KR20210158580A - Novel Bifidobacterium longum TSB-L1 with xylooligosaccharide specific availability - Google Patents
Novel Bifidobacterium longum TSB-L1 with xylooligosaccharide specific availability Download PDFInfo
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- KR20210158580A KR20210158580A KR1020200077066A KR20200077066A KR20210158580A KR 20210158580 A KR20210158580 A KR 20210158580A KR 1020200077066 A KR1020200077066 A KR 1020200077066A KR 20200077066 A KR20200077066 A KR 20200077066A KR 20210158580 A KR20210158580 A KR 20210158580A
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- bifidobacterium longum
- tsb
- bifidobacterium
- xylooligosaccharide
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- 229940009291 bifidobacterium longum Drugs 0.000 title claims abstract description 88
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Abstract
Description
본 발명은 신규 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)에 관한 것으로, 더욱 상세하게는 자일로올리고당(xylooligosaccharide)을 특이적으로 이용할 수 있으면서 프로바이오틱스 소재로 사용가능한 신규 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)에 관한 것이다.The present invention relates to a novel Bifidobacterium longum TSB-L1 (KCTC 18825P), and more particularly, to a novel Bifidobacterium that can be used as a probiotic material while specifically using xylooligosaccharide Bifidobacterium longum TSB-L1 (KCTC 18825P).
프로바이오틱스(probiotics)는 건강에 이익을 제공하는 살아있는 미생물을 일컫는 것으로, 장내 균총에서 유해균을 억제하고 유익균을 증가시키는데 중요한 역할을 하게 된다. 대표적인 프로바이오틱스로는 유산균과 비피더스균(Bifidobacterium)이 있으며, 그 외 효모와 사상균 등이 있다. 유산균을 비롯한 세균들이 프로바이오틱스로 인정받기 위해서는 위산과 담즙산에서 살아남아 소장, 대장까지 도달하여 장에서 증식하고 정착하여야 하며 장관 내에서 유용한 효과를 나타내어야 하고, 독성이 없으며 비병원성이어야 한다.Probiotics refer to living microorganisms that provide health benefits, and play an important role in inhibiting harmful bacteria and increasing beneficial bacteria in the intestinal flora. Representative probiotics include lactic acid bacteria and Bifidobacterium, and other yeasts and filamentous fungi. In order for bacteria, including lactic acid bacteria, to be recognized as probiotics, they must survive gastric acid and bile acid, reach the small intestine and large intestine, multiply and settle in the intestine, exhibit useful effects in the intestine, and must be non-toxic and non-pathogenic.
한편, 올리고당은 소화흡수가 어려운 당류로서 현재 프락토올리고당, 대두올리고당, 말토올리고당, 갈락토올리고당, 이소말토올리고당, 자일로올리고당 등이 있다. 그 중 자일로올리고당(xylooligosaccharide)은 자일로오스가 β-1,4-결합으로 2 ~ 10개 이어진 올리고당으로 위, 소장에서 분해되지 않고 대장 내 세균에 의해 분해되는 난소화성 올리고당이다. 현재로서는 자일로올리고당을 자일로오스 단위로 분해하지 않고 특이적으로 이용할 수 있는 균주에 대한 연구가 미비한 실정이다. 이에 자일로올리고당을 효율적으로 이용하면서 프로바이오틱스 활성이 뛰어난 새로운 균주를 개발하는 것이 필요한 실정이다.On the other hand, oligosaccharides are saccharides that are difficult to digest and absorb, and currently include fructooligosaccharides, soybean oligosaccharides, maltooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and xylooligosaccharides. Among them, xylooligosaccharide is an oligosaccharide in which 2 to 10 xylose is linked by β-1,4-linkage, and is an indigestible oligosaccharide that is not degraded in the stomach or small intestine but is degraded by bacteria in the large intestine. Currently, studies on strains that can be specifically used without decomposing xylooligosaccharides into xylose units are insufficient. Accordingly, it is necessary to develop a new strain with excellent probiotic activity while efficiently using xylo-oligosaccharide.
자일로올리고당(xylooligosaccharide)을 특이적으로 이용할 수 있으면서 프로바이오틱스 소재로 사용가능한 신규 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P) 및 이를 유효성분으로 함유하는 조성물을 제공하고자 한다.An object of the present invention is to provide a novel Bifidobacterium longum TSB-L1 (KCTC 18825P) that can be used as a probiotic material while specifically using xylooligosaccharide and a composition containing the same as an active ingredient.
본 발명은 자일로올리고당 이용능이 우수한 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)을 제공한다.The present invention provides an excellent Bifidobacterium longum TSB-L1 (KCTC 18825P) with excellent xylooligosaccharide utilization.
한편, 본 발명에 있어서, 상기 비피도박테리움 롱검(Bifidobacterium longum)은, 바람직하게 내산성, 내담즙성, 장관세포 부착능 중 선택되는 하나 이상의 성질을 갖는 것일 수 있다.Meanwhile, in the present invention, the Bifidobacterium longum ( Bifidobacterium longum ) may preferably have one or more properties selected from acid resistance, bile resistance, and intestinal cell adhesion ability.
또한, 본 발명은 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)을 유효성분으로 함유하는 것을 특징으로 하는 식품 조성물을 제공한다.In addition, the present invention provides a food composition comprising Bifidobacterium longum TSB-L1 (KCTC 18825P) as an active ingredient.
본 발명에서 선별한 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 내산성, 내담즙성 및 장관세포 부착능이 우수하고, 자일로올리고당을 자일로오스 단위로 분해하지 않고 특이적으로 이용할 수 있다. Bifidobacterium longum TSB-L1 selected in the present invention has excellent acid resistance, bile resistance and intestinal cell adhesion, and can be specifically used without decomposing xylooligosaccharides into xylose units.
또한, 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 장내 유익균의 증가 및 유지에 탁월하여 장내 균총을 개선함으로써 장 건강에 도움을 줄 수 있다.In addition, the Bifidobacterium longum ( Bifidobacterium longum ) TSB-L1 of the present invention is excellent in increasing and maintaining beneficial intestinal bacteria, thereby improving the intestinal flora, thereby contributing to intestinal health.
도 1은 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 16s rRNA 유전자 염기서열 분석 결과를 나타낸 사진이다.
도 2는 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 자일로올리고당 이용률을 나타낸 결과 그래프이다. 비교를 위해 다른 비피도박테리움 균주를 사용하였다.1 is a photograph showing the 16s rRNA gene sequencing result of Bifidobacterium longum TSB-L1 of the present invention.
Figure 2 is a graph showing the results of the xylo-oligosaccharide utilization of Bifidobacterium longum (Bifidobacterium longum) TSB-L1 of the present invention. Another Bifidobacterium strain was used for comparison.
프로바이오틱스(probiotics)는 건강에 이익을 제공하는 살아있는 미생물을 일컫는 것으로, 장내 균총에서 유해균을 억제하고 유익균을 증가시키는데 중요한 역할을 하게 된다. 프로바이오틱스로 인정받기 위해서는 위산과 담즙산에서 살아남아 소장, 대장까지 도달하여 장에서 증식하고 정착하여야 하며 장관 내에서 유용한 효과를 나타내어야 하고, 독성이 없으며 비병원성이어야 한다.Probiotics refer to living microorganisms that provide health benefits, and play an important role in inhibiting harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as a probiotic, it must survive gastric acid and bile acid, reach the small intestine and large intestine, multiply and settle in the intestine, exhibit useful effects in the intestine, and must be non-toxic and non-pathogenic.
한편, 자일로올리고당(xylooligosaccharide)은 자일로오스가 β-1,4-결합으로 2 ~ 10개 이어진 올리고당으로 위, 소장에서 분해되지 않고 대장 내 세균에 의해 분해되는 난소화성 올리고당이다. 현재로서는 자일로올리고당을 자일로오스 단위로 분해하지 않고 특이적으로 이용할 수 있는 균주에 대한 연구가 미비한 실정이다. 이에 자일로올리고당을 효율적으로 이용하면서 프로바이오틱스 활성이 뛰어난 새로운 균주를 개발하는 것이 필요한 실정이다.On the other hand, xylooligosaccharide is an oligosaccharide in which 2 to 10 xylose is linked by β-1,4-linkage, and is an indigestible oligosaccharide that is not degraded in the stomach and small intestine but is degraded by bacteria in the large intestine. Currently, studies on strains that can be specifically used without decomposing xylooligosaccharides into xylose units are insufficient. Accordingly, it is necessary to develop a new strain with excellent probiotic activity while efficiently using xylo-oligosaccharide.
이에 본 발명은 자일로올리고당 이용능이 우수한 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)을 제공한다.Accordingly, the present invention provides a Bifidobacterium longum TSB-L1 (KCTC 18825P) having excellent xylooligosaccharide utilization.
본 발명에서는 건강한 성인의 분변으로부터 균주를 선별하고자, 자일로올리고당을 함유한 배지에 접종하여 자일로올리고당을 선택적으로 가장 빠르게 이용하는 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1을 선별, 분리하였다.In the present invention, in order to select a strain from the feces of healthy adults, Bifidobacterium longum TSB-L1 was selected and isolated by inoculating a medium containing xylooligosaccharide and selectively using xylooligosaccharide the fastest.
한편, 본 발명에 있어서, 상기 비피도박테리움 롱검(Bifidobacterium longum)은, 바람직하게 내산성, 내담즙성, 장관세포 부착능 중 선택되는 하나 이상의 성질을 갖는 것일 수 있다.Meanwhile, in the present invention, the Bifidobacterium longum ( Bifidobacterium longum ) may preferably have one or more properties selected from acid resistance, bile resistance, and intestinal cell adhesion ability.
한편, 하기 실험에 의하면, 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 다른 비피도박테리움 균주들에 비해 자일로올리고당의 이용률이 현저하게 뛰어나는 것을 확인하였고, 다른 비피도박테리움 균주들에 비해 내산성, 내담즙성 및 장관세포 부착능도 매우 뛰어나다는 것을 확인하였다. 또한, 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1과 자일로올리고당을 함유한 설탕의 섭취는 장내 유익균의 증가를 유도하면서, 유익균의 증식을 지속적으로 유지시켰다. 이를 통해 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 자일로올리고당 이용능이 우수한 프로바이오틱스로 소재로서 활용 가능성을 확인할 수 있었다.On the other hand, according to the following experiment, it was confirmed that the Bifidobacterium longum TSB-L1 of the present invention has significantly superior utilization of xylooligosaccharide compared to other Bifidobacterium strains, and other Bifidobacterium It was confirmed that the acid resistance, bile resistance, and intestinal cell adhesion ability were also very excellent compared to the lysium strains. In addition, the intake of sugar containing Bifidobacterium longum TSB-L1 and xylooligosaccharide of the present invention induces an increase in beneficial bacteria in the intestine, while continuously maintaining the growth of beneficial bacteria. Through this, it was confirmed that the Bifidobacterium longum TSB-L1 of the present invention could be used as a material as a probiotic with excellent xylo-oligosaccharide utilization.
또한, 본 발명은 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P)을 유효성분으로 함유하는 것을 특징으로 하는 식품 조성물을 제공한다.In addition, the present invention provides a food composition comprising Bifidobacterium longum TSB-L1 (KCTC 18825P) as an active ingredient.
한편, 본 발명의 식품 조성물에 있어서, 상기 식품 조성물은, 일예로 일반 음료, 빵류, 스낵류, 과자류, 젤리, 아이스크림류, 알코올성 음료, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나의 제형이거나, 이와 같은 제형에 첨가될 수 있다.On the other hand, in the food composition of the present invention, the food composition is, for example, a formulation selected from general beverages, breads, snacks, confectionery, jelly, ice cream, alcoholic beverages, vitamin complexes, and other health supplements, or , can be added to such formulations.
이하, 본 발명의 내용을 하기 실시예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the content of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of technical ideas equivalent thereto.
[실시예 1: 본 발명의 비피도박테리움 롱검 TSB-L1 선별 및 분리동정][Example 1: Selection and isolation of Bifidobacterium longum TSB-L1 of the present invention]
1) 자일로올리고당 특화 균주 비피도박테리움 롱검 TSB-L1 균주 선별1) Selection of xylo-oligosaccharide-specialized strain Bifidobacterium longum TSB-L1 strain
건강한 성인의 분변을 멸균 혐기수에 연속 희석한 후 각 희석액을 MRS 고체 배지에 부어서 혐기 조건으로 37℃에서 3일간 배양하였다. 이후, 생성된 콜로니를 다시 BL 배지에 BCP(Bromocresol purple, 0.17g/L)를 첨가한 비피도박테리움 선택배지에서 골라낸 후 같은 조건에서 3일간 배양하였다. 자일로올리고당을 함유한 프리바이오틱 최소한천배지 (2% agar, 0.5% peptone, 0.5% Sodium acetate, 0.02% MgSO4·7H2O, 0.1% tween 80, 0.1% Monopotassium phosphate, 0.3% Dipotassium phosphate, 0.05% Cysteine hydrochloride, 0.6% 자일로올리고당)에 접종 후 최종적으로 자일로올리고당을 선택적으로 가장 빠르게 이용하는 비피도박테리움 'TSB-L1'을 선별하였다. 상기 선별한 균체를 MRS 액체배지 : 글리세롤(Glycerol)이 4 : 1 비율로 포함된 냉동보관용액(Freezing solution)에 1 ml 첨가한 뒤, 크라이오튜브(Cryo-tube)에 넣어 -70℃에서 냉동보관 후 이후의 테스트를 위한 스타터로 사용하였다.The feces of healthy adults were serially diluted in sterile anaerobic water, and then each dilution was poured into MRS solid medium and cultured at 37°C for 3 days under anaerobic conditions. Thereafter, the generated colonies were again picked from the Bifidobacterium selective medium supplemented with BCP (Bromocresol purple, 0.17 g/L) in the BL medium, and then cultured for 3 days under the same conditions. Prebiotic minimal medium containing xylooligosaccharide (2% agar, 0.5% peptone, 0.5% Sodium acetate, 0.02% MgSO 4 7H 2 O, 0.1% tween 80, 0.1% Monopotassium phosphate, 0.3% Dipotassium phosphate, After inoculation with 0.05% Cysteine hydrochloride, 0.6% xylo-oligosaccharide), Bifidobacterium 'TSB-L1', which selectively and fastest uses xylo-oligosaccharide, was selected. After adding 1 ml of the selected cells to a freezing solution containing MRS liquid medium: glycerol in a 4 : 1 ratio, it is put into a cryo-tube and frozen at -70°C. After storage, it was used as a starter for subsequent testing.
2) 자일로올리고당 특화 균주 비피도박테리움 롱검 TSB-L1 균주의 동정2) Identification of the xylo-oligosaccharide-specific strain Bifidobacterium longum TSB-L1 strain
상기에서 선별한 균주 TSB-L1의 동정을 위하여 16S rRNA 유전자 분석을 실시하였다. 선별 균주 TSB-L1을 MRS 액체배지에 접종하여 37℃에서 24시간 배양한 후 원심분리하여 균체를 회수하고 Accuprep 지놈 추출 키트(Bioneer, Korea)를 이용하여 지놈 DNA를 추출하였다. 추출한 DNA는 유니버셜 프라이머인 27F 및 1492R 프라이머로 합성하여 유전자 단편을 증폭시켰고 코스모진텍에 의뢰하여 염기서열을 분석한 후, 결과를 NCBI(National Center for Biotechnology Information, Bethesda, MD, USA)의 BLAST를 사용하여 GeneBank에 등록된 염기서열과 상동성을 비교하였다. 사용한 16S rRNA 염기서열은 하기 표 1과 같이 나타냈다.16S rRNA gene analysis was performed to identify the strain TSB-L1 selected above. Selected strain TSB-L1 was inoculated in MRS broth, cultured at 37°C for 24 hours, centrifuged to recover cells, and genomic DNA was extracted using Accuprep genome extraction kit (Bioneer, Korea). The extracted DNA was synthesized with the universal primers 27F and 1492R primers to amplify the gene fragment. After analyzing the nucleotide sequence at Cosmogene Tech, the results were analyzed by BLAST of NCBI (National Center for Biotechnology Information, Bethesda, MD, USA). was used and compared with the nucleotide sequence registered in GeneBank for homology. The 16S rRNA base sequence used is shown in Table 1 below.
분석 결과, 도 1과 같이 선별 균주 TSB-L1은 비피도박테리움 롱검(Bifidobacterium longum)과 100% 일치하는 것을 확인할 수 있었다. As a result of the analysis, it was confirmed that the selected strain TSB-L1 was 100% consistent with Bifidobacterium longum as shown in FIG. 1 .
이에 본 발명의 발명자들은 선별 균주를 새로운 '비피도박테리움 롱검(Bifidobacterium longum) TSB-L1'로 명명하였고, 한국생명공학연구원 생물자원센터에서 2020년 6월 15일자로 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (기탁번호: KCTC18825P)의 기탁이 승인되었다.The present inventors have selected strains of new 'Bifidobacterium ronggeom (Bifidobacterium longum) TSB-L1' was named, Korea Research Institute of Bioscience and Biotechnology BP in biological resource centers to June 15th 2020 betting Te Solarium ronggeom (Bifidobacterium longum ) TSB-L1 (Accession No.: KCTC18825P) was approved.
[실험예 1: 신규 비피도박테리움 롱검 TSB-L1의 자일로올리고당 이용능력 측정][Experimental Example 1: Measurement of xylo-oligosaccharide utilization ability of novel Bifidobacterium longum TSB-L1]
본 실험예에서는 선별 균주 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 자일로올리고당 이용능력을 평가하고자 하였다.In this experimental example, the xylo -oligosaccharide utilization ability of the selected strain Bifidobacterium longum TSB-L1 was evaluated.
프리바이오틱 최소배지(0.5% peptone, 0.5% Sodium acetate, 0.02% MgSO4·7H2O, 0.1% tween 80, 0.1% Monopotassium phosphate, 0.3% Dipotassium phosphate, 0.05% Cysteine hydrochloride, 1.0% 자일로올리고당)에 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 배양액을 2% 접종한 다음 37℃에서 48시간 동안 혐기 조건으로 배양 후 600nm에서 흡광도 및 HPLC를 통해 자일로올리고당의 선택적 이용능력을 확인하였다. 비교를 위해 표준균주인 비피도박테리움 락티스(Bifidobacterium lactis) KCTC5854, 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128, 비피도박테리움 비피덤(Bifidobacterium bifidum) KCTC3202, 비피도박테리움 브레브(Bifidobacterium breve) KCTC3220와 이미 제품화된 상용균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 대해서도 동일하게 실험을 수행하였다.Prebiotic minimal medium (0.5% peptone, 0.5% Sodium acetate, 0.02% MgSO 4 7H 2 O, 0.1% tween 80, 0.1% Monopotassium phosphate, 0.3% Dipotassium phosphate, 0.05% Cysteine hydrochloride, 1.0% xylooligosaccharide) Bifidobacterium longum ( Bifidobacterium longum ) After inoculating 2% of a culture solution of TSB-L1, incubated in anaerobic conditions at 37 ° C. for 48 hours, absorbance at 600 nm and the selective availability of xylooligosaccharides were confirmed through HPLC. For comparison, the standard strain Bifidobacterium lactis ( Bifidobacterium lactis ) KCTC5854, Bifidobacterium ronggeom (Bifidobacterium longum) KCTC3128, Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum) KCTC3202, Bifidobacterium breather bracket (Bifidobacterium breve) KCTC3220 and already in the commercialized commercial strain Bifidobacterium ronggeom (Bifidobacterium longum ) The same experiment was performed for Customprobiotics (USA).
실험결과, 표 2와 같이 대조군과 비교하여 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1이 자일로올리고당을 가수분해하여 축적된 자일로오스(xylose) 없이 자일로올리고당을 선택적으로 가장 우수하게 이용하였음을 확인하였다. 이에 반해 비피도박테리움 락티스(Bifidobacterium lactis) KCTC5854와 비피도박테리움 비피덤(Bifidobacterium bifidum) KCTC3202의 경우 자일로올리고당을 가수분해하여 축적된 자일로오스가 배양액에 축적되어 기질을 선택적으로 이용하지 못하는 것으로 추정되었다. As shown in Table 2, compared with the control group, Bifidobacterium longum TSB-L1 hydrolyzes xylo-oligosaccharide to selectively use xylo-oligosaccharide without accumulated xylose. It was confirmed that In contrast, Bifidobacterium lactis In the case of KCTC5854 and Bifidobacterium bifidum KCTC3202, it was estimated that the xylose accumulated by hydrolysis of xylooligosaccharide was accumulated in the culture medium and thus the substrate could not be selectively used.
균주
strain
OD600
OD 600
a : xylose, b: xylobiose, c : xylotriose, d: xylotetraosea: xylose, b: xylobiose, c: xylotriose, d: xylotetraose
또한, 자일로올리고당 이용률을 측정한 결과, 도 2와 같이 다른 비피도박테리움 균주들의 경우 5.8 ~ 25.7% 사이의 자일로올리고당 이용률인 것에 반해 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1이 53%로 현저하게 우수한 것을 확인하였다.In addition, as a result of measuring the xylo-oligosaccharide utilization rate, as shown in FIG. 2, in the case of other Bifidobacterium strains, the xylo-oligosaccharide utilization rate was between 5.8 and 25.7%, whereas Bifidobacterium longum TSB-L1 had 53 %, it was confirmed that it was remarkably excellent.
[실험예 2: 신규 비피도박테리움 롱검 TSB-L1의 내산성 및 내담즙성 측정][Experimental Example 2: Measurement of acid resistance and bile resistance of novel Bifidobacterium longum TSB-L1]
본 실험예에서는 선별 균주 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 내산성 및 내담즙성을 평가하고자 하였다. 비교를 위해 표준균주인 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128과 상업균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 대해서 동일하게 실험을 수행하였다.In this experimental example, the acid resistance and bile resistance of the selected strain Bifidobacterium longum TSB-L1 were evaluated. For comparison, the same experiment was performed with the standard strain Bifidobacterium longum KCTC3128 and the commercial strain Bifidobacterium longum Customprobiotics (USA).
먼저 내산성 실험은 소화관 조건과 유사한 환경에서 측정하기 위해 pH 3.0으로 조정된 MRS 배지에 최종농도가 1,000 U/ml이 되도록 펩신을 첨가한 인공 위액에서 실시하였다. MRS 액체 배지에서 37℃에서 18시간 동안 배양한 균체를 원심분리하여 균주를 침전시키고, 다시 멸균 식염수(0.85% NaCl)로 2회 세척 후 균체 현탁액을 대조균 배지와 인공 위액배지에 각각 약 107 CFU/ml 수준으로 접종시키고, 37℃에서 배양시키면서 배양 초기 및 3시간 후에 생존 균수를 측정하였다. 총균수는 KH2PO4, Na2HPO4, Cystein hydrochloride, tween 80 등이 함유된 인산완충액(pH 6.8)으로 희석하여 측정하였고, 내산성은 '초기 생균수/pH 처리후 생균수'의 비율로 나타내었다. 그 결과, 표 3과 같이 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 생리적 pH와 가까운 pH 3.0의 강산성으로 3시간 처리하여도 90% 이상의 생존율을 나타내었다. 이는 표준균주 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128뿐만 아니라 상용균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 비해서도 더 나은 내산성을 나타내는 결과였다. First, the acid resistance test was performed in artificial gastric juice in which pepsin was added to a final concentration of 1,000 U/ml in MRS medium adjusted to pH 3.0 to measure in an environment similar to the conditions of the digestive tract. The strains were precipitated by centrifugation of the cells cultured at 37°C for 18 hours in MRS liquid medium, washed twice with sterile saline (0.85% NaCl) again, and the cell suspension was placed in a control medium and artificial gastric juice medium, respectively, about 10 7 Inoculated at a CFU/ml level, and incubated at 37° C., the number of viable bacteria was measured at the beginning of the culture and after 3 hours. The total number of bacteria was measured by diluting it with a phosphate buffer solution (pH 6.8) containing KH 2 PO 4 , Na 2 HPO 4 , Cystein hydrochloride, tween 80, etc. indicated. As a result, as shown in Table 3, Bifidobacterium longum TSB-L1 exhibited a survival rate of 90% or more even after 3 hours of treatment with a strong acidity of pH 3.0 close to physiological pH. This was a result showing better acid resistance than the standard strain Bifidobacterium longum KCTC3128 as well as the commercial strain Bifidobacterium longum Customprobiotics (USA).
다음으로, 내담즙성 실험은 인공 담즙에서 시행하였고, 상기 내산성 평가에서 사용된 MRS 액체 배지에 황소 담즙을 0.1, 0.3, 0.5% 첨가한 후, 상기 내산성 평가방법과 동일한 방법으로 하여 유산균을 접종 한 후 생장여부를 확인하였다. 그 결과, 표 3과 같이 장관 내 담즙산염 농도는 0.1% 내외인데 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 0.3%의 담즙이 첨가되어도 생육이 가능하여 매우 우수한 내담즙성을 나타내는 것을 확인하였다. 이는 표준균주 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128에 비해서 매우 우수하며, 상용균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 대해 유사한 수준의 내담즙성을 나타내는 결과였다. Next, the bile tolerance test was performed in artificial bile, and after adding 0.1, 0.3, 0.5% of bull bile to the MRS liquid medium used in the acid resistance evaluation, lactic acid bacteria were inoculated in the same manner as the acid resistance evaluation method. After growth was confirmed. As a result, as shown in Table 3, although the concentration of bile salts in the intestinal tract is around 0.1%, it was confirmed that Bifidobacterium longum TSB-L1 was able to grow even when 0.3% of bile was added, showing very good bile resistance. did This was very superior to the standard strain Bifidobacterium longum KCTC3128, and it was a result of showing a similar level of bile resistance to the commercial strain Bifidobacterium longum Customprobiotics (USA).
상기와 같은 결과를 통해 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 상용균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 비해서도 더 나은 내산성과 유사한 수준의 내담즙성을 가지고 있어 장까지 도달할 확률이 매우 높기 때문에, 식품 또는 건강기능식품으로 섭취할 경우 유산균의 다양한 효능을 효과적으로 발휘할 수 있다.Through the above results, Bifidobacterium longum TSB-L1 has better acid resistance and similar level of bile resistance compared to the commercial strain Bifidobacterium longum Customprobiotics (USA). Since the probability of reaching the intestine is very high, various effects of lactic acid bacteria can be effectively exhibited when consumed as food or health functional food.
[실험예 3: 신규 비피도박테리움 롱검 TSB-L1의 장관세포 부착능 측정][Experimental Example 3: Measurement of intestinal cell adhesion ability of novel Bifidobacterium longum TSB-L1]
본 실험예에서는 선별 균주 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 장관세포 부착능을 평가하고자 하였다. 비교를 위해 표준균주인 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128과 상업균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 대해서 동일하게 실험을 수행하였다.In this experimental example, it was attempted to evaluate the intestinal cell adhesion ability of the selected strain Bifidobacterium longum TSB-L1. For comparison, the same experiment was performed with the standard strain Bifidobacterium longum KCTC3128 and the commercial strain Bifidobacterium longum Customprobiotics (USA).
장 상피세포 부착능 시험을 위한 동물세포로서 HT-29를 한국 세포주은행(KCLB)에서 분양받아 사용하였으며, 열 비활성화된 10% 우태아혈청(FBS), 1% L-글루타민, 페니실린 G(100 IU/mL), 그리고 스트렙토마이신(100 mg/mL)이 첨가된 RPMI 1640(Welgene, KOREA) 배지를 이용하여 5% CO2 존재 하에서 37℃에서 배양하였다. HT-29 세포는 1.0 x 105 CFU/mL의 수가 되도록 24 well-plate에 분주하였고 격일로 배지를 교환하며 완전하게 단일층을 형성할 때까지 배양하였다. 완전 단일층을 형성한 HT-29 세포에 PBS 완충용액을 이용하여 3회 세척하고 항생제가 첨가되지 않은 RPMI 1640 배지 0.5 mL를 첨가하였다. 각각 약 1.0 x 109 CFU/mL의 농도가 되도록 RPMI 1640에 현탁한 다음 상기 well-plate에 접종하고, 5% CO2 존재 하에서 37℃에서 2시간 배양을 실시하고 PBS 완충용액을 사용하여 3회 세척을 실시하였다. 세척이 완료된 후 멸균된 스크래퍼(Scraper)를 이용하여 부착되어 있는 세포를 떼어내고 펩톤수를 이용하여 연속희석법으로 MRS-agar에 평판 도말하고 37℃에서 24 시간동안 배양하여 균수를 측정하였다. As animal cells for intestinal epithelial cell adhesion test, HT-29 was purchased from the Korea Cell Line Bank (KCLB), and heat-inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, and penicillin G (100 IU). /mL), and streptomycin (100 mg/mL) was added using RPMI 1640 (Welgene, KOREA) medium and cultured at 37° C. in the presence of 5% CO 2 . HT-29 cells were seeded in a 24 well-plate so as to have a number of 1.0 x 10 5 CFU/mL, and the medium was exchanged every other day and cultured until a complete monolayer was formed. The HT-29 cells formed a complete monolayer were washed three times with PBS buffer, and 0.5 mL of RPMI 1640 medium without antibiotics was added. Each was suspended in RPMI 1640 to a concentration of about 1.0 x 10 9 CFU/mL, and then inoculated in the well-plate, incubated for 2 hours at 37° C. in the presence of 5% CO 2 and 3 times using PBS buffer. Washing was carried out. After washing was completed, the adherent cells were removed using a sterile scraper, spread on MRS-agar by continuous dilution using peptone water, and cultured at 37°C for 24 hours to measure the number of bacteria.
그 결과, 표 4와 같이 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 표준균주 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128와 상용균주인 비피도박테리움 롱검(Bifidobacterium longum) Customprobiotics (USA)에 비해 2시간 경과후, 장 상피세포 부착능이 우수한 것으로 나타났으며, 특히 비피도박테리움 롱검(Bifidobacterium longum) KCTC3128에 비해서는 현저히 높은 것으로 나타났다. As a result, as shown in Table 4, Bifidobacterium longum TSB-L1 was added to the standard strain Bifidobacterium longum KCTC3128 and the commercial strain Bifidobacterium longum Customprobiotics (USA). After 2 hours, the intestinal epithelial cell adhesion was excellent, and in particular, it was significantly higher than that of Bifidobacterium longum KCTC3128.
이를 일반적으로 미생물의 농도를 나타내는 로그 값을 취해 부착률을 계산해본 결과, 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 약 82%[(8.91 Log/7.30 Log) x 100], 2차 결과는 약 84%[(8.94 Log/7.49 Log) x 100]로 다른 비피도박테리움에 비해 매우 높은 수준이었으며, 이를 통해 본 발명의 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1은 장 상피세포에 부착하여 장내 환경을 개선하는 것에 탁월함을 확인할 수 있었다.As a result of calculating the adhesion rate by taking a log value representing the concentration of microorganisms in general, Bifidobacterium longum TSB-L1 is about 82% [(8.91 Log/7.30 Log) x 100], the secondary result was about 84% [(8.94 Log / 7.49 Log) x 100], which was very high compared to other Bifidobacterium, and through this, Bifidobacterium longum TSB-L1 of the present invention was applied to intestinal epithelial cells. It was confirmed that it was excellent in improving the intestinal environment by attaching it.
(%)adhesion rate
(%)
(CFU/mL)live cell count
(CFU/mL)
(CFU/mL)live cell count
(CFU/mL)
[실험예 4: 신규 비피도박테리움 롱검 TSB-L1의 인체 내 효능 평가][Experimental Example 4: Evaluation of Efficacy in the Human Body of Novel Bifidobacterium Longum TSB-L1]
본 실험예에서는 선별 균주 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1의 인체 내 효능을 평가하고자 하였다.In this experimental example, the efficacy of the selected strain Bifidobacterium longum TSB-L1 in the human body was evaluated.
국제 임상시험기준에 준하여 임상시험 실시 기관인 국민대학교 IRB 심의절차를 거쳐 승인(KMU2019-209)을 받은 후, 건강한 성인 남녀 25명를 대상으로 하였다. In accordance with international clinical trial standards, 25 healthy adult males and females were subject to approval (KMU2019-209) after going through the deliberation process of Kookmin University IRB, a clinical trial institution.
사용된 프로바이오틱스 균주는 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1로 제조원 ㈜씨티씨바이오에서 위탁 생산하였고 1일 섭취량 (10,000,000,000 CFU/일)은 건강기능식품공전에 제시된 1일 섭취량의 범주에 해당하는 함량으로 제공하였다. 사용된 프리바이오틱스는 대한제당㈜에서 판매하고 있는 자일로올리고당 함유 설탕으로 프리바이오틱스 10g을 통한 자일로올리고당 섭취량은 1일 0.7 g으로 건강기능식품의 개별인정형 원료로 허용된 1일 최대 섭취량 보다 낮은 안전한 수준으로 제공하였다. The probiotic strain used was Bifidobacterium longum TSB-L1, which was consigned to the manufacturer CTC Bio, and the daily intake (10,000,000,000 CFU/day) corresponds to the category of daily intake presented in the Health Functional Foods Ordinance. content was provided. The prebiotic used is xylo-oligosaccharide-containing sugar sold by Daehan Jedang Co., Ltd. The intake of xylo-oligosaccharide through 10 g of prebiotics is 0.7 g per day. provided at a lower safe level.
시료 섭취 방법에 따라 신바이오틱스(프로바이오틱스+프리바이오틱스)를 2주 섭취 후, 프리바이오틱스만을 4주 더 섭취하여 총 6주간 섭취하는 군을 BL2XO6군으로, 신바이오틱스(프로바이오틱스+프리바이오틱스)를 2주간 섭취하는 군은 BL2XO2군으로 분류하였고 배변 수거는 모든 실험군에서 시료 섭취 전, 시료 섭취 후 2주, 4주, 6주 총 4회에 걸쳐 수거하였다. 균총분석은 KRIBS 한국의과학연구원(대전, 대한민국)에 의뢰하였고 비피도박테리움 속(Bifidobacterium spp.), 클로스트리디움 속(Clostridium spp.), 비피도박테리움 롱검(Bifidobacterium longum)을 분석하였다.According to the sample intake method, the group that consumes synbiotics (probiotics + prebiotics) for 2 weeks, then consumes only prebiotics for 4 more weeks for a total of 6 weeks is the BL2XO6 group, and synbiotics (probiotics + prebiotics) ) was classified into the BL2XO2 group, and defecation was collected 4 times in all experimental groups before and 2 weeks, 4 weeks, and 6 weeks after sample intake. The flora analysis was requested by KRIBS Korea Institute of Medical Sciences (Daejeon, Korea), and Bifidobacterium spp. , Clostridium spp. , and Bifidobacterium longum were analyzed.
그 결과, 표 5와 같이 신바이오틱스 섭취 후 비피도박테리움 롱검(Bifidobacterium longum)은 증식되었고, 유해균인 클로스트리디움 속(Clostridium spp.)의 증식은 억제되었으며 이후 자일로올리고당만을 섭취하는 BL2XO6군에서 비피도박테리움(Bifidobacterium)은 지속적으로 증가하는 양상을 보였으며 유해균인 클로스트리디움 속(Clostridium spp.)의 증식은 둔화되었다. As a result, as shown in Table 5, after ingestion of synbiotics, Bifidobacterium longum was proliferated, and the proliferation of harmful bacteria, Clostridium spp. In Bifidobacterium ( Bifidobacterium ) showed a continuous increase, and the growth of the harmful bacteria Clostridium spp. was slowed.
반면, 신바이오틱스 섭취후 자일로올리고당을 섭취하지 않은 BL2XO2군의 경우 프로바이오틱스의 영향력이 지속되면서 비피도박테리움 롱검(Bifidobacterium longum)은 증가하였으나 그 증가율이 BL2XO6군에 비해 낮았으며, 비피도박테리움(Bifidobacterium)은 시료 섭취전보다도 감소하는 추이를 보였다. On the other hand, in the BL2XO2 group, which did not consume xylooligosaccharide after ingestion of synbiotics, the effect of probiotics continued and Bifidobacterium longum increased, but the rate of increase was lower than that of the BL2XO6 group, and Bifidobacterium ( Bifidobacterium ) showed a decreasing trend compared to before sample intake.
이를 통해 본 발명의 유산균 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1과 자일로올리고당을 함유한 설탕의 섭취는 장내 유익균의 증가를 유도할 뿐만 아니라, 자일로올리고당 함유 설탕만을 섭취하는 기간 동안에도 비피도박테리움 롱검(Bifidobacterium longum)과 같은 유익균의 증식을 지속적으로 유지시켜 장 건강에 도움을 줄 수 있을 것으로 판단되었다. Through this, ingestion of sugar containing the lactic acid bacterium Bifidobacterium longum TSB-L1 and xylooligosaccharide of the present invention not only induces an increase in beneficial intestinal bacteria, but also during the period when only xylooligosaccharide-containing sugar is consumed. It was determined that it could help intestinal health by continuously maintaining the growth of beneficial bacteria such as Bifidobacterium longum.
기탁기관명 : 한국생명공학연구원Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC18825PAccession number: KCTC18825P
수탁일자 : 20200615Deposit date: 20200615
<110> TS Corporation
<120> Novel Bifidobacterium longum TSB-L1 with xylooligosaccharide
specific availability
<130> YP-20-087
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1214
<212> RNA
<213> Bifidobacterium longum
<400> 1
gaagacctga cccatacacc ggaatagctc ctggaaacgg gtggtaatgc cggatgctcc 60
agttgatcgc atggtcttct gggaaagctt tcgcggtatg ggatggggtc gcgtcctatc 120
agcttgacgg cggggtaacg gcccaccgtg gcttcgacgg gtagccggcc tgagagggcg 180
accggccaca ttgggactga gatacggccc agactcctac gggaggcagc agtggggaat 240
attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt gagggatgga ggccttcggg 300
ttgtaaacct cttttatcgg ggagcaagcg agagtgagtt tacccgttga ataagcaccg 360
gctaactacg tgccagcagc cgcggtaata cgtagggtgc aagcgttatc cggaattatt 420
gggcgtaaag ggctcgtagg cggttcgtcg cgtccggtgt gaaagtccat cgcttaacgg 480
tggatccgcg ccgggtacgg gcgggcttga gtgcggtagg ggagactgga attcccggtg 540
taacggtgga atgtgtagat atcgggaaga acaccaatgg cgaaggcagg tctctgggcc 600
gttactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 660
cacgccgtaa acggtggatg ctggatgtgg ggcccgttcc acgggttccg tgtcggagct 720
aacgcgttaa gcatcccgcc tggggagtac ggccgcaagg ctaaaactca aagaaattga 780
cgggggcccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta 840
cctgggcttg acatgttccc gacggtcgta gagatacggc ttcccttcgg ggcgggttca 900
caggtggtgc atggtcgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 960
agcgcaaccc tcgccccgtg ttgccagcgg attatgccgg gaactcacgg gggaccgccg 1020
gggttaactc ggaggaaggt ggggatgacg tcagatcatc atgcccctta cgtccagggc 1080
ttcacgcatg ctacaatggc cggtacaacg ggatgcgacg cggcgacgcg gagcggatcc 1140
ctgaaaaccg gtctcagttc ggatcgcagt ctgcaactcg actgcgtgaa ggcggagtcg 1200
ctagtaatcg cgaa 1214
<110> TS Corporation
<120> Novel Bifidobacterium longum TSB-L1 with xylooligosaccharide
specific availability
<130> YP-20-087
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1214
<212> RNA
<213> Bifidobacterium longum
<400> 1
gaagacctga cccatacacc ggaatagctc ctggaaacgg gtggtaatgc cggatgctcc 60
agttgatcgc atggtcttct gggaaagctt tcgcggtatg ggatggggtc gcgtcctatc 120
agcttgacgg cggggtaacg gcccaccgtg gcttcgacgg gtagccggcc tgagagggcg 180
accggccaca ttgggactga gatacggccc agactcctac gggaggcagc agtggggaat 240
attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt gagggatgga ggccttcggg 300
ttgtaaacct cttttatcgg ggagcaagcg agagtgagtt tacccgttga ataagcaccg 360
gctaactacg tgccagcagc cgcggtaata cgtagggtgc aagcgttatc cggaattatt 420
gggcgtaaag ggctcgtagg cggttcgtcg cgtccggtgt gaaagtccat cgcttaacgg 480
tggatccgcg ccgggtacgg gcgggcttga gtgcggtagg ggagactgga attcccggtg 540
taacggtgga atgtgtagat atcgggaaga acaccaatgg cgaaggcagg tctctgggcc 600
gttactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 660
cacgccgtaa acggtggatg ctggatgtgg ggcccgttcc acgggttccg tgtcggagct 720
aacgcgttaa gcatcccgcc tggggagtac ggccgcaagg ctaaaactca aagaaattga 780
cgggggcccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta 840
cctgggcttg acatgttccc gacggtcgta gagatacggc ttcccttcgg ggcgggttca 900
caggtggtgc atggtcgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 960
agcgcaaccc tcgccccgtg ttgccagcgg attatgccgg gaactcacgg gggaccgccg 1020
gggttaactc ggaggaaggt ggggatgacg tcagatcatc atgcccctta cgtccagggc 1080
ttcacgcatg ctacaatggc cggtacaacg ggatgcgacg cggcgacgcg gagcggatcc 1140
ctgaaaaccg gtctcagttc ggatcgcagt ctgcaactcg actgcgtgaa ggcggagtcg 1200
Claims (3)
Bifidobacterium longum TSB-L1 (KCTC 18825P) with excellent xylooligosaccharide utilization.
상기 비피도박테리움 롱검(Bifidobacterium longum)은,
내산성, 내담즙성, 장관세포 부착능 중 선택되는 하나 이상의 성질을 갖는 것을 특징으로 하는 비피도박테리움 롱검(Bifidobacterium longum) TSB-L1 (KCTC 18825P).
According to claim 1,
The Bifidobacterium longum ( Bifidobacterium longum ) is,
Bifidobacterium longum TSB-L1 (KCTC 18825P), characterized in that it has one or more properties selected from acid resistance, bile resistance, and intestinal cell adhesion ability.
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Title |
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대한민국 등록특허 제10-1595014호(등록일자: 2016.02.11)에는, 갈락토올리고당 생성 유산균주를 이용한 발효유의 제조방법에 관한 것으로, 원유에 L.reuteri 및 상업적 스타터를 동시에 접종하여 발효시키는 것을 특징으로 하는 갈락토올리고당 생성 유산균주를 이용한 발효유의 제조방법에 관한 것이다. |
류보경 등, Kor. J. Microbiol. Biotechnol., 제30권, 제4호, 380-387면(2002)* * |
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