KR102622613B1 - Lacticaseibacillus paracasei WiKim0136 strain with excellent chiroinositol producing ability and uses for lowering blood sugar thereof - Google Patents
Lacticaseibacillus paracasei WiKim0136 strain with excellent chiroinositol producing ability and uses for lowering blood sugar thereof Download PDFInfo
- Publication number
- KR102622613B1 KR102622613B1 KR1020220135794A KR20220135794A KR102622613B1 KR 102622613 B1 KR102622613 B1 KR 102622613B1 KR 1020220135794 A KR1020220135794 A KR 1020220135794A KR 20220135794 A KR20220135794 A KR 20220135794A KR 102622613 B1 KR102622613 B1 KR 102622613B1
- Authority
- KR
- South Korea
- Prior art keywords
- strain
- chiroinositol
- paracasei
- wikim0136
- formula
- Prior art date
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- CDAISMWEOUEBRE-UHFFFAOYSA-N inositol Chemical compound OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 title claims abstract description 121
- 235000000346 sugar Nutrition 0.000 title abstract description 19
- 239000008280 blood Substances 0.000 title abstract description 18
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- 241000193830 Bacillus <bacterium> Species 0.000 claims description 42
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- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 13
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- 229910052799 carbon Inorganic materials 0.000 claims description 7
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 claims description 6
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- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 claims description 6
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- C—CHEMISTRY; METALLURGY
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 키로이노시톨 생성능이 우수한 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주 및 이의 혈당강하 용도에 관한 것으로, 본 발명에 따른 락티카제이 바실러스 파라카제이 WiKim0136 균주는 내열성이 우수하며, 위산과 담즙산에 영향을 받지 않으며, 혈당강하에 도움을 줄 수 있는 키로이노시톨 생성능이 우수하여 프로바이오틱스 균주로 이용될 수 있다.The present invention relates to the Lacticaseibacillus paracasei WiKim0136 strain, which has excellent chiroinositol production ability, and its use in lowering blood sugar. The Lacticaseibacillus paracasei WiKim0136 strain according to the present invention has excellent heat resistance and gastric acid It is not affected by bile acids and has an excellent ability to produce chiroinositol, which can help lower blood sugar levels, so it can be used as a probiotic strain.
Description
본 발명은 키로이노시톨 생성능이 우수한 락티카제이 바실러스 파라카제이 WiKim0136 균주 및 이의 혈당강하 용도에 관한 것이다.The present invention relates to the Lacticajay Bacillus paracasei WiKim0136 strain, which has excellent chiroinositol production ability, and its use in lowering blood sugar levels.
전 세계적으로 인구 노령화와 비만 인구의 증가, 운동 부족, 식습관 변화, 생활수준의 향상 등에 따라 당뇨의 유병인구는 급격하게 증가하는 추세이다.Worldwide, the number of people with diabetes is rapidly increasing due to population aging, increase in obesity, lack of exercise, changes in eating habits, and improvement in living standards.
이 중, 당뇨병 (Diabetes)은 인슐린의 분비 결함 또는 인슐린 작용의 결함에서 기인한 만성 고혈당증 (Hyperglycemia)의 특징을 나타내는 다중적 병인의 대사장애를 말하며, 혈중의 포도당이 비정상적으로 높은 상태가 장기간 지속될 경우 만성적인 대사장애와 이에 따른 만성적 혈관손상으로 다양한 합병증이 발생한다.Among these, diabetes refers to a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia resulting from defects in insulin secretion or insulin action, and occurs when abnormally high blood glucose persists for a long period of time. Various complications occur due to chronic metabolic disorders and subsequent chronic vascular damage.
당뇨병은 대표적인 성인성 대사질환으로 세계인구의 약 5 %가 앓고 있으며, 그로 인한 인명적, 경제적 손실은 실로 막대하다. 당뇨병 환자의 대부분은 경구용 치료제를 복용하고 있으나, 현재까지 안전한 치료제가 개발되어 있지 않은 실정이다. 인슐린 저항성 (Insulin resistance)이 가장 중요한 기전적 원인으로 알려져 있지만, 정확한 기전은 아직 확실하지 않으며, 다만 유전적인 소인과 환경의 복합적인 원인이 작용하는 것으로 밝혀져 있다.Diabetes is a representative adult metabolic disease that affects approximately 5% of the world's population, and the resulting human and economic losses are truly enormous. Most diabetic patients are taking oral treatments, but no safe treatments have been developed to date. Insulin resistance is known to be the most important mechanistic cause, but the exact mechanism is not yet clear, but it has been revealed that a combination of genetic predisposition and environment plays a role.
당뇨병은 전 세계적으로 3번째로 심각한 질병으로 2010년까지 약 2억 5천만명의 당뇨병환자가 예상되며, 국내의 경우에도 계속적인 증가가 예견되고 있다. 국내 사망원인 중 7번째로 높으며, 전체 당뇨병환자의 90 % 이상을 차지하고 있는 인슐린 비의존성 당뇨병 (Non-insulin dependent diabetes, NIDDM)은 주로 40대 이후에 발병되어 성인형 당뇨병이라 불리며 인슐린을 충분히 생성시키지 못하거나 적절히 이용하지 못함으로써 야기되는 대사 장애이다.Diabetes is the third most serious disease worldwide, and the number of diabetes patients is expected to be approximately 250 million by 2010, and continued growth is expected in Korea as well. Non-insulin dependent diabetes (NIDDM), which is the 7th leading cause of death in Korea and accounts for more than 90% of all diabetic patients, usually develops after the age of 40 and is called adult-onset diabetes, which means it does not produce enough insulin. It is a metabolic disorder caused by the inability to use or not utilize it properly.
NIDDM의 발병 원인은 명확히 밝혀져 있지는 않지만, 서양화된 식생활 및 생활방식 등의 환경적 요인과 비만 및 운동 부족과 같은 유전적인 요인이 모두 작용하는 것으로 생각된다. NIDDM은 보통 식이요법 및 운동요법으로 치료를 먼저 시도하고, 이 방법에 의한 치료 효과가 충분치 않은 경우, 일반적으로 약물들이 이용되는데 많은 경우 인슐린이 사용된다.Although the cause of NIDDM is not clearly known, it is thought that both environmental factors, such as Westernized diet and lifestyle, and genetic factors, such as obesity and lack of exercise, play a role. NIDDM is usually treated first with diet and exercise therapy, and if the treatment effect of these methods is not sufficient, drugs are generally used, and in many cases, insulin is used.
당뇨병의 치료방법으로는 크게 식사 요법, 운동 요법, 약물 요법 등이 사용되고 있으며, 이 중에 약물요법의 경우 화학물질을 사용하기 때문에 여러 부작용 (불편함, 거부감, 저혈당 유발 등)이 야기되는 문제점을 지니고 있다.Treatment methods for diabetes include diet therapy, exercise therapy, and drug therapy. Among these, drug therapy has the problem of causing various side effects (discomfort, rejection, hypoglycemia, etc.) because it uses chemicals. there is.
또한, 인슐린은 식사요법과 경구 혈당강하제로 혈당이 조절되지 않는 환자에게 필요하나, 인슐린은 단백질이므로 소화관에서는 가수 분해되어 비활성화되므로 구강으로는 섭취할 수 없고 정맥이나 피하 내에 주사하여야 한다는 단점이 있다.In addition, insulin is necessary for patients whose blood sugar cannot be controlled by diet and oral hypoglycemic agents, but since insulin is a protein, it is hydrolyzed and inactivated in the digestive tract, so it cannot be ingested orally and has the disadvantage of having to be injected intravenously or subcutaneously.
한편, 아이스플랜트 (Mesembryanthemum crystallinum, common ice plant)는 석죽목 번행초과 메셈브리안테움속에 속하는 염생식물로써 원산지는 남아프리카 나미브 사막으로 사막기후에 적응하기 위해 평소에는 C3 광합성 수행하나 극심한 건조나 염해 조건에서는 CAM 광합성으로 전환되는 선택적 CAM (facultative CAM) 식물이다. 표면에 투명한 결정체 (bladder cell)에는 이노시톨류, 베타카로틴과 풍부한 미네랄을 함유하고 있으며 당뇨병에 효과가 있는 피니톨류의 성분이 높은 것으로 알려져 있다.Meanwhile, the ice plant ( Mesembryanthemum crystallinum , common ice plant) is a halophyte belonging to the genus Mesembryanthemum of the order Mesembryanthemum. Its origin is the Namib Desert in South Africa. It normally performs C3 photosynthesis to adapt to the desert climate, but under extreme dry or salty conditions, it is a halophyte. It is a selective CAM (facultative CAM) plant that switches to CAM photosynthesis. Transparent crystals (bladder cells) on the surface contain inositol, beta-carotene, and abundant minerals, and are known to be high in pinitol, which is effective in treating diabetes.
아이스플랜트의 효능에 대한 연구는 2000년도 초반 함유 성분 중 피니톨을 다량 포함하는 것으로 알려지면서 본격화되었다. 그리고, 아이스플랜트는 주로 혈당강하, 혈압강하, 중성지방 축적 억제 등의 생리적 효능을 가지고 있는 것으로 연구되고 있다. Research on the efficacy of ice plant began in earnest in the early 2000s when it was known that it contains a large amount of pinitol among its ingredients. Additionally, ice plant is mainly being studied to have physiological effects such as lowering blood sugar, lowering blood pressure, and suppressing neutral fat accumulation.
특히, 아이스플랜트에 다수 함유되어 있는 물질인 피니톨은 생체 대사과정에서 카이로-이노시톨로 변환되며, 인슐린과 유사하게 혈중 글루코오스를 세포 내로 이동시키는 것에 관여하는 핵심 물질 (Key molecules)로 알려져 있으며, 식물성 인슐린의 역할을 한다.In particular, pinitol, a substance contained in large quantities in ice plants, is converted into chiro-inositol during biological metabolism, and is known as a key molecule involved in moving blood glucose into cells, similar to insulin, and is a vegetable insulin. plays the role of
D-키로-이노시톨 (D-chiro-inositol, cis-1,2,4-trans-3,5,6-cyclohexol)은 마이오-이노시톨 (myo-inositol, cis-1,2,3,5-trans-4,6-cyclohexanehexol)의 스테레오이소머로서 3번 하이드록실 그룹이 에피머화된 형태이다. 키로이노시톨은 주로 진핵생물에서 발견되며, 마이오-이노시톨의 에피머화에 의해 생합성된다. 키로이노시톨은 인슐린 신호전달계의 주요 성분으로 주사 및 경구투여 시 인슐린 작용성을 크게 개선하는 물질로 알려져 있다. 이는 현재 의약품으로는 등록되어 있지는 않지만 인슐린 비의존형 당뇨환자의 혈당강하 및 부작용 개선에 큰 효과를 나타낸다는 것이 보고되어 왔다. 키로이노시톨은 자연계에 극히 미량만 존재하여 그 생산에 어려움이 있다.D-chiro-inositol (cis-1,2,4-trans-3,5,6-cyclohexol) is myo-inositol (cis-1,2,3,5- It is a stereoisomer of trans-4,6-cyclohexanehexol) in which the 3-hydroxyl group is epimerized. Chiroinositol is mainly found in eukaryotes and is biosynthesized by epimerization of myo-inositol. Chiroinositol is a major component of the insulin signaling system and is known to be a substance that significantly improves insulin action when administered by injection or oral administration. Although this is not currently registered as a medicine, it has been reported to be highly effective in lowering blood sugar levels and improving side effects in patients with non-insulin-dependent diabetes. Chiroinositol exists in extremely small quantities in nature, making its production difficult.
현재 키로이노시톨을 합성은 화학합성 또는 미생물 발효에 의한 방법이 개발되어있다. 키로이노시톨은 주로 피니톨 (D-pinitol)이나 카스가마이신 (Kasugamycin)의 염산 가수분해에 의해 화학적인 합성을 진행할 수 있는데, 원료인 피니톨이나 카스가마이신이 고가이며, 부산물의 분리가 용이하지 않아 경제성이 낮은 실정이다. 키로이노시톨을 효율적으로 생산하는 방법으로 마이오-이노시톨 탈수소효소 (Myo-inositol dehydrogenase) 효소생성 미생물을 이용하여 마이오-이노시톨로부터 생물전환하는 방법이 있으나, 아직까지 초기 연구단계에 있는 실정이다.Currently, methods for synthesizing chiroinositol using chemical synthesis or microbial fermentation have been developed. Chiroinositol can be chemically synthesized mainly through hydrochloric acid hydrolysis of pinitol (D-pinitol) or kasugamycin. However, the raw materials pinitol or kasugamycin are expensive and the by-products are not easy to separate, so it is not economical. This is a low situation. An efficient way to produce chiroinositol is to bioconvert it from myo-inositol using microorganisms that produce the enzyme Myo-inositol dehydrogenase, but it is still in the early research stage.
락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei)는 국내 식품의약품안전처의 건강기능식품 공전 (2021년)에 따른 기준 및 규격으로 허가된 프로바이오틱스 (Probiotics)로 사용가능한 균주로 장내미생물 군총의 균형 및 개선을 통해 장내유익균 증식과 유해균 억제에 도움을 주며 배변활동을 원활하게 유지할 수 있도록 도움을 줄 수 있다고 알려져 있다. 마늘, 고춧가루 등 다양한 종류와 성분의 양념이 혼합 및 함유된 우리 고유의 김치에는 원래부터 염분이 많고 강산성 환경에서 서식하여 위나 소장의 산성 소화액에서도 생존력이 강한 살아있는 식물성 유산균이 존재하며, 락티카제이 바실러스 카제이 (L. casei), 락티카제이 바실러스 파라카제이 (L. paracasei), 락티플렌티바실러스 플란타룸 (L. plantarum)는 김치유래 프로바이오틱스 유산균으로 위산과 담즙산 환경에서 높은 생존성과 항균 활성을 보이며 담즙산 분해, 혈중 콜레스테롤 수치 감소, 장 세포 보호, 대장염 및 과민성 대장증후군 억제, 면역력 증가 등의 효과로 유제품 유래 유산균보다 프로바이오틱 효과가 우수하다고 알려져 있다. 또한 락티카제이 바실러스 파라카제이 유산균은 마이오-이노시톨 디하이드로게나아제 (Myo-inositol dehydrogenase) 효소를 생산하여 마이오-이노시톨을 키로이노시톨로 전환할 수 있다. Lacticaseibacillus paracasei is a strain that can be used as a probiotic approved in accordance with the standards and specifications of the Health Functional Food Code of the Ministry of Food and Drug Safety (2021) to balance and improve the intestinal microbial community. It is known to help proliferate beneficial bacteria in the intestines, suppress harmful bacteria, and help maintain smooth bowel movements. Our unique kimchi, which contains a mixture of various types and ingredients of seasoning such as garlic and red pepper powder, contains live vegetable lactic acid bacteria that are naturally high in salt and live in a strongly acidic environment and have strong survival even in acidic digestive juices in the stomach and small intestine, and Bacillus Lacticajay. Casei ( L. casei ), Lacticasei, Bacillus paracasei ( L. paracasei ), and L. plantarum are probiotic lactic acid bacteria derived from kimchi and have high survival and antibacterial activity in gastric acid and bile acid environments. It is known to have a better probiotic effect than dairy-derived lactic acid bacteria due to its effects such as decomposing bile acid, reducing blood cholesterol levels, protecting intestinal cells, suppressing colitis and irritable bowel syndrome, and increasing immunity. In addition, Bacillus paracasei lactic acid bacteria can convert myo-inositol to chiroinositol by producing the enzyme Myo-inositol dehydrogenase.
따라서, 마이오-이노시톨 함량이 높은 아이스플랜트를 김치 유산균 락티카제이 바실러스 파라카제이 WiKim0136 균주로 발효하여 고농도의 키로이노시톨로 전환하는 기술의 개발이 시급히 요구 되고 있다.Therefore, there is an urgent need for the development of technology to convert ice plants with high myo-inositol content into high-concentration chiroinositol by fermenting them with the kimchi lactic acid bacteria Lacticajay and Bacillus paracasei WiKim0136 strains.
이에 본 발명자들은 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주를 이용하여 키로이노시톨을 높은 수율로 생산할 수 있고, 상기 생산된 키로이노시톨은 혈당강화 효과가 월등히 우수한 것을 확인하였다.Accordingly, the present inventors confirmed that chiroinositol can be produced in high yield using the Lacticaseibacillus paracasei WiKim0136 strain, and that the produced chiroinositol has a significantly excellent blood sugar enhancing effect.
이에, 본 발명의 목적은 키로이노시톨 생성능이 우수한 락티카제이 바실러스 파라카제이 WiKim0136 균주를 제공하는 것이다.Accordingly, the purpose of the present invention is to provide Lacticajay Bacillus paracasei WiKim0136 strain with excellent chiroinositol production ability.
본 발명의 다른 목적은 락티카제이 바실러스 파라카제이 WiKim0136 균주, 균주의 배양물, 배양물의 농축물, 배양물의 건조물 또는 균주의 배양 상등액을 포함하는 키로이노시톨 생성용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for producing chiroinositol containing the Lacticajay Bacillus paracasei WiKim0136 strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, or a culture supernatant of the strain.
본 발명의 또 다른 목적은 락티카제이 바실러스 파라카제이 WiKim0136 균주를 배양하는 배양 단계를 포함하는 키로이노시톨 생산방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing chiroinositol, which includes a culturing step of culturing Lacticajay Bacillus paracasei WiKim0136 strain.
본 발명의 또 다른 목적은 락티카제이 바실러스 파라카제이 WiKim0136 균주를 배양하여 생성된 키로이노시톨을 포함하는 식품 조성물에 관한 것이다.Another object of the present invention relates to a food composition containing chiroinositol produced by culturing Lacticajay Bacillus paracasei WiKim0136 strain.
본 발명의 또 다른 목적은 락티카제이 바실러스 파라카제이 WiKim0136 균주의 키로이노시톨 생산 용도에 관한 것이다.Another object of the present invention relates to the use of Bacillus paracasei WiKim0136 strain for producing chiroinositol.
본 발명은 키로이노시톨 생성능이 우수한 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주 및 이의 혈당강하 용도에 관한 것이다.The present invention relates to the Lacticaseibacillus paracasei WiKim0136 strain, which has excellent chiroinositol production ability, and its use in lowering blood sugar.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 예는 키로이노시톨 (Chiroinositol) 생성능이 우수한 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주에 관한 것이다.An example of the present invention relates to the Lacticaseibacillus paracasei WiKim0136 strain, which has excellent chiroinositol production ability.
본 명세서 상 용어 “키로이노시톨”은 피니톨 (Pinitol)을 산분해 시켜 메틸기를 제거함으로써 생산될 수 있는 물질로서, 체내에서 통상적으로 발견되는 물질로 인슐린 신호전달체계에 관여하는 매개체 중 하나로 알려져 있다.As used herein, the term “chiroinositol” is a substance that can be produced by removing the methyl group through acid decomposition of Pinitol. It is a substance commonly found in the body and is known as one of the mediators involved in the insulin signaling system.
본 발명에 있어서 키로이노시톨은 거울형 이성질체인 D-키로이노시톨, L-키로이노시톨의 두 가지 형태로 존재할 수 있으며, 하기의 화학식 I 및 화학식 II을 갖는다.In the present invention, chiroinositol can exist in two forms: enantiomeric D-chiroinositol and L-chiroinositol, and has the following formulas (I) and (II).
[화학식 I][Formula I]
[화학식 II][Formula II]
본 명세서 상 용어 “피니톨 (Pinitol)”은 수용성 탄수화물류의 일종으로 콩류나 솔잎 등에 포함되어 있는 성분 중의 하나이다. 피니톨은 1987년 이후 전 세계 각국에서 동물실험등에서 혈당조절 효과가 있는 것으로 밝혀졌다. 피니톨은 섭취시 체내에서 키로이노시톨로 전환된다. 또한, 혈당조절 기능이 저하된 실험동물에 피니톨을 섭취시키면 혈당조절 기능이 유의적으로 개선됨을 확인하였다.In this specification, the term “Pinitol” is a type of water-soluble carbohydrate and is one of the ingredients contained in legumes, pine needles, etc. Pinitol has been found to be effective in controlling blood sugar levels in animal experiments in countries around the world since 1987. When consumed, pinitol is converted to chiroinositol in the body. In addition, it was confirmed that when pinitol was ingested in experimental animals with reduced blood sugar control function, blood sugar control function was significantly improved.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주는 2022년 9월 26일자로 대한민국 KCTC (Korean Collection For Type Cultures)에 기탁하여 수탁번호 KCTC 15108BP로 기탁된 것이다.In the present invention, the Lacticajay Bacillus paracasei WiKim0136 strain was deposited with KCTC (Korean Collection For Type Cultures), Republic of Korea, on September 26, 2022, with accession number KCTC 15108BP.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주의 16s rRNA 서열은 서열번호 3의 염기서열을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the 16s rRNA sequence of the Bacillus paracasei WiKim0136 strain may include the base sequence of SEQ ID NO: 3, but is not limited thereto.
본 발명의 다른 일 예는 락티카제이 바실러스 파라카제이 WiKim0136 균주, 상기 균주의 배양물, 상기 배양물의 농축물, 상기 배양물의 건조물 및 상기 균주의 배양 상등액으로 이루어진 군으로부터 선택된 1종 이상을 포함하는 키로이노시톨 생산용 조성물에 관한 것이다.Another example of the present invention includes at least one selected from the group consisting of Lacticajay Bacillus paracasei WiKim0136 strain, a culture of the strain, a concentrate of the culture, a dried material of the culture, and a culture supernatant of the strain. It relates to a composition for producing chiroinositol.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주는 수탁번호 KCTC 15108BP로 기탁된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the Lacticajay Bacillus paracasei WiKim0136 strain may be deposited under the accession number KCTC 15108BP, but is not limited thereto.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주의 16s rRNA 서열은 서열번호 3의 염기서열을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the 16s rRNA sequence of the Bacillus paracasei WiKim0136 strain may include the base sequence of SEQ ID NO: 3, but is not limited thereto.
본 발명에 있어서 조성물은 하기 화학식 I 및/또는 화학식 2의 화합물을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the composition may include a compound of the following formula (I) and/or formula (2), but is not limited thereto.
[화학식 I][Formula I]
[화학식 II][Formula II]
본 발명의 또 다른 일 예는 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주를 배양하는 배양 단계를 포함하는 키로이노시톨 생산방법에 관한 것이다.Another example of the present invention relates to a method for producing chiroinositol including a culturing step of culturing the Lacticaseibacillus paracasei WiKim0136 strain.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주는 수탁번호 KCTC 15108BP로 기탁된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the Lacticajay Bacillus paracasei WiKim0136 strain may be deposited under the accession number KCTC 15108BP, but is not limited thereto.
본 발명에 있어서 락티카제이 바실러스 파라카제이 WiKim0136 균주의 16s rRNA 서열은 서열번호 3의 염기 서열을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the 16s rRNA sequence of the Bacillus paracasei WiKim0136 strain may include the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.
본 발명에 있어서 키로이노시톨은 하기 화학식 I 및/또는 화학식 II의 화합물인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, chiroinositol may be a compound of the following formula (I) and/or formula (II), but is not limited thereto.
[화학식 I][Formula I]
[화학식 II][Formula II]
본 발명에 있어서 배양 단계는 균주를 pH 3 내지 9, pH 3 내지 8, pH 3 내지 7, pH 3 내지 6, pH 3 내지 5, pH 4 내지 9, pH 4 내지 8, pH 4 내지 7, pH 4 내지 6, pH 4 내지 5, 예를 들어, pH 4.0으로 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by cultivating the strain at pH 3 to 9, pH 3 to 8, pH 3 to 7, pH 3 to 6, pH 3 to 5, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH It may include culturing at pH 4 to 6, pH 4 to 5, for example, pH 4.0, but is not limited thereto.
본 발명에 있어서 배양 단계는 균주를 0.1 내지 1.0 %, 0.1 내지 0.9 %, 0.1 내지 0.8 %, 0.1 내지 0.7 %, 0.1 내지 0.6 %, 0.1 내지 0.5 %, 0.2 내지 1.0 %, 0.2 내지 1.0 %, 0.2 내지 0.9 %, 0.2 내지 0.8 %, 0.2 내지 0.7 %, 0.2 내지 0.6 %, 0.2 내지 0.5 %, 0.3 내지 1.0 %, 0.3 내지 0.9 %, 0.3 내지 0.8 %, 0.3 내지 0.7 %, 0.3 내지 0.6 %, 0.3 내지 0.5 %, 0.4 내지 1.0 %, 0.4 내지 0.9 %, 0.4 내지 0.8 %, 0.4 내지 0.7 %, 0.4 내지 0.6 %, 예를 들어, 0.5 % 담즙염 (bile salt)를 함유한 배지에서 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is 0.1 to 1.0%, 0.1 to 0.9%, 0.1 to 0.8%, 0.1 to 0.7%, 0.1 to 0.6%, 0.1 to 0.5%, 0.2 to 1.0%, 0.2 to 1.0%, 0.2%. to 0.9%, 0.2 to 0.8%, 0.2 to 0.7%, 0.2 to 0.6%, 0.2 to 0.5%, 0.3 to 1.0%, 0.3 to 0.9%, 0.3 to 0.8%, 0.3 to 0.7%, 0.3 to 0.6%, 0.3 Culturing in a medium containing 0.5%, 0.4 to 1.0%, 0.4 to 0.9%, 0.4 to 0.8%, 0.4 to 0.7%, 0.4 to 0.6%, for example, 0.5% bile salt It may include, but is not limited to this.
본 발명에 있어서 배양 단계는 균주를 20 내지 45 ℃, 20 내지 40 ℃, 25 내지 45 ℃, 25 내지 40 ℃, 30 내지 45 ℃, 30 내지 40 ℃, 35 내지 45 ℃, 35 내지 40 ℃, 예를 들어, 40 ℃에서 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by culturing the strain at 20 to 45°C, 20 to 40°C, 25 to 45°C, 25 to 40°C, 30 to 45°C, 30 to 40°C, 35 to 45°C, 35 to 40°C, e.g. For example, it may include culturing at 40°C, but is not limited thereto.
본 발명에 있어서 배양 단계는 락티카제이 바실러스 파라카제이 WiKim0136 균주를 0.05 내지 3.0 %(v/v), 0.05 내지 2.8 %(v/v), 0.05 내지 2.5 %(v/v), 0.05 내지 2.3 %(v/v), 0.05 내지 2.0 %(v/v), 0.05 내지 1.8 %(v/v), 0.05 내지 1.5 %(v/v), 0.05 내지 1.3 %(v/v), 0.05 내지 1.0 %(v/v), 0.05 내지 0.8 %(v/v), 0.05 내지 0.5 %(v/v), 0.05 내지 0.3 %(v/v), 예를 들어, 0.1 %(v/v) 접종하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by cultivating Lacticajay Bacillus paracasei WiKim0136 strain at 0.05 to 3.0% (v/v), 0.05 to 2.8% (v/v), 0.05 to 2.5% (v/v), 0.05 to 2.3. % (v/v), 0.05 to 2.0 % (v/v), 0.05 to 1.8 % (v/v), 0.05 to 1.5 % (v/v), 0.05 to 1.3 % (v/v), 0.05 to 1.0 % (v/v), 0.05 to 0.8 % (v/v), 0.05 to 0.5 % (v/v), 0.05 to 0.3 % (v/v), for example 0.1 % (v/v). It may include steps, but is not limited thereto.
본 발명에 있어서 배양 단계는 락티카제이 바실러스 파라카제이 WiKim0136 균주를 1 내지 10일, 1 내지 7일, 1 내지 4일, 2 내지 10일, 2 내지 7일, 2 내지 4일, 예를 들어, 3일 동안 배양하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by culturing the Lacticajay Bacillus paracasei WiKim0136 strain for 1 to 10 days, 1 to 7 days, 1 to 4 days, 2 to 10 days, 2 to 7 days, 2 to 4 days, for example. , may include culturing for 3 days, but is not limited thereto.
본 발명에 있어서 배양 단계는 배지에 아이스플랜트 분말을 1.0 내지 20.0 % (w/w), 1.0 내지 18.0 %(v/v), 1.0 내지 15.0 %(v/v), 1.0 내지 13.0 %(v/v), 1.0 내지 11.0 %(v/v), 3.0 내지 20.0 %(v/v), 3.0 내지 18.0 %(v/v), 3.0 내지 15.0 %(v/v), 3.0 내지 13.0 %(v/v), 3.0 내지 11.0 %(v/v), 5.0 내지 20.0 %(v/v), 5.0 내지 18.0 %(v/v), 5.0 내지 15.0 %(v/v), 5.0 내지 13.0 %(v/v), 5.0 내지 11.0 %(v/v), 7.0 내지 20.0 %(v/v), 7.0 내지 18.0 %(v/v), 7.0 내지 15.0 %(v/v), 7.0 내지 13.0 %(v/v), 7.0 내지 11.0 %(v/v), 예를 들어, 10 %(w/w) 첨가하는 단계를 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culturing step is performed by adding 1.0 to 20.0% (w/w), 1.0 to 18.0% (v/v), 1.0 to 15.0% (v/v), 1.0 to 13.0% (v/) of ice plant powder in the medium. v), 1.0 to 11.0 % (v/v), 3.0 to 20.0 % (v/v), 3.0 to 18.0 % (v/v), 3.0 to 15.0 % (v/v), 3.0 to 13.0 % (v/ v), 3.0 to 11.0 % (v/v), 5.0 to 20.0 % (v/v), 5.0 to 18.0 % (v/v), 5.0 to 15.0 % (v/v), 5.0 to 13.0 % (v/ v), 5.0 to 11.0 % (v/v), 7.0 to 20.0 % (v/v), 7.0 to 18.0 % (v/v), 7.0 to 15.0 % (v/v), 7.0 to 13.0 % (v/ v), it may include adding 7.0 to 11.0% (v/v), for example, 10% (w/w), but is not limited thereto.
본 발명의 또 다른 일 예는 다음 단계를 포함하는 키로이노시톨 생상방법에 관한 것이다:Another example of the present invention relates to a method for producing chiroinositol comprising the following steps:
락티카제이 바실러스 파라카제이 WiKim0136 균주를 배지에서 배양하는 배양 단계; 및A culture step of culturing the Bacillus paracasei WiKim0136 strain in a medium; and
배지에서 배양물을 분리하는 분리 단계.Separation step to separate the culture from the medium.
본 발명에 있어서 배지는 갈락토스 (galctose), 글루코스 (glugose), 프락토스 (fructose), 만노스 (mannose), 만니톨 (mannitol), N-아세틸글르코사민 (N-acetylglucosamime), 아미그달린 (amygdalin), 알부틴 (arbutin), 에스큘린 (esculin), 살리신 (salicin), D-셀로비오스 (D-cellobiose), D-락토스 (D-lactose), D-트레할로스 (D-trehalose), D-멜레지토스 (D-melezitose), 젠티오비오스 (gentiobiose), D-타가토스 (D-tagatose) 및 글루콘산칼륨 (potassium gluconate)으로 이루어진 군에서 선택된 1종 이상의 탄소원을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the medium contains galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamime, amygdalin, and arbutin ( arbutin, esculin, salicin, D-cellobiose, D-lactose, D-trehalose, D-melezitose (D- It may include, but is not limited to, one or more carbon sources selected from the group consisting of melezitose, gentiobiose, D-tagatose, and potassium gluconate.
본 발명은 키로이노시톨 생성능이 우수한 락티카제이 바실러스 파라카제이 (Lacticaseibacillus paracasei) WiKim0136 균주 및 이의 혈당강하 용도에 관한 것으로, 본 발명에 따른 락티카제이 바실러스 파라카제이 WiKim0136 균주는 내열성이 우수하며, 위산과 담즙산에 영향을 받지 않으며, 혈당강하에 도움을 줄 수 있는 키로이노시톨 생성능이 우수하여 프로바이오틱스 균주로 이용될 수 있다.The present invention relates to the Lacticaseibacillus paracasei WiKim0136 strain, which has excellent chiroinositol production ability, and its use in lowering blood sugar. The Lacticaseibacillus paracasei WiKim0136 strain according to the present invention has excellent heat resistance and gastric acid It is not affected by bile acids and has an excellent ability to produce chiroinositol, which can help lower blood sugar levels, so it can be used as a probiotic strain.
도 1은 본 발명의 일 실시예에 따른 다양한 김치 유래 유산균 17종의 마이오-이노시톨 분해 활성을 비교한 결과를 나타낸 그래프이다.
도 2는 본 발명의 일 실시예에 따른 균주의 49개의 탄소원에 대한 당이용성을 비교한 결과를 나타낸 도이다.
도 3은 본 발명의 일 실시예에 따른 락티카제이 바실러스 파라카제이 WiKim0136 균주의 16S rRNA 유전자의 염기서열 분석을 통해 나타낸 계통도이다.
도 4는 본 발명의 일 실시예에 따른 지표성분 3종의 성분인 피니톨, 마이오이노시톨 및 키로이노시톨의 동시분석을 나타낸 GC/FID 크로마토그래프이다.
도 5는 본 발명의 일 실시예에 따른 발효 조건에 따른 키로이노시톨의 함량을 비교하여 그 결과를 나타낸 그래프이다.
도 6은 본 발명의 일 실시예에 따른 락티카제이 바실러스 파라카제이 WiKim0136 균주의 내산성, 내담즙성 및 내열성을 비교하여 그 결과를 나타낸 그래프이다.Figure 1 is a graph showing the results of comparing the myo-inositol decomposition activity of 17 different types of lactic acid bacteria derived from kimchi according to an embodiment of the present invention.
Figure 2 is a diagram showing the results of comparing sugar availability for 49 carbon sources of strains according to an embodiment of the present invention.
Figure 3 is a schematic diagram showing the base sequence analysis of the 16S rRNA gene of Lacticajay Bacillus paracasei WiKim0136 strain according to an embodiment of the present invention.
Figure 4 is a GC/FID chromatograph showing simultaneous analysis of three indicator components, pinitol, myo-inositol, and chiroinositol, according to an embodiment of the present invention.
Figure 5 is a graph showing the results of comparing the content of chiroinositol according to fermentation conditions according to an embodiment of the present invention.
Figure 6 is a graph showing the results of comparing the acid resistance, bile resistance, and heat resistance of the Lacticajay Bacillus paracasei WiKim0136 strain according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1. 균주의 선발Example 1. Selection of strains
다양한 김치 유래 유산균 17종을 대상으로 마이오-이노시톨 분해 활성을 비교하였다. 구체적으로, 김치 유산균을 분리하기 위해 다양한 종류의 김치 (14종류)를 수집하였고, 김치 파쇄액을 MRS 고체배지에 도말 및 배양하여 미생물 콜로니를 순수 분리하였다. 김치 유산균 200종을 분리하였으며, 마이오-이노시톨 어세이 키트 (Megazyme, Ireland) 분석을 통해 17종 김치 유산균을 1차 선발하였고, 각각의 김치 유산균의 마이오-이노시톨 분해 활성을 비교한 후, 그 결과를 도 1에 나타내었다.The myo-inositol decomposition activity of 17 different types of lactic acid bacteria derived from kimchi was compared. Specifically, to isolate kimchi lactic acid bacteria, various types of kimchi (14 types) were collected, and the kimchi crushed liquid was spread and cultured on MRS solid medium to pure isolate microbial colonies. 200 types of kimchi lactic acid bacteria were isolated, and 17 types of kimchi lactic acid bacteria were initially selected through analysis of a myo-inositol assay kit (Megazyme, Ireland). After comparing the myo-inositol decomposition activity of each kimchi lactic acid bacteria, the The results are shown in Figure 1.
도 1에서 확인할 수 있듯이, 17종의 김치 유래 유산균 중 락티카제이 바실러스 파라카제이 종이 마이오-이노시톨 분해 활성에 가장 우수한 것을 확인할 수 있었다. 단백질을 검색할 수 있는 오픈 데이터베이스 (Uniprot.org)에서 각 유산균 종의 효소를 검색한 결과, 락티카제이 바실러스 파라카제이는 마이오-이노시톨을 분해하여 acetyl-coA를 합성할 수 있는 iol operon을 가지고 있었으며, 락토바실러스 플란타럼 (Lactobacillus plantarum), 락토바실러스 펜토서스 (Lactobacillus pentosus)는 inositol transporter와 inositol phosphate phosphatase만 가지고 있었다.As can be seen in Figure 1, it was confirmed that among 17 types of kimchi-derived lactic acid bacteria, Lacticajay Bacillus paracasei species was the most excellent in myo-inositol decomposition activity. As a result of searching the enzymes of each lactic acid bacteria species in an open database (Uniprot.org) where you can search proteins, Lacticase Bacillus paracasei has an iol operon that can decompose myo-inositol and synthesize acetyl-coA. Lactobacillus plantarum and Lactobacillus pentosus had only inositol transporter and inositol phosphate phosphatase.
따라서, 마이오-이노시톨 분해능이 우수한 김치미생물로 락티카제이 바실러스 파라카제이 균주를 최종 선발하였다.Therefore, the Bacillus paracasei strain Lacticajay was finally selected as a kimchi microorganism with excellent myo-inositol decomposition ability.
실시예 2. 선발 균주의 동정Example 2. Identification of selection strains
2-1. 균주의 당이용성 검사2-1. Sugar availability test of strains
최종 선발한 김치유산균 락티카제이 바실러스 파라카제이 균주를 생화학적 분석방법인 API CHL 50 kit을 이용해 균주의 49개 탄소원에 대한 당이용성을 확인하였다. 배양기간 동안 발효된 탄소원은 산을 생성하고 pH를 낮추는데, API 50 CHL 배지에는 pH indicator가 들어 있어서 색의 변화로 양성(노란색)과 음성(보라색)을 판독하였으며, 균주의 49개 탄소원에 대한 당이용성을 확인하여, 그 결과를 표 1 및 도 2에 나타내었다.The sugar availability of 49 carbon sources of the finally selected Kimchi lactic acid bacteria Lacticajay and Bacillus paracasei strains was confirmed using the API CHL 50 kit, a biochemical analysis method. The carbon source fermented during the cultivation period produces acid and lowers the pH. API 50 CHL medium contains a pH indicator to read positive (yellow) and negative (purple) by color change, and the sugars for 49 carbon sources of the strain. Usability was confirmed, and the results are shown in Table 1 and Figure 2.
상기 표 1 및 도 2에서 확인할 수 있듯이, 리보오스 (ribose), 갈락토스 (galctose), 글루코스 (glugose), 프락토스 (fructose), 만노스 (mannose), 만니톨 (mannitol), 솔비톨 (sorbitol), N-아세틸글루코사민 (N-acetylglucosamime), 살리신 (salicin), D-락토스 (D-lactose), D-사카로스 (D-saccharose), D-트레할로스 (D-trehalose), 이눌린 (inulin), D-멜레지토스 (D-melezitose), D-투라노스 (D-turanose), D-타가토스 (D-tagatose) 등을 탄소원으로 이용할 수 있음을 확인하였다.As can be seen in Table 1 and Figure 2, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetyl Glucosamine (N-acetylglucosamime), salicin, D-lactose, D-saccharose, D-trehalose, inulin, D-melezitose It was confirmed that D-melezitose, D-turanose, D-tagatose, etc. can be used as carbon sources.
2-2. 균주의 분자생물학적 분석 및 계통도 분석2-2. Molecular biological analysis and phylogenetic analysis of strains
최종 선발한 김치유산균 락티카제이 바실러스 파라카제이 균주를 16s rDNA의 염기서열 분석을 통해 분자생물학적으로 동정하였다. 균주는 MRS 배지에 접종한 다음 30 ℃에서 2일간 배양하였다. 그 다음, 수확한 균주는 멸균된 생리식염수에 균체를 2회 세척한 후, DNeasy tissue kit (Qiagen, Valecia, CA, USA)를 사용하여 DNA를 추출하였다. 추출된 DNA의 16S rDNA 유전자 증폭을 위해 하기 표 2의 공통 프라이머 (universal primer)를 사용하였다.The final selected kimchi lactic acid bacteria lacticase and Bacillus paracasei strains were identified molecularly through 16s rDNA nucleotide sequence analysis. The strain was inoculated into MRS medium and then cultured at 30°C for 2 days. Next, the harvested strains were washed twice in sterilized saline solution, and then their DNA was extracted using the DNeasy tissue kit (Qiagen, Valecia, CA, USA). To amplify the 16S rDNA gene of the extracted DNA, the universal primers shown in Table 2 below were used.
PCR 반응을 위해, 0.4 mM의 dNTP, 0.5 units의 Taq polymerase 및 4 mM의 Mg2+가 함유된 Takara Perfect Premix (Takara, Japan) 10 ㎕에 DNA template (20㎍/mL) 1㎕, 1.0 μM의 정방향 프라이머와 1.0 μM의 역방향 프라이머를 각각 1 ㎕씩 넣고 나머지는 증류수를 첨가하여 총 부피가 20 ㎕가 되도록 제조하였다.For PCR reaction, 10 μl of Takara Perfect Premix (Takara, Japan) containing 0.4 mM dNTP, 0.5 units of Taq polymerase, and 4 mM Mg 2+ was added with 1 μl of DNA template (20 μg/mL) and 1.0 μM of DNA template. 1 μl each of the forward primer and 1.0 μM reverse primer were added, and the remainder was added with distilled water to make a total volume of 20 μl.
PCR 증폭은 Mastercycler gradient (Eppendorf, Hamburg, Germany)로 수행하였으며, PCR 반응은 95 ℃에서 5분 (initial denaturation), 94 ℃에서 45초(denaturation), 52 ℃에서 45초 (annealing), 72 ℃에서 1분 (extension)을 30 사이클 실시하였고, 72 ℃에서 5분간 최종 extension을 수행하였다.PCR amplification was performed using a Mastercycler gradient (Eppendorf, Hamburg, Germany), and the PCR reaction was at 95°C for 5 minutes (initial denaturation), 94°C for 45 seconds (denaturation), 52°C for 45 seconds (annealing), and 72°C. 30 cycles of 1 minute (extension) were performed, and a final extension was performed at 72°C for 5 minutes.
증폭된 16 sRNA 유전자 PCR 산물은 마크로젠 (대전, 대한민국)에 염기서열 분석을 의뢰하여 염기서열을 얻었다.The amplified 16 sRNA gene PCR products were sequenced by Macrogen (Daejeon, Korea).
상기 분리 균주인 락티카제이 바실러스 파라카제이 균주의 16s rRNA 코딩 염기서열로 총 1,300 bp의 염기서열을 결정하였으며, 그 결과를 하기 표 3에 나타내었다.A total of 1,300 bp of nucleotide sequence was determined as the 16s rRNA coding nucleotide sequence of the isolated Bacillus paracasei strain, and the results are shown in Table 3 below.
상기 수행한 염기서열을 기초로 염기서열의 상동성 검사는 등록된 정보 (Genebank database)를 대상으로 블라스트 프로그램 (Blast program, http://www.ncbi.nlm.nhi.gov)에 의해 실행하였다. 그 결과, 락티카제이 바실러스 파라카제이 균주는 Lacticaseibacillus paracasei와 98% 동일함을 확인하였다.Based on the base sequence performed above, the homology test of the base sequence was performed using the Blast program (http://www.ncbi.nlm.nhi.gov) against registered information (Genebank database). As a result, it was confirmed that the Lacticaseibacillus paracasei strain was 98% identical to Lacticaseibacillus paracasei .
부분적인 16 rRNA의 염기서열 (서열번호 3)은 GenBank에 Accession number OP579146 번으로 등록되었다. 락티카제이 바실러스 파라카제이 균주는 하기 표 4 및 도 3과 같이 유전적 유사성을 나타내었다.The partial nucleotide sequence of 16 rRNA (SEQ ID NO: 3) was registered in GenBank under accession number OP579146. Lacticase Bacillus paracasei strains showed genetic similarity as shown in Table 4 and Figure 3 below.
따라서, 상기 균주를 락티카제이 바실러스 파라카제이 WiKim0136로 명명하였고, 2022년 9월 26일자로 대한민국 KCTC (Korean Collection For Type Cultures)에 기탁하여 수탁번호 KCTC 15108BP를 부여 받았다.Therefore, the strain was named Lacticajay Bacillus paracasei WiKim0136, and was deposited with the Korean Collection For Type Cultures (KCTC), Republic of Korea, on September 26, 2022, and given the accession number KCTC 15108BP.
실시예 3. 키로이노시톨 전환능 분석Example 3. Chiroinositol conversion ability analysis
3-1. 아이스플랜트를 첨가한 배지에서 균주의 생장 관찰3-1. Observation of strain growth in medium containing ice plant
배지 (아이스플랜트 분말 10 %(w/v)과 Glucose 2 %(w/v))를 121 ℃, 15분 동안 멸균하였다. 그 다음, 1 % Celluclast, 1 % viscozyme을 첨가 후, 50 ℃에서 효소 반응을 진행하였고, 121 ℃, 10분 동안 열처리를 하여 효소 반응을 불활성화하여 균주를 접종할 배지를 준비하였다. 그 다음, 배지에 키로이노시톨 최대 생산을 위해 1차 발효는 바실러스 서브틸리스 HD15를 3.9x108 CFU/mL 접종하고 37 ℃, 2일동안 진탕배양 (shaking incubtion)을 진행하였다. 그 다음, 2차 발효로 락티카제이 바실러스 파라카제이 WiKim0136 균주를 0.1 %(v/v) 접종하여 3일, 6일, 9일 동안 발효한 후 생균수를 측정하였다. 접종 후 3일차에는 6.1x108 CFU/ml, 6일차 1.7x108 CFU/ml, 9일차 4.0x107 CFU/ml으로 생균수가 확인되었다.The medium (10% (w/v) ice plant powder and 2% (w/v) glucose) was sterilized at 121°C for 15 minutes. Next, after adding 1% Celluclast and 1% viscozyme, the enzyme reaction was performed at 50°C, and the enzyme reaction was inactivated by heat treatment at 121°C for 10 minutes to prepare a medium to inoculate the strain. Next, in order to maximize chiroinositol production in the medium, the primary fermentation was performed by inoculating 3.9x10 8 CFU/mL of Bacillus subtilis HD15 and shaking incubation at 37°C for 2 days. Next, in secondary fermentation, 0.1% (v/v) of Lacticajay Bacillus paracasei WiKim0136 strain was inoculated and fermented for 3, 6, and 9 days, and then the number of viable bacteria was measured. After vaccination, the viable cell count was confirmed to be 6.1x10 8 CFU/ml on the 3rd day, 1.7x10 8 CFU/ml on the 6th day, and 4.0x10 7 CFU/ml on the 9th day.
3-2. 가스 크로마토 그래피 분석법3-2. Gas chromatographic analysis method
하기 표 5의 조건으로 GC-FID (Gas Chromatography-Flame Ionization Detector)를 수행하여 3종의 지표 성분인 피니톨, 키로이노시톨 및 마이오-이노시톨을 동시 분석하여, 그 결과를 도 4 내지 5 및 표 6에 나타내었다.GC-FID (Gas Chromatography-Flame Ionization Detector) was performed under the conditions in Table 5 below to simultaneously analyze the three indicator components, pinitol, chiroinositol, and myo-inositol, and the results are shown in Figures 4 to 5 and Table 6. shown in
(0.32 mm i.d.x30 m, 0.25 um)HP-5 capillary column
(0.32 mm idx30 m, 0.25 um)
(Inject Temp.)injection temperature
(Inject Temp.)
(Column Temp.)column temperature
(Column Temp.)
도 4 내지 5 및 표 6에서 확인할 수 있듯이, 락티카제이 바실러스 파라카제이 WiKim0136 균주로 3일동안 발효한 시료에서 가장 높은 키로이노시톨 생성량인 약 302.74 mg/kg를 나타내었으며, 무처리군의 키로이노시톨 함량은 약 105.98 mg/kg으로 무처리군 대비 락티카제이 바실러스 파라카제이 WiKim0136 균주를 이용하여 아이스플랜트 발효시 약 2.86배 증가한 키로이노시톨 생성능을 나타내었다.As can be seen in Figures 4 to 5 and Table 6, the sample fermented for 3 days with the Lacticajay Bacillus paracasei WiKim0136 strain showed the highest chiroinositol production of about 302.74 mg/kg, and the chiroinositol production in the untreated group was The content was about 105.98 mg/kg, showing a 2.86-fold increase in chiroinositol production capacity during ice plant fermentation using the Lacticajay Bacillus paracasei WiKim0136 strain compared to the untreated group.
균주 발효 9일차로 진행되면서 키로이노시톨 함량이 감소하였는데, 이는 락티카제이 바실러스 파라카제이 WiKim0136 균주의 생균수도 감소하기 때문에 생균수 감소에 따른 유효성분의 감소에 기인하는 것으로 확인할 수 있다.As the strain fermentation progressed to the 9th day, the chiroinositol content decreased. This can be confirmed to be due to a decrease in the active ingredient due to the decrease in the number of viable bacteria, as the number of viable bacteria of the Lacticajay Bacillus paracasei WiKim0136 strain also decreased.
따라서, 락티카제이 바실러스 파라카제이 WiKim0136 균주는 혈당강하를 위한 키로이노시톨 생성에 우수한 아이스플랜트 발효용 종균임을 확인할 수 있었다.Therefore, it was confirmed that Lacticajay Bacillus paracasei WiKim0136 strain is an excellent starter for ice plant fermentation in producing chiroinositol for lowering blood sugar.
실시예 4. 균주의 내산성, 내담즙성 및 내열성 조사Example 4. Investigation of acid resistance, bile resistance and heat resistance of strains
프로바이오틱스 유산균주는 장관에 도달하기 위해서 위액에 대한 내산성 뿐만 아니라, 췌장에서 십이지장까지 분비되는 담즙산에 대한 내성 또한 중요하다. 또한, 프로바이오틱스 유산균주는 제제화 공정에서 고온의 환경에 노출되면 생균활성이 감소되므로, 유산균의 열 안정성은 프로바이오틱스 선정에 중요한 기준 중 하나이다. 이에, 락티카제이 바실러스 파라카제이 WiKim0136 균주의 프로바이오틱스 특성인 내산성, 내담즙성 및 내열성을 확인하였다.In order for probiotic lactic acid bacteria to reach the intestinal tract, it is important not only to have acid resistance to gastric juice, but also to have resistance to bile acids secreted from the pancreas to the duodenum. In addition, the probiotic activity of probiotic lactic acid bacteria is reduced when exposed to a high temperature environment during the formulation process, so the thermal stability of lactic acid bacteria is one of the important criteria for selecting probiotics. Accordingly, the probiotic characteristics of the Lacticajay Bacillus paracasei WiKim0136 strain, namely acid resistance, bile resistance, and heat resistance, were confirmed.
내산성 평가로 락티카제이 바실러스 파라카제이 WiKim0136 균주를 pH 4.0으로 조정한 MRS 배지에서 30 ℃로 2시간 동안 배양한 결과, 약 97 %의 생존률 (p<0.05)로 내산성을 보유하고 있음을 확인하였다.As a result of acid resistance evaluation, the Lacticajay Bacillus paracasei WiKim0136 strain was cultured for 2 hours at 30°C in MRS medium adjusted to pH 4.0, and it was confirmed that it had acid resistance with a survival rate of about 97% ( p<0.05 ). .
또한, 내담즙성 평가로 락티카제이 바실러스 파라카제이 WiKim0136 균주를 0.5 % 담즙염 (bile salt)를 함유한 MRS 액체배지에 24시간 동안 배양한 결과, 약 90 %의 생존율을 유지하며 담즙산에 큰 영향을 받지 않아 우수한 안정성을 나타내었다.In addition, as a bile resistance evaluation, the Lacticajay Bacillus paracasei WiKim0136 strain was cultured for 24 hours in MRS liquid medium containing 0.5% bile salt, and the results showed that it maintained a survival rate of about 90% and was highly resistant to bile acids. It was not affected, showing excellent stability.
내열성 평가로 락티카제이 바실러스 파라카제이 WiKim0136 균주를 40 ℃에서 1시간씩 처리한 결과, 97 %의 생존율을 나타내며 강한 내열성을 나타내었다.As a result of heat resistance evaluation, the Lacticajay Bacillus paracasei WiKim0136 strain was treated at 40°C for 1 hour, showing a survival rate of 97% and strong heat resistance.
이를 통해서, 락티카제이 바실러스 파라카제이 WiKim0136 균주는 산성, 담즙 및 고온의 환경에서도 생존율이 높았으며, 우수한 프로바이오틱스 효과를 나타내는 것을 확인할 수 있었다.Through this, it was confirmed that the Lacticajay Bacillus paracasei WiKim0136 strain had a high survival rate even in acidic, biliary, and high-temperature environments and exhibited excellent probiotic effects.
Claims (13)
[화학식 I]
[화학식 II]
.The composition of claim 4, wherein the composition comprises at least one compound selected from the group consisting of Formula I and Formula II below:
[Formula I]
[Formula II]
.
[화학식 I]
[화학식 II]
.The method of producing chiroinositol according to claim 8, wherein the chiroinositol is selected from the group consisting of Formula I and Formula II below.
[Formula I]
[Formula II]
.
서열번호 3의 16s rRNA 서열을 가지며 수탁번호 KCTC 15108BP로 기탁된 락티카제이 바실러스 파라카제이 WiKim0136 균주를 배지에서 배양하는 배양 단계; 및
배지에서 배양물을 분리하는 분리 단계.Method for producing chiroinositol comprising the following steps:
A culture step of culturing the Lacticajay Bacillus paracasei WiKim0136 strain, which has the 16s rRNA sequence of SEQ ID NO: 3 and deposited under the accession number KCTC 15108BP, in a medium; and
Separation step to separate the culture from the medium.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122189A1 (en) * | 2010-11-15 | 2012-05-17 | Hsia-Ching Hsiao | Lactobacillus Mutant |
| KR20140040509A (en) * | 2012-09-26 | 2014-04-03 | 주식회사 디와이내츄럴 | Method for preparing d- chiro-inositol by using the microorganism |
| KR20140078381A (en) * | 2012-12-17 | 2014-06-25 | 주식회사 디와이내츄럴 | Method for producing d-chiro-inositol by using the resting cell conversion |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120122189A1 (en) * | 2010-11-15 | 2012-05-17 | Hsia-Ching Hsiao | Lactobacillus Mutant |
| KR20140040509A (en) * | 2012-09-26 | 2014-04-03 | 주식회사 디와이내츄럴 | Method for preparing d- chiro-inositol by using the microorganism |
| KR20140078381A (en) * | 2012-12-17 | 2014-06-25 | 주식회사 디와이내츄럴 | Method for producing d-chiro-inositol by using the resting cell conversion |
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| Hwang, H. et al., 2021 한국생물공학회 추계학술발표대회 및 국제심포지엄, poster P0123 (2021.10.06.~09.)* * |
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