JPH0517831B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a novel microorganism, Streptomyces sp. Y-125, and a method for producing an amylase inhibitor therefrom. (Background Art) In general, most of carbohydrates, which are one of the three major nutrients necessary for the human body, are ingested in the form of disaccharides or polysaccharides, and then immediately before being absorbed into the body, carbohydrates are processed by amylase or maltase. Or, it is decomposed by the action of glycoside hydrolase such as satucalase, and if an excessive amount of sugar is produced in the body at this time, it causes diabetes, obesity, hyperlipidemia, gastritis, gastric ulcer, duodenal ulcer, etc. Therefore, in order to prevent and treat these diseases, excessive amounts of sugar can be prevented by suppressing the action of various glycoside hydrolases existing in the body using glycoside hydrolase inhibitors. Methods are known to prevent the production of glycoside hydrolase (U.S. Pat. Nos. 4,307,194 and 4,451,455), and such glycoside hydrolase inhibitors are inhibited by actinomycetes, particularly Streptomyces. It is known as a substance that is often produced. It has been reported that conventional glycoside hydrolases have a superior inhibitory effect on amylase depending on their molecular weight, whereas those with a low molecular weight have an excellent inhibitory effect on maltase or satucarase. What is obtained by the method of the present invention is an amylase inhibitor that has a relatively low molecular weight but exhibits a specific inhibitory effect on amylase. Conventionally known methods for producing amylase inhibitors include non-biological methods, i.e. physical adsorption of low molecular weight substances such as salicylic acid or abiscine to non-specifically inhibit the enzyme. There are also methods of manufacturing using polymeric substances that inhibit, denature, or precipitate the enzyme. However, these conventional amylase inhibitors only affect salivary amylase;
It has little effect on pancreatic amylase (E. Kneen, RMStandtest, "Arch.
Biochem . It is reported that the degree of activity is relatively low because it becomes inactive by (US Pat. No. 4,282,318). Therefore, the present inventors discovered a new microorganism with high activity that produces an amylase inhibitor, and conducted extensive research on its isolation and utilization. As a result, the amylase inhibitor produced by the new microorganism was found to We have discovered that this substance has better activity and stability than inhibitors, leading to the present invention. An object of the present invention is to provide a novel strain that produces amylase inhibitors and a method for effectively producing amylase inhibitors in high yield using the same. (Disclosure of the Invention) The microorganism of the present invention, Streptomyces sp.
Y-125 is a new bacterial strain isolated and cultured from soil samples collected in Chuncheon, Gangwon-do, and Osan, Gyeonggi-do, Republic of Korea.
Entrusted to the Korea Advanced Institute of Science and Technology under accession number KCTC8387P on July 5, 1988, and designated as the American Type Culture Collection on March 31, 1989.
(ATCC) under accession number ATCC53890. Below, the new strain Streptomyces sp.Y−125
We will explain the phylogenetic characteristics of The characteristics were measured using the International Streptomyces project.
The methods recommended by the Bergey's Manual of Determinative Bacteriology (ISP) and the Bergey's Manual of Determinative Bacteriology (ISP)
This was carried out in accordance with the method described in "Manual of Determinative Bacteriology" Volume 2 (1986). Below is data comparing characteristics with the following five known Streptomyces microorganisms: Microorganism 1: Streptomyces carpus (St.
(J. Antibiotics) 35: 1156-1159
(1982)) Microorganism 2: St. Corchorusii (Agricultural and Biological Chemistry (Agric.
Biol.chem.) 49(1), 107-110 (1985)) Microorganism 3: amylostatics, a subspecies of St. diastaticus (Agricultural and biological chemistry,
41(6), 919-924 (1977)) Microorganism 4: St. griscosporeus (Agricultural and Biological Chemistry,
45(11), 2599-2604 (1981)) Microorganism 5: St. spiritus (U.S. Patent No. 4254256) The following properties were measured: (1) ISP No. 2 = yeast/malt agar medium (2) ISP No. 3 = oatmil agar medium (3) ISP No. 4 = starch/inorganic salt agar medium (4) ISP No. 5 = Glycerin/Asparagine Agar (5) ISP No. 6 = Peptide/Yeast/Iron Agar (6) ISP No. 7 = Tyrosine Agar (7) SM = Skim Milk Agar (8) NT = Nutrition Agar medium (9) GA = glucose-asparagine agar medium (Note) ISP = International Streptomyces project (International
Streptomuces Project) medium The new strain of Streptomuces of the present invention
Since sp. , starch/inorganic salt agar medium (ISP No.4), tyrosine agar medium (ISP
No. 7), and the growth state and color can be clearly observed especially on oatmeal agar medium (ISP No. 3). The hue of the back side is tan, and this hue is not affected by pH. A yellow pigment was produced on the tyrosine agar medium, a pale yellow pigment was produced on the skim milk agar medium, and no soluble pigment was produced on the other media. Table 2 shows comparative data between Streptomyces sp. Y-125 and the above-mentioned five known microorganisms of the genus Streptomyces.

【table】

【table】

[Table] +: Generated; -: Not generated; ±: Unclear.
×: No data.
(2) Morphological characteristics Observation of the morphological characteristics of Streptomyces sp. Ta. In addition, sporophytes are produced at the edges of aerial hyphae, and the aerial hyphae are wavy in shape, and the spores are spherical or rod-shaped. The surface of the spore is soft and smooth, and its size is 0.7~1.2×
It was 1.2 to 1.8 μm. At this time, the morphological characteristics were investigated using an optical microscope, and the surface and size of the spores were observed using a scanning electron microscope. Table 3 shows comparative data between Streptomyces sp. Y-125 and the above-mentioned five known microorganisms of the genus Streptomyces.

[Table] (3) Physiological characteristics The following Table 4 shows comparative data between Streptomyces sp. Y-125 and the above-mentioned five known microorganisms of the genus Streptomyces. The degree of carbon utilization, that is, the assimilability of the carbon source shown in Table 4 is the result of measuring the degree of carbon utilization using a Pridham-Godlieb agar medium to which a sterilized carbon source was added to give a final concentration of 1.0%. The culture temperature was 30℃.
Measurements were taken 14 days later.

[Table] (Note) −: Not used; +: Used;
±: Unclear; ×: No data As shown in Table 4, Streptomyces sp.
Y-125 hydrolyzed starch but did not liquefy (i.e. hydrolyze) gelatin, coagulate and peptonize skimmed milk, reduce nitrate to nitrite, or degrade cellulose. Furthermore, these strains grew at 20 to 80°C, particularly actively multiplied at 25 to 30°C, were aerobic, and produced melanin-like pigments on tyrosine agar (ISP No. 7). As mentioned above, the Streptomyces of the present invention
Since sp. 2, pp. 1383-1418 (1986)). The new strain of the present invention, Streptomyces sp.
The amylase inhibitor produced by Y-125 is a substance composed of polysaccharides and amino acids, and its activity is
It was extremely high at over 12,000 AIU/mg, and showed excellent inhibitory activity even when heated at 100°C for 120 minutes. One unit of amylase inhibitor (1 AIU) is defined as the amount of inhibitor when a unit of amylase is inhibited by 50%, and one unit of amylase is converted from starch per minute.
Defined as the enzyme amount when 1 μM of glucose is produced, the glucose produced was measured for reducing sugar using 3,5-dinitrosalicylic acid, and a standard curve was obtained using maltose.The activity of the amylase inhibitor according to the present invention was It was measured using the following method. That is, 100mM Tris-HCl buffer containing 5mM calcium chloride (PH
7.0) in 2 x 10 ^-2 % amylase solution (10~
Dissolve 0.5 ml of 20 AIU/ml), add 0.5 ml of inhibitor solution (0-300 μg) or culture medium, mix and incubate at 37°C for 10 minutes, then
Add 2 ml of 1.5% soluble starch solution in 100 mM Tris-HCl buffer and incubate for 10 minutes. Next, 2 ml of 3,5-dinitrosalicylic acid was added, heated for 5 minutes, cooled thoroughly, and diluted with distilled water for 10 minutes.
After dilution, the absorbance was measured at 546 nm. The control test was carried out in the same manner as above except that 0.5 ml of distilled water was added instead of the inhibitor solution, and the blank test was carried out using 10 ml of distilled water instead of the amylase solution.
It was carried out in the same manner as described above, except that 0.5 ml of mM Tris-HCl buffer (PH 7.0) was added and 0.5 ml of distilled water was added instead of the inhibitor solution. Inhibition rate (%) was determined from the measured value of absorbance based on the following formula: Inhibition rate (%) = (B-T)-C/B-C×100 In the above formula, T, C, and B each represent an inhibitory substance. The absorbance obtained in the test, control test, and blank test is shown. The amylase inhibitor according to the present invention has extremely high stability against pH, being stable in the pH range of 2.0 to 12.0, and therefore its activity does not decrease during the decomposition process. The amylase inhibitor according to the present invention has a molecular weight of 700 to 1500 as measured by Sephadex G-25 (trade name), and is sufficient for amylase, maltase, and satucalase despite its relatively low molecular weight. It was found that the inhibitory effect on pancreatic amylase was particularly superior to that on salivary amylase. Streptomyces sp.Y-125 of the present invention
(KCTC8387P, ATCC53890) The amylase inhibitor produced by the bacterial species can be used as an active ingredient and by containing it in a pharmaceutically acceptable carrier or diluent, it can be used to treat obesity, diabetes, gastritis, hyperlipidemia, and gastric ulcers. , can be used as a prophylactic and therapeutic agent for duodenal ulcer, and Streptomyces sp. Y-125 (KCTC8387P, ATCC53890) of the present invention
Amylase inhibitors produced by the bacterial species are useful as food additives. Below, a method for culturing Streptomyces sp. Y-125, a new strain of the present invention, and a method for separating amylase inhibitors from the culture will be described in detail. (1) Cultivation of the strain A dilute aqueous hydrochloric acid solution is added to a medium containing yeast extract, defatted soybeans, a nitrogen source such as peptone, soluble starch, a carbon source such as glucose, and an inorganic salt such as sodium chloride. PH
After adjusting the temperature to 6.0 to 7.0 and sterilizing at 121℃ for 15 minutes, the bacteria were inoculated and incubated at 25 to 30℃ under aerobic conditions.
Culture was carried out for ~4 days. Table 5 below shows the activity of the inhibitors against various carbon sources (1% concentration). Table 6 shows the activity of inhibitors against nitrogen sources. As can be seen from Tables 5 and 6, the carbon source is soluble starch, and the nitrogen source is yeast extract and polypeptone.
The activity was highest when it was a mixture of Antifoaming agents may also be used at this time, and antifoaming agents are generally useful in preventing excessive foaming. A commonly used antifoam agent such as a silicone antifoam agent was used.

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[Table] (2) Isolation of amylase inhibitors The process of separating the culture fluid from the bacterial cells from the bacterial strain culture can be carried out using centrifugation or a filtration method using a filter aid such as Celite (trade name). This can be done using conventional methods such as The step of separating the amylase inhibitor from the culture solution thus separated can be carried out using conventional methods, such as freeze-drying the culture solution, salting out, or precipitation with an organic solvent.
This can be done using methods such as adsorption, as well as adsorption with ion exchange resins, gel filtration. These methods will be explained in more detail below. (b) Centrifuge the bacterial strain culture medium (30,000 ~
40,000 rpm) to separate the bacterial cells and the culture solution, and then the culture solution was heated under reduced pressure (10 to 70°C) at 50 to 70°C.
50mmHg) and concentrate to 1/5 to 1/10. The precipitate is then filtered off. The obtained concentrate is freeze-dried as required. (b) A hydrophilic solvent such as methanol or acetone is added as an organic solvent to the centrifuged culture solution or concentrate to precipitate inhibitory substances. Impurities precipitate at low concentrations, with concentrations of 60-70%
It is easy to remove impurities if the (c) Separating the precipitate using salts such as ammonium sulfate or sodium chloride. The precipitate is centrifuged or directly washed with an organic solvent, dialyzed, and dried. (d) Adsorption by ion exchange resin: This method is the most suitable method for separating amylase inhibitors when they have polarity, and is based on changes in ionic strength or pH. The amylase inhibitor is separated by elution and gel filtration using its molecular weight. In addition to the above-mentioned separation methods, it is also possible to use methods such as precipitation of impurities due to thermal denaturation, permeability of molecular membranes based on molecular weight, and ultrafiltration. (Example) Next, the present invention will be described with reference to an example. Example 1 0.1% soluble starch in 2 thin flasks,
PH7.0 containing 1% glucose, 0.5% gravy, 0.5% peptone, and 0.3% sodium chloride
After adding 400 ml of culture medium and sterilizing it at 121°C for 15 minutes, Streptomyces sp.
20 ml of Y-125 bacterial solution was inoculated and cultured at 28°C for 4 days. After culturing, this culture fluid was separated into bacterial cells and culture fluid by centrifugation, and 270 ml of the resulting culture fluid with a concentration of 15 AIU/ml was heated at 40 to 60°C under reduced pressure (10 to 50 mm
Hg) to obtain 30 ml of concentrated solution. Add 90% ethanol to this concentrate, centrifuge the generated precipitate, then dissolve this concentrate in distilled water,
After that, freeze-dry for 36 hours to obtain 2×10 ⁴ AIU/g.
0.07g of amylase inhibitor was obtained. Example 2 2 Five flasks were each filled with a pH 7.0 medium containing 1% soluble starch, 0.5% peptone, 0.5% meat juice, and 0.3% sodium chloride.
Pour 400 ml of Streptomyces sp.
20 ml of each bacterial solution was inoculated and cultured at 28°C for 4 days. Next, this culture was separated into bacterial cells and a culture solution by centrifugation, and 1500% of the culture solution with a concentration of 85AIU/ml was obtained.
ml of this culture under reduced pressure at 50-60 °C.
Concentrate to 150ml, filter to remove impurity precipitate,
Thereafter, it was treated with 60% ethanol, the precipitate formed was removed by filtration, and the filtrate was concentrated. This concentrate was freeze-dried for 24 hours to give a concentration of 3×10 ⁵ AIU/g.
6.4g of amylase inhibitor was obtained. Example 3 2 Using 10 flasks, Example 2
Culture was carried out in the same manner as above to obtain 3000 ml of culture solution.
This culture solution was concentrated to 1/10 under reduced pressure at 60-70°C, then treated with 90% ethanol, the formed precipitate was filtered off, the filtrate was concentrated again, and the formed concentrate was mixed with distilled water. and then lyophilized for 24 hours to obtain 10.2 g of amylase inhibitor with 4.7×10 ⁵ AIU/g. Example 4 2% soluble starch, 0.5% polypeptone, 0.5% meat juice, and 0.3% soluble starch in 5 thin flasks.
Pour 400 ml of a medium with a pH of 7.0 containing % sodium chloride into each plate, sterilize it at 121°C for 15 minutes, and then inoculate 20 ml of Streptomyces sp. The cells were cultured at 28° C. for 3 days, and then the culture was separated into bacterial cells and a culture medium by centrifugation. 218 AIU/ml culture solution obtained in this way 1550
ml was concentrated to 1/10 under reduced pressure at 60-70°C,
The impurity precipitate was removed by filtration, and then the concentrate was treated with 90% methanol, the formed precipitate was filtered out again, and then the filtrate was concentrated to obtain a concentrate. This concentrate was dissolved in distilled water and freeze-dried for 36 hours, resulting in 8.2 g, which is 5.5 × 10 ⁵ AIU/g.
amylase inhibitor was obtained. Example 5 2 In 5 flasks, 1% corn starch, 0.5% polypeptone, 0.5% meat juice,
Pour 400 ml of PH7.0 medium containing 0.3% sodium chloride into each plate, sterilize them at 121°C for 15 minutes, and inoculate each with 20 ml of Streptomyces sp. and 28℃
The cells were cultured for 4 days. Next, this culture solution was separated into bacterial cells and culture solution by centrifugation, and 1600 of culture solution with 186 AIU/ml was obtained.
ml was obtained, the culture solution was concentrated to 1/10, and the precipitate was removed by filtration. The concentrated solution was then treated with 90% ethanol, the resulting precipitate was filtered off again, and then the filtrate was concentrated to obtain a concentrate. This concentrate was dissolved in distilled water and freeze-dried for 42 hours.
9.8 g of amylase inhibitor, which is 5×10 ⁵ AIU/g, was obtained. Example 6 Culture was carried out under the same conditions as in Example 5 except that a medium containing 1% corn starch was used. The culture solution had a concentration of 220 AIU/ml, and in this way, 12.0 g of amylase inhibitor was obtained. (5.8×
10 ⁵ AIU/g) was obtained. Example 7 Five 2 flasks containing 2.5% soluble starch, 0.5% polypeptone, 0.5% yeast extract, and 0.3% sodium chloride, pH 6.0
Add 400 ml of culture medium adjusted to the above and sterilize it at 121°C for 15 minutes, then inoculate 20 ml of Streptomyces sp. did. Next, this culture was separated into bacterial cells and a culture solution by centrifugation to obtain a culture solution with a concentration of 2750 AIU/ml.
Obtained 1550ml. This culture solution was concentrated under reduced pressure at 60 to 70°C to obtain 200 ml of a concentrated solution, which was filtered to remove impurity precipitates and then treated with 90% ethanol. was concentrated. The concentrate was freeze-dried for 24 hours, resulting in 70×
An inhibitor of 10 ⁵ AIU/g was obtained. After this inhibitory substance was dissolved again in distilled water, gel filtration was performed using a Sephadex G-10 column (1.8 x 30 cm). At this time, distilled water was used for elution, and only the active part was collected and freeze-dried.
0.8 g of amylase inhibitor which is 2.1×10 ³ AIU/mg
I got it. Example 8 2 Using 10 flasks, Example 7
Cultured in the same manner as above, the yield was 3000 AIU/ml.
3000 ml of culture solution was obtained. The culture solution was then concentrated to 300 ml, then treated with 90% ethanol, the precipitate formed was filtered off, and the filtrate was concentrated to obtain a concentrate. This concentrate was redissolved in distilled water and then lyophilized for 24 hours to yield 7.1 x 10 ⁵ AIU/g of inhibitor.
15.5g was obtained. This was dissolved in distilled water again, gel-filtered through a Cephadex G-10 column (2.1 x 30 cm), and only the active portion was collected and freeze-dried. As a result, 1.02 g of amylase inhibitor with a concentration of 2700 AIU/mg was obtained. I got it. Example 9 Culture was carried out in the same manner as in Example 8 except that 0.1% silicone A (manufactured by Sigma, trade name) was added as an antifoaming agent. As a result, inhibition was 7.3 × 10 ⁵ AIU/g. 16.4 g of material was obtained. Further, using a Sephadex G-10 column (2.1 x 30 cm), 1.2 g of an amylase inhibitor having a concentration of 3100 AIU/mg was obtained in the same manner as in Example 8. Example 10 2.5% soluble starch, 0.5% polypeptone, 0.5% yeast extract,
Add 400 ml each of a medium containing 0.3% sodium chloride and 0.05% antifoaming agent and adjusted to pH 6.0.
After sterilizing this at 121℃ for 15 minutes, 20ml of the previously cultured bacterial solution was inoculated and cultured at 28℃ for 3 days. The resulting culture was centrifuged to obtain 3000ml of a culture solution with a concentration of 5250AIU/ml. Ta. This culture solution was heated to 60 to 70°C under reduced pressure (10 to 50 mm).
Concentrate to 1/10 under Hg), filter to remove impurities, treat it with 90% ethanol, filter out the formed precipitate, and concentrate the filtrate until ethanol is completely removed. . The concentrate was then freeze-dried to 7.5×10 ⁵ AIU/
We obtained amylase inhibitor g. As a result of gel filtration through a Sephadex G-10 column (1.8 x 40 cm), 1.70 g of an amylase inhibitor with a concentration of 3300 AIU/mg was obtained. Obtained inhibitor
Transfer 1.70 g to a Cephadex G-25 column (2.6 x 60
As a result of gel filtration through cm), 15200AIU/mg
350 mg of inhibitor was obtained. Characteristics of changes in blood sugar level Animal experiments were conducted using the amylase inhibitor obtained in Example 10. The animals used were mice weighing 17 to 20 g. Hyperglycemia was induced in groups of five mice, and changes in blood glucose levels of these mice were measured at intervals of 5, 15, and 30 minutes. To induce hyperglycemia, add 2.5 g of starch or 2.5 g of maltose/
Orally administered at a dose of Kg mice or 5.0
Sutucarose was orally administered to g/Kg mice. Blood sugar levels were measured using glucose oxidase.

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[Table] Toxicity experiment A toxicity experiment was conducted using the amylase inhibitor obtained in Example 10. The animals used were mice weighing 17 to 20 g, and groups of 5 were treated with amylase inhibitors.
A dose of 3000-3000000 AIU/Kg mouse was used. As a result of observation, no dead mice were observed.
Also, no special symptoms were found. Test on body weight change After administering the amylase inhibitors obtained in Example 10 by mixing them into mouse feed at different concentrations, changes in the body weight of mice were measured. The weight of the mouse used was 17
~20g, and as a result of observing changes in body weight for about 60 days in groups of 5 animals, it was found that amylase inhibitors
In the case of 3000 AIU/g, a gradual decrease in body weight was observed after 42 days, and amylase inhibitor
In the case of 9000 AIU/g, a decrease in body weight was observed after 21 days. Table 7 below shows the weight loss (%) of the mice.

[Table] The amylase inhibitor obtained in Example 10 exhibited the following properties. (1) Molecular weight In order to measure the molecular weight of amylase inhibitors, amylase inhibitors are
As a result of gel filtration using a 25 column and development by thin layer chromatography, the molecular weight was
It turned out to be between 700 and 1500. At this time, a mixture of ethyl acetate:methanol:water=27:15:23 was used as a developing solvent. (2) PH stability An amylase inhibitor was added to a solution with a pH of 2 to 12, and after incubation at 37°C for 30 minutes, the activity against human salivary amylase was measured. As a result, it was stable in all acidic, neutral, and basic conditions (see Figure 1). (3) Thermostability 1% amylase inhibitor was added to a neutral solution with a pH of 7.0, heated at 100°C for 30 to 120 minutes, then cooled, and the activity against human salivary amylase was measured. As a result, it was completely stable when heated for 30 minutes, and showed 85% activity even when heated for 120 minutes (see Figure 2). (4) Inhibitory effect on other amylases In addition to human salivary amylase, we tested the inhibitory properties against amylase produced by microorganisms such as bacteria and fungi. showed less than 50% inhibition, but 90% inhibition against animal amylases such as porcine pancreatic amylase and human salivary amylase.
The above-mentioned remarkable inhibitory effects were demonstrated.

[Table] (5) Chemical properties of the amylase inhibitor The chemical properties of the amylase inhibitor according to the present invention are as shown in Table 9 below. It was also similar to maltopentose in that its absorption spectrum was similar to the absorption wavelength characteristics of oligosaccharides (see Figure 3). It was also found that the measured reducing sugar values increased significantly after hydrolysis with hydrochloric acid. From the above results, it can be seen that the amylase inhibitor according to the present invention is composed of 3 to 6 sugar compounds and is not a pure sugar but a heterooligoaccharide. Furthermore, the amylase inhibitor according to the present invention is thought to be in the form of a sugar and an amino acid bonded together.
Moreover, it is soluble only in water and insoluble in methanol, ethanol, etc.

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【table】 [Brief explanation of the drawing]

FIG. 1 is a graph showing the stability of an example of an amylase inhibitor produced by the method of the present invention, FIG. 2 is a graph showing the thermostability of an example of an amylase inhibitor produced by the method of the present invention, and FIG. FIG. 2 is a diagram showing an infrared spectrum of an example of an amylase inhibitor produced by the method of the invention.

Claims (1)

[Scope of Claims] 1. Produces white aerial mycelium on a yeast malt agar medium, produces corrugated aerial mycelia carrying spores on an oatmilk agar medium, and assimilates carbon sources for D-glucose. Streptomyces sp. Y-125 (KCTC8387P,
ATCC53890). 2 Streptomyces sp.Y−125
(KCTC8387P, ATCC53890) strains were cultured,
A method for producing an amylase inhibitor, which comprises separating the amylase inhibitor from the culture.