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WO2017222142A1 - Bacillus licheniformis strain ny1505 for mass producing α-glucosidase inhibitor - Google Patents

Bacillus licheniformis strain ny1505 for mass producing α-glucosidase inhibitor Download PDF

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WO2017222142A1
WO2017222142A1 PCT/KR2017/001754 KR2017001754W WO2017222142A1 WO 2017222142 A1 WO2017222142 A1 WO 2017222142A1 KR 2017001754 W KR2017001754 W KR 2017001754W WO 2017222142 A1 WO2017222142 A1 WO 2017222142A1
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strain
bacillus licheniformis
glucosidase inhibitor
glucosidase
intestine
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PCT/KR2017/001754
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French (fr)
Korean (ko)
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이해익
김희웅
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강원대학교 산학협력단
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Priority to JP2018565720A priority Critical patent/JP6785324B2/en
Publication of WO2017222142A1 publication Critical patent/WO2017222142A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/10—Bacillus licheniformis

Definitions

  • the present invention relates to a Bacillus licheniformis NY1505 strain, Bacillus licheniformis NY1505, a new strain that produces a large amount of an ⁇ -glucosidase inhibitor.
  • ⁇ -glucosidase inhibitors are distributed in some plants and microorganisms, and are substances that inhibit the degradation of oligosaccharides into monosaccharides. Inhibition of ⁇ -glucosidase activity inhibits the smooth absorption of sugars in the intestine, which is expected to have a secondary effect such as obesity treatment and suppression of post-prandial blood sugar. Therefore, it is necessary to economically mass-produce ⁇ -glucosidase inhibitors. Is gradually increasing.
  • ⁇ -glucosidase inhibitors of plant origin include aza sugars such as 1-deoxynojirimycin in mulberry leaves, genistein and dyed zein in soybeans. isoflavones such as daidzein, and some flavone glycosides are known, and the ⁇ -glucosidase inhibitors of microbial origin are pseudooligosaccharides derived from Actinoplanes strains. , 1-deoxynojirimycin produced by microorganisms such as Streptomyces or Bacillus , and tris base are known. ⁇ -glucosidase inhibitors such as acarbose, boglibose and the like are already commercially available as diabetes treatments.
  • ⁇ -glucosidase inhibitors of natural origin used as foods include mulberry leaves for plants, and some natto for microorganisms.
  • these primary processed products or extracts are produced in large quantities, there are limitations in product diversification since they have factors that reduce palatability such as smell and color.
  • secondary metabolites produced by plants or microorganisms there is a disadvantage in that a certain amount must be taken several times a day to achieve a desired purpose.
  • intestinal bacteria such as lactic acid bacteria have the property of inhabiting and proliferating in the intestine. Therefore, enteric bacteria that produce secondary metabolites such as ⁇ -glucosidase inhibitors are expected to proliferate in the intestine and constantly produce and supply ⁇ -glucosidase inhibitors to the intestinal environment.
  • enteric bacteria that produce secondary metabolites such as ⁇ -glucosidase inhibitors are expected to proliferate in the intestine and constantly produce and supply ⁇ -glucosidase inhibitors to the intestinal environment.
  • some natto bacteria such as Bacillus subtilis , which are known in the art, are known to produce ⁇ -glucosidase inhibitors, but because Bacillus subtilis is an aerobic bacterium, it is difficult to inhabit the intestine.
  • Patent Document 1 Korean Patent Publication No. 10-2014-0123847 (2014.10.23.)
  • Patent Document 2 Korean Patent Publication No. 10-2007-0028997 (2007.03.13.)
  • Non-Patent Document 1 Overseas Papers (Zhu YP, Li XT, Teng C, Sun BG, "Enhanced production of ⁇ -glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2 using response surface methodology" FOOD AND BIOPRODUCTS PROCESSING Vol. 91 No. 3 264p ⁇ 270p)
  • Non-Patent Document 2 Domestic papers (Kim Hyun-soo, Lee Jae-yeon, Hwang Kyo-yeol, Yong-seok Cho, Young-sik Park, Kyung-don Kang, Soo-il Sung, "Isolation and Identification of Bacillus Strain Producing Alpha-glucosidase Inhibitor 1-deoxynojirimycin” Korean J. Microbiol.Biotechnol. Vol. 39, No. 1, 49-55 (2011))
  • the inventors of the present invention intensively tried to select enteric microorganisms with strong ⁇ -glucosidase inhibitory activity.
  • GRAS bacteria which can be anaerobic, form spores and proliferate in the intestine
  • Bacillus licheniformis NY1505 strain that produces a large amount of ⁇ -glucosidase inhibitor
  • the microbial fermentation product or the spores containing the spores reduced the weight loss and blood sugar drop. It was confirmed that it can be usefully used for food, medicine, feed, etc. for the purpose.
  • an object of the present invention is to provide a description of the Bacillus licheniformis NY1505 strain, a new strain that produces a large amount of an ⁇ -glucosidase inhibitor.
  • the present invention provides a Bacillus licheniformis NY1505 strain ( Bacillus licheniformis NY1505, Accession No. KCTC13021BP) that produces a high ⁇ -glucosidase inhibitor.
  • the strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.
  • the strain may be anaerobic or aerobic.
  • the strain may form spores and may be excreted within 2 weeks from the organism after the strain is administered.
  • the strain may produce an ⁇ -glucosidase inhibitor in the intestine of the animal, and the ⁇ -glucosidase inhibitor produced in the intestine may be excreted within 2 weeks from the organism after the strain is administered.
  • the present invention also provides a food composition comprising the strain described above.
  • the food composition may include the strain in the form of spores, it may be included in 10 6 to 10 10 spores per kg.
  • the food composition may further comprise a fermentation product of the strain, the fermentation may be soybean fermentation.
  • Bacillus licheniformis NY1505 strain according to the present invention a new strain that can be inhabited even under anaerobic conditions, produces and supplies an ⁇ -glucosidase inhibitor in the intestine, thereby maintaining an effective concentration and exhibiting excellent weight loss and hypoglycemic effect.
  • the ⁇ -glucosidase inhibitor produced by the administered strain or strains can be safely used as it exhibits the characteristics of temporarily inhabiting without sticking to the intestine.
  • ⁇ - glucosidase inhibitor a separate fermentation cost and processing cost for the production of the ⁇ - glucosidase inhibitor is not required, so that the production cost can be reduced, and problems such as fermentation odor do not occur, and thus it is applicable to various types of food.
  • Figure 1 is a graph showing the effect of the administration of Bacillus licheniformis NY1505 strain spores on the body weight of the mouse when fed a high carbohydrate diet (control group, high carbohydrate diet group and high carbohydrate diet + spore group).
  • Figure 2 is a graph showing the effect of the administration of Bacillus licheniformis NY1505 spores on the body weight of mice when fed a high-fat diet (control, high fat diet and high fat diet + spore group).
  • Figure 3 is a graph showing the change in the number of strains contained in the feces of the mouse (high carb diet + spore group and high fat diet + spore group).
  • FIG. 4 is a graph showing the inhibitory activity of ⁇ -glucosidase contained in feces of mice (high carbohydrate diet + spore group and high fat diet + spore group).
  • FIG. 6 is a schematic diagram showing the taxonomic location of Bacillus rickenformis NY1505 strain.
  • the present invention provides a Bacillus licheniformis NY1505 strain (Accession No. KCTC13021BP) which produces a high ⁇ -glucosidase inhibitor.
  • the strain can be obtained by separating the bacteria that form heat-resistant spores by using straw and hay as a sample, and selecting strains capable of growing in vivo and producing high ⁇ -glucosidase inhibitors.
  • the strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.
  • the strain may have both anaerobic properties capable of producing ⁇ -glucosidase inhibitors in a high yield in the intestine and aerobic properties capable of producing ⁇ -glucosidase inhibitors in vitro.
  • the strain can form spores, and exhibits the characteristics of temporarily inhabiting without sticking in the intestine, and can be safely used because it is possible to remove the administered strain as needed.
  • the strain may be excreted in vitro after two weeks, and more preferably one week after the administration to the organism.
  • the strain can produce the ⁇ -glucosidase inhibitor in the intestine, can maintain a constant effective concentration of the ⁇ -glucosidase inhibitor, the ⁇ -glucosidase inhibitor produced in the intestine is excreted You can use it safely.
  • the ⁇ -glucosidase inhibitor may be excreted in vitro after two weeks after the strain is administered to the organism.
  • the food containing the strain can be produced in a high yield of the intestinal ⁇ -glucosidase inhibitor, can be discharged to the outside of the organism can be effectively used for the purpose of weight loss and blood sugar lowering. .
  • the present invention provides a food composition comprising the strain described above.
  • the food composition may include a strain, spores of the strain or fermentation of the strain, which are cultured by a culturing method of Bacillus rickenformis commonly used.
  • the food composition may comprise the spores of the strain, the food composition comprises the strain with 10 6 to 10 10 spores per kg the strain contains a large amount of the ⁇ -glucosidase inhibitor in the intestine Can produce.
  • the food composition may further include a fermentation product of the strain, and the fermentation product may be a soybean fermentation product inoculated with the strain soybean.
  • the culture was centrifuged to measure ⁇ -glucosidase inhibitor activity from the supernatant (aerobic culture in Table 1).
  • ⁇ -glucosidase inhibitor activity from the supernatant (aerobic culture in Table 1).
  • approximately 60 anaerobic bacteria are first selected as ⁇ -glucosidase inhibitor-producing bacteria, and then, strains capable of anaerobic growth at 50 ° C. and magnetizing propionic acid are selected.
  • Three species with high -glucosidase inhibitory activity were selected secondarily, and the culture solution was centrifuged to measure ⁇ -glucosidase inhibitor activity from the supernatant (anaerobic culture of Table 1).
  • Beans were soaked at room temperature for 12 hours and then boiled at 121 ° C. for 40 minutes. After inoculating 10 3 spores per gram before cooling the boiled beans, 50g each was put in a foamed styrene container for natto production, covered with a vinyl film, and incubated at 37 ° C. for 20 hours. After incubating the cultured natto for 12 hours, ⁇ -glucosidase inhibitory activity was measured and sensory evaluation was performed (natto in Table 1).
  • ⁇ -glucosidase was extracted from the small intestine of pigs and partially purified to use as an enzyme source.
  • ⁇ -glucosidase enzyme solution was appropriately diluted with 0.5 M phosphate buffer (pH 7.0) to 24 units / ml, and the substrate was p-nitrophenyl ⁇ -D-glucopyranoside (p-nitrophenyl).
  • ⁇ -D-glucopyranoside (pNPG) was used. 30 ⁇ l of the culture supernatant and 50 ⁇ l of the enzyme were mixed in a test tube and preliminarily insulated at 37 ° C. for 10 minutes, and then 50 ⁇ l of 3mM pNPG was added and reacted at 37 ° C. for 20 minutes.
  • ⁇ -glucosidase inhibition unit ( ⁇ -glucosidase inhibition unit, AGI unit) was defined as the amount of inhibitor when 100% inhibition of ⁇ -glucosidase used for activity measurement in the following equation (1).
  • Bacillus licheniformis To determine whether the NY1505 strain produces useful substances in the animal intestine, Bacillus licheniformis NY1505 strains are administered by mixing and administering Bacillus licheniformis NY1505 spores to the feed of mice, and ⁇ -glucosidase in the intestine. The production of inhibitors was examined.
  • mice Bacillus licheniformis to produce ⁇ -Glucosidase inhibitors
  • the diet was mixed with the spores of the strain NY1505 (10 8 spores / kg feed) was fed to the mice and observed changes in body weight.
  • commercial standard feed and water were freely fed and male mice (00 weeks old) were used after preliminary breeding for one week.
  • the light and dark cycles were designed at 08:00 am to 20:00 pm and at 20:00 pm to 08:00 am, and were bred under the environment of 22.5 ⁇ 0.5 ° C. and humidity of 50 to 60%. .
  • Bacillus licheniformis a feed containing the spores of NY1505 strain was prepared by mixing 10 8 spores per 1kg each guided obesity. Mice were divided into 5 groups (8 animals each), and were divided into a control group, a high carbohydrate diet group, a high carbohydrate diet + spore group, a high fat diet group, and a high fat diet + spore group, respectively, for 7 weeks.
  • Weight of the high carbohydrate diet benefit mice (body weight, unit of gram) to a result of analyzing the effect of the administration of Bacillus licheniformis NY1505 bacteria spores on, as shown in Figure 1, high-carbohydrate food group is a marked weight gain compared to the control group However, the high carbohydrate feed group containing spores of Bacillus licheniformis NY1505 strain showed similar weight gain pattern as the control group.
  • Bacillus licheniformis As a result of analyzing the change of the strain excreted into the stool by measuring the number of spores contained in the stool of the mice administered spores NY1505, as shown in Figure 3, 10 5 spores per 1g of feed is mixed and per You eat about 5g of feed per day, so you eat about 5 x 10 5 spores per day. Three weeks after spore administration, about 10 7 Bacillus licheniformis per gram of feces Since NY1505 was detected, it was found that this strain was vigorously proliferated and excreted in the intestine. When spores were administered for 7 weeks, the number of germs was significantly reduced at 8 weeks after only 1 week without spores. Therefore, when the administration of the strain is stopped, it is expected that the change in the intestinal flora can be confirmed that there is an advantage that the removal of the strain in the intestine as needed.
  • Bacillus licheniformis As a result of analyzing the inhibitory activity of ⁇ -glucosidase contained in the stool of the mouse administered with the NY1505 spores, as shown in FIG. 4, the Bacillus licheniformis NY1505 strain proliferated vigorously in the intestine so as to be excreted into the stool. It was confirmed that the glucosidase inhibitor was produced. Bacillus licheniformis proliferated in the intestine at the stool two weeks after the spore-administered mice The NY1505 strain was excreted in feces and activity was detected.
  • the strain Bacillus licheniformis NY1505 according to the present invention does not use the bioactive substance produced by fermentation, but the strain continuously produces and supplies the bioactive substance in the body, thereby producing the ⁇ -glucosidase inhibitor. It is possible to reduce the production cost because it does not require separate fermentation cost and processing cost, and it can be confirmed that there is an advantage of maintaining a constant effective concentration by continuously producing in the body. There is no problem and can be used as an additive to a variety of processed foods, it was confirmed that it can be applied to various types of food.
  • Bacillus licheniformis isolated as above The NY1505 strain showed a light reddish brown color at the center of the colonies and irregular edges in LB agar plate medium (FIG. 5).
  • the temperature range of the growth of bacteria was good in the range of 25 to 50 °C, cell growth was not confirmed above 60 °C.
  • Microscopic observation of the cell growth using LB liquid medium showed that gram-positive short rods were similar to Bacillus rickenformis, a comparative strain.
  • the isolated strain was Gram-positive, as shown in Table 4 below, the cell size was found to be approximately 0.5 ⁇ 0.7 ⁇ 1.5 ⁇ 1.8 ⁇ m, synthesized the above results, Bacillus rickenformis The taxonomic location of the NY1505 strain is shown in FIG. 6.
  • Bacillus licheniformis NY1505 strain according to the present invention can be used in food compositions because it produces and supplies an ⁇ -glucosidase inhibitor in the intestine.

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Abstract

The present invention provides Bacillus licheniformis strain NY1505 (accession number KCTC13021BP) for a high yield of an α-glucosidase inhibitor. Bacillus licheniformis strain NY1505, according to the present invention, is a novel strain which can survive in an anaerobic condition, and enables production and supply of an α-glucosidase inhibitor in the intestine and thus maintains a fixed level of effective concentration and shows excellent weight loss and blood glucose lowering effects. And since the Bacillus licheniformis strain NY1505 survives temporarily and does not stay in the intestine, the strain that has been administered when necessary or the α-glucosidase inhibitor produced by means of the strain can be discharged and thus can be safely used. Moreover, separate fermentation cost and processing cost are not required for the production of the α-glucosidase inhibitor and thus the production cost can be reduced. And the present invention does not have the problems of fermentation odor and the like and thus can be applied to various forms of food products.

Description

알파글루코시다아제 저해제를 다량 생산하는 바실러스 리케니포르미스 NY1505 균주Bacillus rickeniformis NY1505 strain producing large amounts of alphaglucosidase inhibitors
본 발명은 α-글루코시다아제 저해제를 다량 생산하는 신균주인 바실러스 리케니포르미스 NY1505 균주(Bacillus licheniformis NY1505)에 관한 것이다.The present invention relates to a Bacillus licheniformis NY1505 strain, Bacillus licheniformis NY1505, a new strain that produces a large amount of an α-glucosidase inhibitor.
α-글루코시다아제 저해제(α-glucosidase inhibitors)는 일부의 식물이나 미생물에 분포하며, 올리고당을 단당류로의 분해를 억제하는 작용을 하는 물질이다. α-글루코시다아제 활성의 저해는 장내에서 당류의 원활한 흡수를 저해함으로써 식후 혈당상승 억제는 물론이고 비만치료와 같은 부수적인 효과가 기대가 되므로 α-글루코시다아제 저해제를 경제적으로 대량 생산하기 위한 필요성은 점차 증대되고 있는 실정이다. α-glucosidase inhibitors are distributed in some plants and microorganisms, and are substances that inhibit the degradation of oligosaccharides into monosaccharides. Inhibition of α-glucosidase activity inhibits the smooth absorption of sugars in the intestine, which is expected to have a secondary effect such as obesity treatment and suppression of post-prandial blood sugar. Therefore, it is necessary to economically mass-produce α-glucosidase inhibitors. Is gradually increasing.
식물 기원의 α-글루코시다아제 저해제로는 뽕잎에 함유된 1-데옥시노지리마이신(1-deoxynojirimycin)과 같은 아자당(aza sugar)류, 콩에 함유된 제니스테인(genistein), 다이드제인(daidzein)과 같은 이소플라본(isoflavone), 일부의 플라본 배당체(flavone glycoside) 등이 알려져 있으며, 미생물 기원의 α-글루코시다아제 저해제로는 악티노플라네스속(Actinoplanes) 균주 기원의 유사올리고당(pseudooligosaccharide), 스트렙토미세스속(Streptomyces) 또는 바실러스속(Bacillus) 등의 미생물이 생산하는 1-데옥시노지리마이신(1-deoxynojirimycin), 트리스 염(tris base) 등이 알려져 있으며, 의약품으로는 아카보오스(acarbose), 보글리보오스(voglibose) 등과 같은 α-글루코시다아제 저해제들이 이미 당뇨병 치료제로서 시판되고 있다. Α-glucosidase inhibitors of plant origin include aza sugars such as 1-deoxynojirimycin in mulberry leaves, genistein and dyed zein in soybeans. isoflavones such as daidzein, and some flavone glycosides are known, and the α-glucosidase inhibitors of microbial origin are pseudooligosaccharides derived from Actinoplanes strains. , 1-deoxynojirimycin produced by microorganisms such as Streptomyces or Bacillus , and tris base are known. α-glucosidase inhibitors such as acarbose, boglibose and the like are already commercially available as diabetes treatments.
최근, 혈당강하에 효과가 있는 α-글루코시다아제 저해제를 식품으로 섭취하기 위해 식용 가능한 천연물에 대한 연구가 활발하게 진행되고 있다. 식품으로 사용되는 천연물 기원의 α-글루코시다아제 저해제는 식물의 경우 뽕잎, 미생물의 경우 일부의 낫토를 대표적인 예로 들 수 있다. 이들의 1차 가공품 또는 추출물은 대량으로 생산이 되고 있으나 공통적으로 냄새, 색택과 같은 기호성을 떨어뜨리는 인자를 가지고 있으므로 제품의 다양화에 제한이 있다. 또한, 이들은 식물 또는 미생물이 생산한 이차대사산물을 이용하는 경우이므로 하루에 수회 일정량을 복용하여야 소기의 목적을 달성할 수 있는 단점이 있다.Recently, researches on edible natural products for the ingestion of α-glucosidase inhibitors, which are effective in lowering blood sugar, have been actively conducted. Α-glucosidase inhibitors of natural origin used as foods include mulberry leaves for plants, and some natto for microorganisms. Although these primary processed products or extracts are produced in large quantities, there are limitations in product diversification since they have factors that reduce palatability such as smell and color. In addition, since they use secondary metabolites produced by plants or microorganisms, there is a disadvantage in that a certain amount must be taken several times a day to achieve a desired purpose.
한편, 젖산균 등과 같은 장내 세균은 장내에서 서식하며 증식하는 특성을 가지고 있다. 따라서, α-글루코시다아제 저해제와 같은 이차대사산물을 생산하는 장내 세균은 장내에서 증식하며 끊임없이 장내 환경에 α-글루코시다아제 저해제를 생산하여 공급할 것으로 예상된다. 하지만, 종래에 알려진 바실러스 서브틸리스(Bacillus subtilis) 등과 같은 일부의 낫토균은 α-글루코시다아제 저해제를 생산하는 것으로 알려져 있으나, 바실러스 서브틸리스는 호기성 균으로 장내에서 서식하기 힘들기 때문에, 바실러스 서브틸리스의 증식으로 α-글루코시다아제 저해제를 장내에서 공급받기는 어렵고, 낫토의 섭취를 통해 α-글루코시다아제 저해제를 공급받기 위해서는 다량 섭취해야한다는 문제점이 있다. On the other hand, intestinal bacteria such as lactic acid bacteria have the property of inhabiting and proliferating in the intestine. Therefore, enteric bacteria that produce secondary metabolites such as α-glucosidase inhibitors are expected to proliferate in the intestine and constantly produce and supply α-glucosidase inhibitors to the intestinal environment. However, some natto bacteria, such as Bacillus subtilis , which are known in the art, are known to produce α-glucosidase inhibitors, but because Bacillus subtilis is an aerobic bacterium, it is difficult to inhabit the intestine. Due to the proliferation of tilis, it is difficult to supply an α-glucosidase inhibitor in the intestine, and a large amount of α-glucosidase inhibitor is required to receive an α-glucosidase inhibitor through ingestion of natto.
따라서, 장내에서 서식이 가능하고, α-글루코시다아제 저해제를 다량 생성할 수 있어 체중 감량과 혈당강하 등을 위한 목적으로 효과적으로 활용가능한 새로운 균주에 대한 연구 및 개발이 필요하다.Therefore, there is a need for research and development of a new strain that can be intestinally available and can produce a large amount of α-glucosidase inhibitors, which can be effectively utilized for the purpose of weight loss and hypoglycemia.
[선행기술문헌][Preceding technical literature]
[특허문헌][Patent Documents]
(특허문헌 1) 한국공개특허 제10-2014-0123847호 (2014.10.23.)(Patent Document 1) Korean Patent Publication No. 10-2014-0123847 (2014.10.23.)
(특허문헌 2) 한국공개특허 제10-2007-0028997호 (2007.03.13.)(Patent Document 2) Korean Patent Publication No. 10-2007-0028997 (2007.03.13.)
[비특허문헌][Non-Patent Documents]
(비특허문헌 1) 해외논문(Zhu Y.P, Li X.T, Teng C, Sun B.G, "Enhanced production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2 using response surface methodology" FOOD AND BIOPRODUCTS PROCESSING Vol. 91 No. 3 264p ~ 270p)(Non-Patent Document 1) Overseas Papers (Zhu YP, Li XT, Teng C, Sun BG, "Enhanced production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2 using response surface methodology" FOOD AND BIOPRODUCTS PROCESSING Vol. 91 No. 3 264p ~ 270p)
(비특허문헌 2) 국내논문(김현수, 이재연, 황교열, 조용석, 박영식, 강경돈, 성수일, "알파글루코시다아제 저해제 1-deoxynojirimycin을 생산하는 Bacillus 균주의 분리 및 동정" Korean J. Microbiol. Biotechnol. Vol. 39, No. 1, 49-55 (2011))(Non-Patent Document 2) Domestic papers (Kim Hyun-soo, Lee Jae-yeon, Hwang Kyo-yeol, Yong-seok Cho, Young-sik Park, Kyung-don Kang, Soo-il Sung, "Isolation and Identification of Bacillus Strain Producing Alpha-glucosidase Inhibitor 1-deoxynojirimycin" Korean J. Microbiol.Biotechnol. Vol. 39, No. 1, 49-55 (2011))
본 발명의 발명자들은 α-글루코시다아제 저해 활성이 강한 장내 미생물을 선발하고자 예의 연구 노력한 결과, α-글루코시다아제 저해 활성을 나타내는 미생물들 중에서 통성 혐기성이고 포자를 형성하며 장내에서 증식이 가능한 GRAS 균으로서 α-글루코시다아제 저해제를 다량으로 생산하는 신규한 바실러스 리케니포르미스 NY1505(Bacillus licheniformis NY1505) 균주를 분리·동정하였으며, 상기 미생물의 발효물 또는 포자를 포함한 영양세포는 체중 감량과 혈당강하를 목적으로 식품, 의약품, 사료 등에 유용하게 사용될 수 있음을 확인하였다.The inventors of the present invention intensively tried to select enteric microorganisms with strong α-glucosidase inhibitory activity. Among the microorganisms showing α-glucosidase inhibitory activity, GRAS bacteria which can be anaerobic, form spores and proliferate in the intestine As a novel bacterium Bacillus licheniformis NY1505 strain that produces a large amount of α-glucosidase inhibitor, the microbial fermentation product or the spores containing the spores reduced the weight loss and blood sugar drop. It was confirmed that it can be usefully used for food, medicine, feed, etc. for the purpose.
이에, 본 발명에서는 α-글루코시다아제 저해제를 다량 생산하는 신균주인 Bacillus licheniformis NY1505 균주에 관한 기술 내용을 제공하는 것을 그 목적으로 한다.Accordingly, an object of the present invention is to provide a description of the Bacillus licheniformis NY1505 strain, a new strain that produces a large amount of an α-glucosidase inhibitor.
상기한 바와 같은 기술적 과제를 달성하기 위해서 본 발명은, α-글루코시다아제 저해제(α-glucosidase inhibitor)를 고생산하는 바실러스 리케니포르미스 NY1505 균주(Bacillus licheniformis NY1505, 수탁번호KCTC13021BP)를 제공한다.In order to achieve the above technical problem, the present invention provides a Bacillus licheniformis NY1505 strain ( Bacillus licheniformis NY1505, Accession No. KCTC13021BP) that produces a high α-glucosidase inhibitor.
또한, 상기 균주는 서열번호 1의 16s RNA를 포함하는 것을 특징으로 한다.In addition, the strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.
또한, 상기 균주는 혐기성이거나, 또는, 호기성일 수 있다.In addition, the strain may be anaerobic or aerobic.
또한, 상기 균주는 포자를 형성할 수 있으며, 상기 균주가 투여된 후, 생물체로부터 2주 이내에 배설될 수 있다.In addition, the strain may form spores and may be excreted within 2 weeks from the organism after the strain is administered.
또한, 상기 균주는 동물의 장내에서 α-글루코시다아제 저해제를 생산할 수 있으며, 장내에서 생산된 α-글루코시다아제 저해제는 상기 균주가 투여된 후, 생물체로부터 2주 이내에 배설될 수 있다.In addition, the strain may produce an α-glucosidase inhibitor in the intestine of the animal, and the α-glucosidase inhibitor produced in the intestine may be excreted within 2 weeks from the organism after the strain is administered.
또한, 본 발명은, 상기에 기재된 균주를 포함하는 식품 조성물을 제공한다.The present invention also provides a food composition comprising the strain described above.
또한, 상기 식품 조성물에는 상기 균주가 포자의 형태로 포함될 수 있고, 1 kg 당 106 내지 1010개의 포자로 포함될 수 있다.In addition, the food composition may include the strain in the form of spores, it may be included in 10 6 to 10 10 spores per kg.
또한, 상기 식품 조성물은 상기 균주의 발효물을 추가로 포함할 수 있으며, 상기 발효물은 대두 발효물일 수 있다.In addition, the food composition may further comprise a fermentation product of the strain, the fermentation may be soybean fermentation.
본 발명에 따른 Bacillus licheniformis NY1505 균주는, 혐기성 조건에서도 서식이 가능한 신균주로서, 장내에서 α-글루코시다아제 저해제를 생산하여 공급하므로 유효 농도를 일정하게 유지하여 우수한 체중감량 및 혈당강하 효과를 나타낼 수 있고, 장내에 고착하지 않고 일시적으로 서식하는 특성을 나타내 필요에 따라 투여된 균주 또는 균주가 생산한 α-글루코시다아제 저해제는 배출이 가능하여 안전하게 사용할 수 있다. Bacillus licheniformis NY1505 strain according to the present invention, a new strain that can be inhabited even under anaerobic conditions, produces and supplies an α-glucosidase inhibitor in the intestine, thereby maintaining an effective concentration and exhibiting excellent weight loss and hypoglycemic effect. In addition, the α-glucosidase inhibitor produced by the administered strain or strains can be safely used as it exhibits the characteristics of temporarily inhabiting without sticking to the intestine.
또한, α-글루코시다아제 저해제의 생성을 위한 별도의 발효비용 및 가공비가 필요하지 않아 생산비용을 절감할 수 있고, 발효취 등의 문제가 발생하지 않아 다양한 형태의 식품에 적용이 가능하다.In addition, a separate fermentation cost and processing cost for the production of the α- glucosidase inhibitor is not required, so that the production cost can be reduced, and problems such as fermentation odor do not occur, and thus it is applicable to various types of food.
도 1은 고탄수화물 식이의 급여시 Bacillus licheniformis NY1505균주 포자의 투여가 마우스의 체중에 미치는 영향을 나타낸 그래프이다(대조군, 고탄수화물식이군 및 고탄수화물식이 + 포자군).Figure 1 is a graph showing the effect of the administration of Bacillus licheniformis NY1505 strain spores on the body weight of the mouse when fed a high carbohydrate diet (control group, high carbohydrate diet group and high carbohydrate diet + spore group).
도 2는 고지방 식이의 급여시 Bacillus licheniformis NY1505균 포자의 투여가 마우스의 체중에 미치는 영향을 나타낸 그래프이다(대조군, 고지방식이군 및 고지방식이 + 포자군).Figure 2 is a graph showing the effect of the administration of Bacillus licheniformis NY1505 spores on the body weight of mice when fed a high-fat diet (control, high fat diet and high fat diet + spore group).
도 3은 마우스의 분변에 포함된 균주 수의 변화를 나타내는 그래프이다(고탄수화물식이 + 포자군 및 고지방식이 + 포자군).Figure 3 is a graph showing the change in the number of strains contained in the feces of the mouse (high carb diet + spore group and high fat diet + spore group).
도 4는 마우스의 분변에 포함된 α-글루코시다아제의 저해 활성을 나타내는 그래프이다(고탄수화물식이 + 포자군 및 고지방식이 + 포자군).4 is a graph showing the inhibitory activity of α-glucosidase contained in feces of mice (high carbohydrate diet + spore group and high fat diet + spore group).
도 5는 바실러스 리케니포르미스 NY1505 균주의 전자현미경 사진이다.5 is an electron micrograph of the Bacillus rickeniformis NY1505 strain.
도 6은 바실러스 리케니포르미스 NY1505 균주의 분류학상 위치를 나타낸 개략도이다.6 is a schematic diagram showing the taxonomic location of Bacillus rickenformis NY1505 strain.
이하, 본 발명을 상세히 설명하도록 한다.Hereinafter, the present invention will be described in detail.
본 발명은, α-글루코시다아제 저해제(α-glucosidase inhibitor)를 고생산하는 바실러스 리케니포르미스 NY1505 균주(Bacillus licheniformis NY1505, 수탁번호KCTC13021BP)를 제공한다.The present invention provides a Bacillus licheniformis NY1505 strain (Accession No. KCTC13021BP) which produces a high α-glucosidase inhibitor.
상기 균주는 볏짚 및 건초를 시료로 하여 내열성 포자를 형성하는 균을 분리고, 생체내에서 생육이 가능하며 α-글루코시다아제 저해제를 고생산하는 균주를 선별함으로써 얻을 수 있다. The strain can be obtained by separating the bacteria that form heat-resistant spores by using straw and hay as a sample, and selecting strains capable of growing in vivo and producing high α-glucosidase inhibitors.
상기 균주는 하기 서열번호 1의 16s RNA를 포함하는 것을 특징으로 한다.The strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.
서열번호 1:SEQ ID NO 1:
AGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAAAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAA
상기 균주는 α-글루코시다아제 저해제를 장내에서 고수율로 생산가능하는 혐기성 특성과 생체외에서도 α-글루코시다아제 저해제를 생산가능한 호기적 특성을 동시에 가질 수 있다.The strain may have both anaerobic properties capable of producing α-glucosidase inhibitors in a high yield in the intestine and aerobic properties capable of producing α-glucosidase inhibitors in vitro.
상기 균주는 포자를 형성할 수 있으며, 장내에 고착하지 않고 일시적으로 서식하는 특성을 나타내, 필요에 따라 투여된 균주의 제거가 가능해 안전하게 사용할 수 있다. 바람직하게는 상기 균주는 생물체에 투여된 후, 2주 경과하는 시점에 생물체외로 배설될 수 있으며, 보다 바람직하게는 1주 경과하는 시점에 배설될 수 있다.The strain can form spores, and exhibits the characteristics of temporarily inhabiting without sticking in the intestine, and can be safely used because it is possible to remove the administered strain as needed. Preferably, the strain may be excreted in vitro after two weeks, and more preferably one week after the administration to the organism.
또한, 상기한 균주는 장내에서 상기 α-글루코시다아제 저해제를 생산할 수 있어, α-글루코시다아제 저해제의 유효 농도를 일정하게 유지할 수 있으며, 장내에서 생산한 상기 α-글루코시다아제 저해제는 배설이 가능해 안전하게 사용할 수 있다. 바람직하게는 상기 α-글루코시다아제 저해제는 균주가 생물체에 투여된 후, 2주 경과하는 시점에 생물체외로 배설될 수 있다.In addition, the strain can produce the α-glucosidase inhibitor in the intestine, can maintain a constant effective concentration of the α-glucosidase inhibitor, the α-glucosidase inhibitor produced in the intestine is excreted You can use it safely. Preferably, the α-glucosidase inhibitor may be excreted in vitro after two weeks after the strain is administered to the organism.
이에 따라, 상기 균주를 포함하는 식품을 섭취하는 방법에 의해 투여하여 장내에서 α-글루코시다아제 저해제를 고수율로 생산할 수 있어, 생물체외로 배출이 가능해 체중 감량과 혈당강하의 목적으로 효과적으로 사용할 수 있다.Accordingly, by ingesting the food containing the strain can be produced in a high yield of the intestinal α-glucosidase inhibitor, can be discharged to the outside of the organism can be effectively used for the purpose of weight loss and blood sugar lowering. .
본 발명은 상기에 기재된 균주를 포함하는 식품 조성물을 제공한다.The present invention provides a food composition comprising the strain described above.
상기 식품 조성물은 통상적으로 사용되는 바실러스 리케니포르미스의 배양방법에 의해 배양된 균주, 균주의 포자 또는 균주의 발효물을 포함할 수 있다.The food composition may include a strain, spores of the strain or fermentation of the strain, which are cultured by a culturing method of Bacillus rickenformis commonly used.
일례로, 상기 식품 조성물은 상기 균주의 포자를 포함할 수 있고, 상기 식품 조성물은 1 kg 당 106 내지 1010개의 포자로 상기 균주를 포함하여 상기 균주가 장내에서 α-글루코시다아제 저해제를 다량 생산할 수 있다.In one example, the food composition may comprise the spores of the strain, the food composition comprises the strain with 10 6 to 10 10 spores per kg the strain contains a large amount of the α-glucosidase inhibitor in the intestine Can produce.
또 다른 예로, 상기 식품 조성물은 상기 균주의 발효물을 추가로 포함할 수 있으며, 상기 발효물은 상기 균주를 대두에 접종하여 발효한 대두 발효물일 수 있다.As another example, the food composition may further include a fermentation product of the strain, and the fermentation product may be a soybean fermentation product inoculated with the strain soybean.
이하, 본 발명을 실시예를 들어 더욱 상세히 설명하도록 한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
제시된 실시예는 본 발명의 구체적인 예시일 뿐이며, 본 발명의 범위를 제한하기 위한 것은 아니다.The examples presented are merely illustrative of the invention and are not intended to limit the scope of the invention.
<< 실시예Example >>
1. 균주의 탐색1. Search for Strains
한국, 중국, 일본, 미국 등 세계 각지에서 수집한 약 500여점의 볏짚 및 건초를 시료로 하여 미생물을 분리하였다. 각 시료를 멸균 식염수에 소량 가하여 현탁시킨 다음 80℃ 항온 수조에서 20분간 열처리하여 얻은 포자액을 2% 한천을 함유하는 LB 평판배지(1% 트립톤, 0.2% 설탕, 0.5% 효모추출물 및 0.5% NaCl, pH7.0)에 도말하고, 55℃ 배양기에서 2일간 혐기적으로 배양하였으며, 배양 후 균 집락을 형성하는 균체를 분리하였다. α-글루코시다아제 저해제의 생산량을 조사하기 위하여, 분리된 균주를 5 ml의 5% 콩가루를 현탁한 배지에 접종하고 37℃에서 24시간 동안 배양하였다. 배양액을 원심분리하여 상등액으로부터 α-글루코시다아제 저해제 활성을 측정하였다(표 1의 호기적 배양). 상기의 과정으로 약 60종의 통성혐기성 세균을 α-글루코시다아제 저해제 생산균으로 1차 선발 한 후, 50℃에서 혐기적으로 생육이 가능하고 프로피온산(propionic acid)을 자화하는 균주를 선발하여 α-글루코시다아제 저해 활성이 높은 3종을 2차 선발하였으며, 배양액을 원심분리하여 상등액으로부터 α-글루코시다아제 저해제 활성을 측정하였다(표 1의 혐기적 배양). 상기한 3종의 균주는 버지스 매뉴얼(Bergey's Manual of Systematic Bacteriology, 2002)에 의한 분류학적 특성 분석 방법에 따라서 분류하였으며, 그 중 1종이 식용 가능한 균인 바실러스 리케니포르미스(Bacillus licheniformis)로 동정되어 바실러스 리케니포르미스 NY1505(Bacillus licheniformis NY1505)로 명명하고, 상기 균주가 하기 서열번호 1의 16S RNA서열을 갖는 다는 것을 확인하였으며, 상기 서열을 한국생명공학 연구원 생물자원센터(KCTC)에 2016.05.03.일자로 기탁하였다(기탁번호: KCTC13021BP).About 500 rice straws and hay collected from around the world, such as Korea, China, Japan, and the United States, microorganisms were isolated. Each sample was added to a small amount of sterile saline and suspended, followed by heat treatment for 20 minutes in an 80 ° C constant temperature water bath. LB plate medium containing 2% agar (1% tryptone, 0.2% sugar, 0.5% yeast extract and 0.5% NaCl, pH 7.0), and anaerobic culture for 2 days in a 55 ℃ incubator, after the culture was isolated cells forming a colony colonies. To investigate the production of α-glucosidase inhibitors, the isolated strains were inoculated in medium suspended in 5 ml of 5% soy flour and incubated at 37 ° C. for 24 hours. The culture was centrifuged to measure α-glucosidase inhibitor activity from the supernatant (aerobic culture in Table 1). By the above process, approximately 60 anaerobic bacteria are first selected as α-glucosidase inhibitor-producing bacteria, and then, strains capable of anaerobic growth at 50 ° C. and magnetizing propionic acid are selected. Three species with high -glucosidase inhibitory activity were selected secondarily, and the culture solution was centrifuged to measure α-glucosidase inhibitor activity from the supernatant (anaerobic culture of Table 1). Strains of the three species are identified as Burgess Manual (Bergey's Manual of Systematic Bacteriology, 2002) Taxonomic properties were classified according to the analysis method, one member edible gyunin Bacillus Lee Kenny formate miss (Bacillus licheniformis) of which by Bacillus Rickenioformis NY1505 ( Bacillus licheniformis NY1505), it was confirmed that the strain has a 16S RNA sequence of SEQ ID NO: 1, the sequence was deposited to the Korea Biotechnology Research Institute Biological Resource Center (KCTC) dated May 03, 2016 (Accession Number: KCTC13021BP).
서열번호 1:SEQ ID NO 1:
AGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAAAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAA
2. 2. 낫토의Natto 제조 Produce
콩을 상온에서 12시간 동안 불린 후 121℃에서 40분 동안 삶았다. 삶은 콩을 냉각하기 전 g당 103마리의 포자를 접종한 후, 50g씩 낫토 제조용 발포스티렌 용기에 넣고 비닐막을 씌운뒤 37℃에서 20시간 동안 배양하였다. 배양된 낫토를 12시간 동안 냉장보관 후 α-글루코시다아제 저해 활성을 측정 및 관능평가를 수행하였다(표 1의 낫토).Beans were soaked at room temperature for 12 hours and then boiled at 121 ° C. for 40 minutes. After inoculating 10 3 spores per gram before cooling the boiled beans, 50g each was put in a foamed styrene container for natto production, covered with a vinyl film, and incubated at 37 ° C. for 20 hours. After incubating the cultured natto for 12 hours, α-glucosidase inhibitory activity was measured and sensory evaluation was performed (natto in Table 1).
3. α-글루코시다아제 저해 활성 측정3. Measurement of α-glucosidase inhibitory activity
α-글루코시다아제는 돼지의 소장으로부터 추출하여 부분 정제한 것을 효소원으로 사용하였다. α-글루코시다아제 효소액은 24unit/ml가 되게 0.5M 인산염 완충용액(potassium phosphate buffer, pH 7.0)으로 적당히 희석하여 사용하였으며, 기질은 p-니트로페닐 α-D-글루코피라노시드(p-nitrophenyl α-D-glucopyranoside, pNPG)를 이용하였다. 시험관에 배양 상등액 30μl와 효소 50μl를 섞어 37 ℃에서 10분간 예비 보온을 한 후, 3mM pNPG 50μl를 첨가하고 37 ℃에서 20분간 반응시켰다. 반응액에 0.1M Na2CO3를 가하여 반응을 정지하고 405 nm에서 흡광도를 측정하여 하기 수학식 1으로 저해 활성을 측정하였으며, α-글루코시다아제 저해 활성의 측정 결과를 하기의 표 1에 나타내었다. 이때, α-글루코시다아제 저해단위(α-glucosidase inhibition unit, AGI unit)는 하기 수학식 1에서 활성 측정에 사용된 α-glucosidase를 100% 저해할 때의 저해제의 양으로 정의하였다.α-glucosidase was extracted from the small intestine of pigs and partially purified to use as an enzyme source. α-glucosidase enzyme solution was appropriately diluted with 0.5 M phosphate buffer (pH 7.0) to 24 units / ml, and the substrate was p-nitrophenyl α-D-glucopyranoside (p-nitrophenyl). α-D-glucopyranoside (pNPG) was used. 30 μl of the culture supernatant and 50 μl of the enzyme were mixed in a test tube and preliminarily insulated at 37 ° C. for 10 minutes, and then 50 μl of 3mM pNPG was added and reacted at 37 ° C. for 20 minutes. 0.1M Na 2 CO 3 was added to the reaction solution to stop the reaction, and the absorbance was measured at 405 nm. The inhibitory activity was measured by the following Equation 1, and the results of the α-glucosidase inhibitory activity are shown in Table 1 below. It was. In this case, α-glucosidase inhibition unit (α-glucosidase inhibition unit, AGI unit) was defined as the amount of inhibitor when 100% inhibition of α-glucosidase used for activity measurement in the following equation (1).
[수학식 1][Equation 1]
Figure PCTKR2017001754-appb-I000001
Figure PCTKR2017001754-appb-I000001
Figure PCTKR2017001754-appb-T000001
Figure PCTKR2017001754-appb-T000001
표 1에 나타난 바와 같이, 산소의 공급 여부에 관계 없이 균일한 AGI저해 활성을 나타내고 있어, Bacillus licheniformis가 통성 혐기성 세균이므로 혐기적인 조건에서도 생육이 가능하다는 사실을 확인할 수 있었다. 따라서, 동물의 장관 내에서 Bacillus licheniformis는 증식을 하며 유용한 생리 활성 물질을 생산할 수 있을 것으로 확인되었다. As shown in Table 1, uniform AGI inhibitory activity was shown regardless of the supply of oxygen, and it was confirmed that Bacillus licheniformis was a permeable anaerobic bacterium and thus grown under anaerobic conditions. Therefore, Bacillus licheniformis was found to be able to proliferate and produce useful bioactive substances in the intestinal tract of animals.
4. 4. Bacillus Bacillus licheniformislicheniformis NY1505 균주의 식이 실험Dietary Experiments of the NY1505 Strain
Bacillus licheniformis NY1505 균주가 동물의 장관내에서 유용물질을 생산 하는 지의 여부를 확인하기 위해, 마우스의 사료에 Bacillus licheniformis NY1505 포자를 혼합하여 투여함으로서 Bacillus licheniformis NY1505 균주의 장내 증식 여부, 장내에서의 α-글루코시다아제 저해제의 생산 여부 등을 검토하였다. Bacillus licheniformis To determine whether the NY1505 strain produces useful substances in the animal intestine, Bacillus licheniformis NY1505 strains are administered by mixing and administering Bacillus licheniformis NY1505 spores to the feed of mice, and α-glucosidase in the intestine. The production of inhibitors was examined.
먼저, α-Glucosidase저해제를 생산하는 Bacillus licheniformis NY1505 균주의 포자를 혼합한 사료(108포자/kg사료)를 마우스에 급여하며 체중의 변화를 관찰하였다. 이를 위해, 시판되는 표준사료와 물을 자유로 급여하며 수컷 마우스(00주령)를 1주일간 예비 사육한 후 사용하였다. 명암 사이클은 오전 08:00시에서 오후 20:00를 명기, 오후 20:00에서 오전 08:00를 암기로 하였으며, 22.5±0.5℃의 온도 및 50 내지 60%의 습도가 유지되는 환경하에서 사육하였다. 비만 유도식으로 하기 표 2 및 표 3의 고탄수화물 식이와 고지방 식이로 하였으며 Bacillus licheniformis NY1505 균주의 포자가 함유된 사료는 각각의 비만 유도식 1kg당 108포자를 혼합하여 제조하였다. 마우스는 5개의 군(각 8마리)으로 나누어, 대조군, 고탄수화물식이군, 고탄수화물 식이 + 포자군, 고지방식이군, 고지방식이 + 포자군으로 나누어 각각 7주간 사육하였다.First, Bacillus licheniformis to produce α-Glucosidase inhibitors The diet was mixed with the spores of the strain NY1505 (10 8 spores / kg feed) was fed to the mice and observed changes in body weight. To this end, commercial standard feed and water were freely fed and male mice (00 weeks old) were used after preliminary breeding for one week. The light and dark cycles were designed at 08:00 am to 20:00 pm and at 20:00 pm to 08:00 am, and were bred under the environment of 22.5 ± 0.5 ° C. and humidity of 50 to 60%. . Was made to a mast guided in Table 2 and the high carbohydrate diet and high fat diet shown in Table 3 Bacillus licheniformis a feed containing the spores of NY1505 strain was prepared by mixing 10 8 spores per 1kg each guided obesity. Mice were divided into 5 groups (8 animals each), and were divided into a control group, a high carbohydrate diet group, a high carbohydrate diet + spore group, a high fat diet group, and a high fat diet + spore group, respectively, for 7 weeks.
Figure PCTKR2017001754-appb-T000002
Figure PCTKR2017001754-appb-T000002
Figure PCTKR2017001754-appb-T000003
Figure PCTKR2017001754-appb-T000003
(1) 고탄수화물 식이의 급여시 Bacillus licheniformis NY1505균 포자의 투여가 마우스의 체중에 미치는 영향 분석 (1) Bacillus licheniformis at the time of feeding a high-carb diet Analysis of Effects of NY1505 Spores on Body Weight in Mice
고탄수화물 식이 급여된 마우스의 체중(body weight, 단위 gram)에 Bacillus licheniformis NY1505균 포자의 투여가 미치는 영향을 분석한 결과, 도 1에 나타난 바와 같이, 고탄수화물사료군은 대조군에 비하여 뚜렷한 체중 증가를 보이나 Bacillus licheniformis NY1505 균주의 포자가 혼합된 고탄수화물 사료군에서는 대조군과 비슷한 체중 증가 양상을 보이는 것을 확인할 수 있었다.Weight of the high carbohydrate diet benefit mice (body weight, unit of gram) to a result of analyzing the effect of the administration of Bacillus licheniformis NY1505 bacteria spores on, as shown in Figure 1, high-carbohydrate food group is a marked weight gain compared to the control group However, the high carbohydrate feed group containing spores of Bacillus licheniformis NY1505 strain showed similar weight gain pattern as the control group.
(2) 고지방 식이의 급여시 Bacillus licheniformis NY1505균 포자의 투여가 마우스의 체중에 미치는 영향 (2) Bacillus licheniformis during the feeding of high fat diets Effect of Spore of NY1505 Spores on Mouse Weight
고지방 식이 급여된 마우스의 체중에 Bacillus licheniformis NY1505균 포자의 투여가 마우스의 체중에 미치는 영향을 분석한 결과, 도 2에 나타난 바와 같이, 고지방사료군은 대조군에 비하여 뚜렷한 체중 증가를 보이나 Bacillus licheniformis NY1505 균주의 포자가 혼합된 고지방 사료군에서는 대조군과 비슷한 체중 증가 양상을 보이는 것을 확인할 수 있었다. Bacillus licheniformis in Body Weight of High-fat Dieted Mice As a result of analyzing the effect of the administration of NY1505 spores on the body weight of the mouse, as shown in Figure 2, the high fat feed group showed a significant weight gain compared to the control group, but in the high fat feed group mixed with spores of Bacillus licheniformis NY1505 strain It can be seen that the weight gain pattern similar to.
(3) (3) 분변으로With feces 배설되는  Excreted Bacillus Bacillus licheniformislicheniformis NY1505 NY1505 균수의Fungal 변화 change
Bacillus licheniformis NY1505균 포자가 투여된 마우스의 변에 포함된 포자수를 측정하여 변으로 배설되는 균주의 변화를 분석한 결과, 도 3에 나타난 바와 같이, 사료 1g 당 105마리의 포자가 혼합되어 있고 한 마리 당 하루에 약 5g의 사료를 섭취하므로 하루에 약 5 x 105 마리의 포자를 섭취한다. 포자 투여 3주후에는 분변 1g 당 약 107마리의 Bacillus licheniformis NY1505가 검출되므로 장내에서 본 균주가 왕성하게 증식하여 배출됨을 알 수 있었다. 포자를 7주간 투여한 경우, 포자를 투여하지 않은 1주일 만인 8주차에는 균의 숫자가 현격히 감소하므로 장내에 고착하지 않고 일시적으로 서식하는 것으로 판단할 수 있었다. 따라서, 본 균주의 투여를 중단하면 장내 균총의 변화가 예상되므로 필요에 따라 장내에서 본 균주의 제거도 가능한 장점이 있음을 확인할 수 있었다. Bacillus licheniformis As a result of analyzing the change of the strain excreted into the stool by measuring the number of spores contained in the stool of the mice administered spores NY1505, as shown in Figure 3, 10 5 spores per 1g of feed is mixed and per You eat about 5g of feed per day, so you eat about 5 x 10 5 spores per day. Three weeks after spore administration, about 10 7 Bacillus licheniformis per gram of feces Since NY1505 was detected, it was found that this strain was vigorously proliferated and excreted in the intestine. When spores were administered for 7 weeks, the number of germs was significantly reduced at 8 weeks after only 1 week without spores. Therefore, when the administration of the strain is stopped, it is expected that the change in the intestinal flora can be confirmed that there is an advantage that the removal of the strain in the intestine as needed.
(4) 변으로 배설되는 α-글루코시다아제 저해 활성의 변화 (4) Change of α-glucosidase inhibitory activity excreted in feces
Bacillus licheniformis NY1505균 포자가 투여된 마우스의 변에 포함된 α-글루코시다아제의 저해 활성을 분석한 결과, 도 4에 나타난 바와 같이, Bacillus licheniformis NY1505 균주는 변으로 배출될 정도로 장내에서 왕성하게 증식하며 α-글루코시다아제 저해제를 생산하는 것을 확인할 수 있었다. 포자가 투여된 마우스의 2주 경과한 시점의 변에서 장내에서 증식한 Bacillus licheniformis NY1505 균주가 변으로 배출되어 활성이 검출되었다. 또한, 4주차부터는 7주까지는 배출되는 Bacillus licheniformis NY1505 균주의 양이 안정화되는 시기로 배출되는 α-글루코시다아제 저해제의 양도 일정한 것을 확인할 수 있었다. 즉, α-글루코시다아제 저해제는 항상 장내에서 생산되므로 장내에서의 농도가 일정량 유지되며 효율이 높게 작용하는 장점을 가지며 생산된 저해제의 일정량이 배출되는 것임을 알 수 있다. 분변으로 배출되는 α-글루코시다아제 저해제의 활성을 보면 Bacillus licheniformis NY1505 균주로 제조한 낫토의 활성(표 1)과 비교하여 보면 상당한 양의 α-글루코시다아제 저해제가 장내에서 생산되고 그 일부가 배출된다는 사실을 확인할 수 있었다. Bacillus licheniformis As a result of analyzing the inhibitory activity of α-glucosidase contained in the stool of the mouse administered with the NY1505 spores, as shown in FIG. 4, the Bacillus licheniformis NY1505 strain proliferated vigorously in the intestine so as to be excreted into the stool. It was confirmed that the glucosidase inhibitor was produced. Bacillus licheniformis proliferated in the intestine at the stool two weeks after the spore-administered mice The NY1505 strain was excreted in feces and activity was detected. In addition, Bacillus licheniformis , which is released from week 4 to week 7 It was confirmed that the amount of α-glucosidase inhibitor discharged at a time when the amount of the NY1505 strain was stabilized was also constant. That is, since the α-glucosidase inhibitor is always produced in the intestine, the concentration in the intestine is maintained at a certain amount, it can be seen that there is an advantage that the efficiency is high and a certain amount of the inhibitor produced is discharged. Fecal activity of α-glucosidase inhibitors is compared to the activity of natto prepared from Bacillus licheniformis NY1505 strain (Table 1), and a significant amount of α-glucosidase inhibitors are produced in the intestine and some of them are excreted. I could confirm that.
상기와 같은 결과를 통해 본 발명에 따른 Bacillus licheniformis NY1505 균주가 발효에 의해 생성된 생리활성물질을 사용하는 것이 아니라 체내에서 균주가 생리활성물질을 연속적으로 만들어 공급하므로 α-글루코시다아제 저해제의 생성을 위한 별도의 발효비용 및 가공비가 필요하지 않아 생산비용을 절감할 수 있으며 체내에서 연속적으로 생산하므로서 유효한 농도를 일정하게 유지할 수 있는 장점이 있음을 확인할 수 있었고, 유용균의 포자를 이용하므로서 발효취 등의 문제가 발생하지 않으며 다양한 가공식품에 첨가물로 사용할 수 있으므로 다양한 형태의 식품에 적용할 수 있다는 사실을 확인할 수 있었다. Through the above results, the strain Bacillus licheniformis NY1505 according to the present invention does not use the bioactive substance produced by fermentation, but the strain continuously produces and supplies the bioactive substance in the body, thereby producing the α-glucosidase inhibitor. It is possible to reduce the production cost because it does not require separate fermentation cost and processing cost, and it can be confirmed that there is an advantage of maintaining a constant effective concentration by continuously producing in the body. There is no problem and can be used as an additive to a variety of processed foods, it was confirmed that it can be applied to various types of food.
5. 분리된 균주의 형태학적 및 생화학적 특성 규명5. Morphological and Biochemical Characterization of Isolated Strains
(1) 분리된 균주의 형태학적 특성(1) Morphological Characteristics of Isolated Strains
상기와 같이 분리된 Bacillus licheniformis NY1505 균주는 LB 한천 평판배지에서 콜로니의 중앙부분은 옅은 적갈색을 나타내며 가장자리는 불규칙한 외형을 나타내었다(도 5). 균의 생육이 가능한 온도 범위는 25 내지 50 ℃의 범위에서 성장이 양호하였으며, 60 ℃ 이상에서는 균체 성장이 확인되지 않았다. LB 액체배지를 이용한 균체 성장의 대수기에서 현미경 관찰한 결과, 그람 양성의 짧은 막대모양을 나타내면 비교 균주인 바실러스 리케니포르미스와 유사하였다. 또한, 분리한 균주는 하기 표 4에 나타낸 바와 같이, 그람 양성이었고, 세포의 크기는 대략 0.5~0.7 × 1.5~1.8 ㎛인 것을 확인할 수 있었으며, 상기한 결과를 종합하여, 바실러스 리케니포르미스 NY1505 균주의 분류학상 위치를 도 6에 나타내었다. Bacillus licheniformis isolated as above The NY1505 strain showed a light reddish brown color at the center of the colonies and irregular edges in LB agar plate medium (FIG. 5). The temperature range of the growth of bacteria was good in the range of 25 to 50 ℃, cell growth was not confirmed above 60 ℃. Microscopic observation of the cell growth using LB liquid medium showed that gram-positive short rods were similar to Bacillus rickenformis, a comparative strain. In addition, the isolated strain was Gram-positive, as shown in Table 4 below, the cell size was found to be approximately 0.5 ~ 0.7 × 1.5 ~ 1.8 ㎛, synthesized the above results, Bacillus rickenformis The taxonomic location of the NY1505 strain is shown in FIG. 6.
Figure PCTKR2017001754-appb-T000004
Figure PCTKR2017001754-appb-T000004
(2) 분리된 균주의 생화학적 특성(2) Biochemical Properties of Isolated Strains
버지스 매뉴얼(Bergey's Manual of Systematic Bacteriology, 2002)에 의한 바실러스속 균주의 분류학적 특성 분석 방법과 API50CHB 키트(BioMerieux사, France)를 사용하여, 분리된 균주의 생화학적 특성 등을 조사한 결과, 분리한 균주는 그람 양성의 원통형으로 포자는 세포 중앙에 형성되며, 질산염의 환원력은 양성이며, 인돌 형성은 음성이었다. 또한, 카제인과 전분을 분해하며, 카탈라아제 양성이며, 호기적 조건 및 혐기적 조건에서 성장하는 것으로 확인되었으며, 하기 표 5에 나타낸 바와 같은 생화학적 특성(API test 결과)을 가짐을 확인할 수 있었다.다. Using the method of taxonomic characterization of Bacillus strains by Bergy's Manual of Systematic Bacteriology (2002) and the biochemical properties of the isolated strains using the API50CHB kit (BioMerieux, France), the isolates were isolated. The gram-positive cylindrical spores were formed in the center of the cell, the reducing power of nitrate was positive, and indole formation was negative. In addition, it was confirmed that the casein and starch were degraded, catalase positive, and grown under aerobic and anaerobic conditions, and had biochemical properties (API test results) as shown in Table 5 below. .
Figure PCTKR2017001754-appb-T000005
Figure PCTKR2017001754-appb-T000005
본 발명에 따른 Bacillus licheniformis NY1505 균주는 장내에서 α-글루코시다아제 저해제를 생산하여 공급하므로 식품조성물에 사용될 수 있다. Bacillus licheniformis NY1505 strain according to the present invention can be used in food compositions because it produces and supplies an α-glucosidase inhibitor in the intestine.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원 한국생물자원센터(KCTC)Name of institution: Korea Biotechnology Research Institute Korea Bioresource Center (KCTC)
수탁번호 : KCTC13021BPAccession number: KCTC13021BP
수탁일자 : 20160503Deposit date: 20160503
[기탁증][Deposit]
Figure PCTKR2017001754-appb-I000002
Figure PCTKR2017001754-appb-I000002

Claims (13)

  1. α-글루코시다아제 저해제(α-glucosidase inhibitor)를 고생산하는 바실러스 리케니포르미스 NY1505 균주(Bacillus licheniformis NY1505, 수탁번호KCTC13021BP). Bacillus licheniformis NY1505, Accession No. KCTC13021BP, which produces high α-glucosidase inhibitor.
  2. 제1항에 있어서, 상기 균주가 서열번호 1의 16s RNA를 포함하는 것을 특징으로 하는 균주.The strain according to claim 1, wherein the strain comprises 16s RNA of SEQ ID NO: 1.
  3. 제1항에 있어서, 상기 균주가 혐기성인 것을 특징으로 하는 균주.The strain of claim 1, wherein said strain is anaerobic.
  4. 제3항에 있어서, 상기 균주가 호기성인 것을 특징으로 하는 균주.4. The strain of claim 3, wherein said strain is aerobic.
  5. 제1항에 있어서, 상기 균주가 포자를 형성하는 것을 특징으로 하는 균주.The strain of claim 1, wherein said strain forms spores.
  6. 제1항에 있어서, 상기 균주가 생물체로부터 투여 후 2주 이내에 배설되는 것을 특징으로 하는 균주.The strain of claim 1, wherein the strain is excreted within two weeks after administration from the organism.
  7. 제1항에 있어서, 상기 균주가 장내에서 α-글루코시다아제 저해제를 생산하는 것을 특징으로 하는 균주.The strain of claim 1, wherein the strain produces an α-glucosidase inhibitor in the intestine.
  8. 제1항에 있어서, 장내에서 생산된 α-글루코시다아제 저해제가 생물체로부터 균주 투여 후 2주 이내에 배설되는 것을 특징으로 하는 균주.The strain according to claim 1, wherein the intestinal produced α-glucosidase inhibitor is excreted within 2 weeks after administration of the strain from the organism.
  9. 제1항 내지 제8항 중 어느 한 항에 기재된 균주를 포함하는 식품 조성물.Food composition containing the strain of any one of Claims 1-8.
  10. 제9항에 있어서, 상기 균주가 포자 형태로 포함되는 것을 특징으로 하는 식품 조성물.The food composition according to claim 9, wherein the strain is included in spore form.
  11. 제10항에 있어서, 상기 균주가 1 kg 당 106 내지 1010개의 포자로 포함되는 것을 특징으로 하는 식품 조성물.The food composition of claim 10, wherein the strain comprises 10 6 to 10 10 spores per kg.
  12. 제9항에 있어서, 상기 식품 조성물이 상기 균주의 발효물을 추가로 포함하는 것을 특징으로 하는 식품 조성물.The food composition of claim 9, wherein said food composition further comprises a fermentation product of said strain.
  13. 제12항에 있어서, 상기 발효물이 대두 발효물인 것을 특징으로 하는 식품 조성물.The food composition according to claim 12, wherein the fermentation product is soybean fermentation product.
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