. Isolate from the strain Of the surfactant  Substrate specificity

As shown in Table 1, Pseudomonas of hydrocarbons such as sunflower oil, setane, kerosene, hexane, and benzene sp . As a result of investigating the substrate specificity of the Z1 production surfactant, the surfactant was found to be inferior in emulsifying activity to commercial emulsifiers such as lecithin and Tween 80. However, the emulsifying activities of edible oils such as sunflower oil were almost similar.

Pseudomonas sp . The substrate specificity of the surfactant isolated from Z1 temperament
Emulsion index (E24) Pseudomonas sp. Z1 Twin 80 lecithin Sunflower oil 86 ± 2.9 88 ± 2.6 92 ± 1.1 Setain 73 ± 1.2 84 ± 3.6 87 ± 0.8 Kerosene 72 ± 2.4 76 ± 1.2 79 ± 2.1 Hexane 41 ± 4.6 69 ± 0.7 72 ± 3.9 benzene 39 ± 5.1 53 ± 1.3 61 ± 1.6

1-4. Isolate from the strain Of the surfactant pH  And thermal stability

The pH stability of the surfactant was investigated. It showed high emulsification index and low surface tension in the neutral range of pH 6 ~ 8, and low emulsification index and high surface tension in other ranges. On the other hand, when the surfactant was heat-treated at a temperature range of 4 ~ 121 ℃, the thermal stability of the surfactant was higher than that of the surfactant in the range of 50 ~ 75 ℃. Appear to be missing Pseudomonas sp . It was found that the thermal stability of the Z1 production surfactant was excellent (see FIGS. 4 and 5).

Example  2. Pseudomonas sp . Z1  Using the extract of the culture Emulsion  Manufacturing and characterization thereof

2-1. Emulsion  Produce

In order to prepare an emulsion using the extract of the culture isolated from the strain of Example 1, the blend ratio of the culture extract to the mixture of 2 mM Bis-tris buffer and olive oil was changed to 20 and 40 mg% according to the formulation shown in Table 2 Emulsions were prepared using lecithin and Tween 80, which are industrially used as emulsifiers. The addition amount of lecithin and tween 80 was 40 mg%. The mixture was sonicated for 15 minutes and then homogenized for 5 minutes using ULTRA-TURRAX T25 (IKA-Werke, Germany). Using a microfluidizer processor (M-110EH-30, At a pressure of 3 times.

Preparation of Emulsifier for Analysis of Lipid Digestion Inhibitory Effect of Extracts from Cultures olive oil 2 mM Bis-tris Buffer Surfactants Production Example 1 200 mg 100 mL Extract of the culture (20 mg%) 20 mg Production Example 2 200 mg 100 mL Extract of the culture (40 mg%) 40 mg Comparative Example 1 200 mg 100 mL Lecithin (40 mg%) 40 mg Comparative Example 2 200 mg 100 mL Tween 80 (40 mg%) 40 mg

2-2. Amount of free fatty acid

Pseudomonas sp . As a result of measuring the amount of free fatty acids produced by lipid digestion of the emulsion prepared by adding the extract of the culture isolated from the Z1 culture medium, the amount of free fatty acid produced during the reaction time of lecithin and tween 80 added emulsion was large, while that of Pseudomonas sp . In the case of the emulsion added with the extract of the culture isolated from Z1, the amount of free fatty acid produced was small, and in particular, Pseudomonas sp . It was judged that the production of the free fatty acid of the emulsion containing 40 mg% of the extract of the Z1 culture was the least and the digestion of the fat particles was significantly suppressed (see FIG. 6).

2-3. Surface tension

Pseudomonas sp . The effect of Z1 cultured extract on the surface tension of emulsions during lipid digestion was found to be similar to that of free fatty acid production. Pseudomonas sp . In the case of the emulsions containing the Z1 cultured extract, the surface tension values did not change after 10 minutes and the values remained stable and similar values, while the surface tension of lecithin and Tween 80 added emulsion continuously decreased. When the lipid is produced by the action of lipase, the free fatty acid formed forms micelles and lowers the surface tension. The surface tension of lecithin and twin 80-added emulsions with high free fatty acids is low, (See Fig. 7).

2-4. Particle size

Pseudomonas sp . As a result of investigating the effect of the extract of Z1 culture on the particle size of the emulsion during the lipid digestion, the particle size of the lipid formed by the emulsion continued to be decreased due to the liberation of the free fatty acid by the action of lipase as the lipid digestion progressed , Especially the lecithin and Tween 80 added emulsion showed a large particle size reduction. Pseudomonas sp . In the case of the emulsions containing the Z1 culture, the particle size did not decrease significantly even at 40 mg% treatment time, indicating that the lipid digestion was not as good as the surface tension of the free fatty acids and the culture medium (See Fig. 8).

Korea Research Institute of Bioscience and Biotechnology KCTC12364BP 20130130

Claims (6)

Pseudomonas sp. Z1 (Accession No. KCTC 12364BP) in a culture medium for at least 3 days; And separating the glycolic surfactant from the culture solution. A pharmaceutical composition for preventing or treating obesity, which comprises as an active ingredient a glycolipid surfactant prepared according to the production method of claim 1. 3. The method of claim 2,
A pharmaceutical composition for the prevention or treatment of obesity, characterized by inhibiting lipid digestion. A health functional food for improving obesity containing the glycolipid surfactant prepared according to the production method of claim 1 as an active ingredient. delete 5. The method of claim 4,
A health functional food for improving obesity characterized by inhibiting lipid digestion.

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