KR102567095B1 - Novel Lactobacillus sp. strain isolated from Sorbaria sorbifolia var. stellipila Maxim. - Google Patents
Novel Lactobacillus sp. strain isolated from Sorbaria sorbifolia var. stellipila Maxim. Download PDFInfo
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- 241000186610 Lactobacillus sp. Species 0.000 title claims abstract description 14
- 241001335386 Sorbaria sorbifolia var. stellipila Species 0.000 title description 2
- 239000000203 mixture Substances 0.000 claims description 52
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- 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
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- C12N1/205—Bacterial isolates
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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Abstract
본 발명의 신규 락토바실러스 종(Lactobacillus sp.) SSD-9 균주는 쉬땅나무에서 분리된 균주로서, 수탁번호 KACC 92363P로 기탁되어 있으며, 우수한 항균 활성, 내산성, 담즙산 내성 및 뮤신 부착능을 나타낼 수 있다.The novel Lactobacillus sp. SSD-9 strain of the present invention is a strain isolated from the Shitang tree, deposited under the accession number KACC 92363P, and exhibits excellent antibacterial activity, acid resistance, bile acid resistance and mucin adhesion ability .
Description
본 발명은 쉬땅나무에서 분리된 신규 락토바실러스 종 균주에 관한 것이다. 구체적으로 대장균, 살모넬라균, 세레우스균, 황색포도상구균, 칸디다 알비칸스 및 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타내며, 우수한 내산성 및 담즙산 내성을 나타내고, 뮤신에 대해 우수한 부착능을 나타낼 수 있는 신규 락토바실러스 종 SSD-9 균주에 관한 것이다.The present invention relates to a novel Lactobacillus sp. Specifically, it exhibits excellent antibacterial activity against Escherichia coli, Salmonella, Cereus, Staphylococcus aureus, Candida albicans and Clavibacter misiganensis, exhibits excellent acid resistance and bile acid resistance, and exhibits excellent adhesion to mucin. It relates to a novel Lactobacillus species SSD-9 strain.
유산균(lactic acid bacteria, LAB)은 다양한 환경, 즉, 식물, 곤충, 토양, 물, 유제품, 발효물, 사일리지(silage), 동물의 위장관, 호흡기관 등에서 발견된다[George et al., 2018; Yu et al., 2020]. 이들의 광범위한 서식지는 다양한 스트레스 조건과 영양적 조건들을 유도하는데, 단백질분해효소(protease) 및 지방분해효소(lipase)의 특이 생리적 및 생화학적 특성, 식물 파이토알렉신(phytoalexin)에 대한 내성, 위장관에서의 생존을 위한 담즙산 내성 등과 같은 특수성을 부여한다. 따라서 LAB는 발효식품, 음료, 사일리지 등의 3,500종 이상의 발효에 관여하여 필수적인 역할을 수행한다[Tamang et al., 2016].Lactic acid bacteria (LAB) are found in a variety of environments: plants, insects, soil, water, dairy products, fermented products, silage, animal gastrointestinal tract, respiratory tract, etc. [George et al., 2018; Yu et al., 2020]. Their extensive habitat induces a variety of stressful and nutritional conditions, including specific physiological and biochemical properties of proteases and lipases, tolerance to plant phytoalexins, endows specificity such as bile acid tolerance for the survival of Therefore, LAB plays an essential role in the fermentation of more than 3,500 kinds of fermented foods, beverages, and silage [Tamang et al., 2016].
자연계의 다양한 유산균들 중 산업적으로 유용한 유산균을 발굴하여 이를 활용하고자 하는 많은 연구들이 이루어지고 있다. 특히 프로바이오틱스로서 유용한 유산균을 발굴하고 이를 프로바이오틱스 제제뿐만 아니라 다양한 식품에 적용함으로써 소비자의 장내 환경을 개선하거나 보호하여 궁극적으로는 건강을 유지할 수 있도록 하기 위한 많은 관련 연구가 이루어지고 있다.Many studies have been conducted to find industrially useful lactic acid bacteria among various lactic acid bacteria in nature and to utilize them. In particular, many related studies have been conducted to improve or protect the intestinal environment of consumers by discovering useful lactic acid bacteria as probiotics and applying them to various foods as well as probiotics preparations so that they can ultimately maintain health.
유용한 다양한 유산균들이 발굴 및 연구되었지만, 여전히 다른 유용한 유산균을 새롭게 발굴하여 이용가능한 유용 유산균의 풀(pool)을 늘리는 것이 필요하며, 특히 보다 우수한 특성을 나타낼 수 있는 유산균의 발굴이 필요하다.Although various useful lactic acid bacteria have been discovered and studied, it is still necessary to newly discover other useful lactic acid bacteria to increase the pool of available useful lactic acid bacteria, and in particular, it is necessary to discover lactic acid bacteria that can exhibit better characteristics.
이에 본 발명자들은 다양한 자연계 시료로부터 유용하고 우수한 특성, 특히 프로바이오틱스로서의 우수한 특성을 나타낼 수 있는 새로운 유산균을 발굴하고자 하였다.Accordingly, the present inventors attempted to discover new lactic acid bacteria capable of exhibiting useful and excellent properties, particularly excellent properties as probiotics, from various natural samples.
따라서 본 발명의 주된 목적은 자연계의 시료로부터 발굴된 유용하고 우수한 특성을 갖는 새로운 유산균을 제공하는데 있다.Therefore, the main object of the present invention is to provide new lactic acid bacteria having useful and excellent properties discovered from natural samples.
본 발명의 다른 목적은 상기 유산균의 특성을 활용한 물품을 제공하는데 있다.Another object of the present invention is to provide an article utilizing the characteristics of the lactic acid bacteria.
본 발명의 한 양태에 따르면, 본 발명은 수탁번호 KACC 92363P로 기탁된 락토바실러스 종(Lactobacillus sp.) SSD-9 균주를 제공한다.According to one aspect of the present invention, the present invention provides a Lactobacillus sp. SSD-9 strain deposited under accession number KACC 92363P.
본 발명의 다른 양태에 따르면, 본 발명은 상기 균주를 포함하는 프로바이오틱스 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a probiotics composition comprising the strain.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 균주 또는 이의 배양물을 포함하는 항균용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides an antimicrobial composition comprising the strain or its culture.
본 발명의 항균용 조성물에 있어서, 상기 항균용 조성물은 대장균(Escherichia coli), 살모넬라균(Salmonella enterica serovar. Typhimurium), 세레우스균(Bacillus cereus), 황색포도상구균(Staphylococcus aureus), 칸디다 알비칸스(Candida albicans) 및 클라비박터 미시가넨시스(Clavibacter michiganensis)로 이루어진 군으로부터 선택된 하나 이상에 대한 항균용인 것이 바람직하다.In the antibacterial composition of the present invention, the antimicrobial composition is Escherichia coli , Salmonella ( Salmonella enterica serovar. Typhimurium), Bacillus cereus , Staphylococcus aureus , Candida albicans ( Candida albicans ) and Clavibacter mishiganensis ( Clavibacter michiganensis ) It is preferably for antibacterial use against at least one selected from the group consisting of.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 균주 또는 이의 배양물을 포함하는 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition comprising the strain or a culture thereof.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 균주 또는 이의 배양물을 포함하는 식물병 방제용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for controlling plant diseases comprising the strain or its culture.
본 발명의 식물병 방제용 조성물에 있어서, 상기 식물병은 토마토 궤양병인 것이 바람직하다.In the composition for controlling plant diseases of the present invention, the plant disease is preferably tomato ulcer disease.
본 발명의 균주는 대장균, 살모넬라균, 세레우스균, 황색포도상구균, 칸디다 알비칸스 및 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타내며, 우수한 내산성 및 담즙산 내성을 나타내고, 뮤신에 대해 우수한 부착능을 나타낼 수 있다. 이에 따라 프로바이오틱스로서 활용될 수 있으며, 항균 및 식물병 방제의 용도로도 활용될 수 있다.The strain of the present invention exhibits excellent antibacterial activity against Escherichia coli, Salmonella, Cereus, Staphylococcus aureus, Candida albicans and Clavibacter misiganensis, exhibits excellent acid resistance and bile acid resistance, and has excellent adhesion to mucin. can represent Accordingly, it can be used as probiotics, and can also be used for antibacterial and plant disease control purposes.
도 1은 본 발명 락토바실러스 종 SS-9 균주의 미생물 수탁증을 나타낸 것이다.
도 2는 본 발명 락토바실러스 종 SS-9 균주의 16S rRNA 유전자 서열을 나타낸 것이다.
도 3은 본 발명 락토바실러스 종 SS-9 균주의 대장균(Escherichia coli), 살모넬라균(Salmonella enterica serovar. Typhimurium), 세레우스균(Bacillus cereus), 황색포도상구균(Staphylococcus aureus) 및 칸디다 알비칸스(Candida albicans)에 대한 최소생장저해농도(MIC)를 나타낸 것이다.
도 4는 본 발명 락토바실러스 종 SS-9 균주의 클라비박터 미시가넨시스(Clavibacter michiganensis)에 대한 항균 활성을 나타낸 것이다.
도 5는 본 발명 락토바실러스 종 SS-9 균주의 배양 온도에 따른 성장력을 표준 균주(락토바실러스 애시도필루스(Lactobacillus acidophilus) KCTC 3164)와 비교하여 나타낸 것이다.
도 6은 본 발명 락토바실러스 종 SS-9 균주의 성장 곡선(배양 온도 : 37℃)을 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 7은 본 발명 락토바실러스 종 SS-9 균주의 배양 시간에 따른(배양 온도 : 37℃) pH 변화를 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 8은 본 발명 락토바실러스 종 SS-9 균주의 배양 시간에 따른(배양 온도 : 37℃) 대장균(E. coli) 및 황색포도상구균(S. aureus)에 대한 MIC를 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 9는 본 발명 락토바실러스 종 SS-9 균주의 배양 시간에 따른(배양 온도 : 37℃) 탄수화물 이용 정도를 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 10은 본 발명 락토바실러스 종 SS-9 균주의 배양 시간에 따른(배양 온도 : 37℃) 젖산 생산능을 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 11은 본 발명 락토바실러스 종 SS-9 균주의 내산성을 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 12는 본 발명 락토바실러스 종 SS-9 균주의 내담즙성을 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다.
도 13은 본 발명 락토바실러스 종 SS-9 균주의 뮤신 부착능을 살모넬라균(S. Typhimurium), 세레우스균(B. cereus) 및 표준 균주(락토바실러스 애시도필루스 KCTC 3164)와 비교하여 나타낸 것이다. PBS, 인산 완충 염수(대조군).Figure 1 shows the microbial accession of the present invention Lactobacillus sp. SS-9 strain.
Figure 2 shows the 16S rRNA gene sequence of the Lactobacillus sp. SS-9 strain of the present invention.
3 is Escherichia coli , Salmonella enterica serovar. Typhimurium, Bacillus cereus , Staphylococcus aureus and Candida albicans of the Lactobacillus species SS-9 strain of the present invention albicans ) shows the minimum growth inhibitory concentration (MIC).
Figure 4 shows the antibacterial activity of the Lactobacillus sp. SS-9 strain of the present invention against Clavibacter michiganensis .
Figure 5 shows the growth potential of the Lactobacillus species SS-9 strain according to the culture temperature of the present invention compared with a standard strain ( Lactobacillus acidophilus KCTC 3164).
Figure 6 shows the growth curve (incubation temperature: 37 ° C.) of the Lactobacillus species SS-9 strain of the present invention compared with a standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 7 shows the change in pH according to the incubation time (incubation temperature: 37 ° C) of the Lactobacillus species SS-9 strain of the present invention compared with the standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 8 shows the MIC for Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) according to the incubation time of the Lactobacillus species SS-9 strain of the present invention (cultivation temperature: 37 ° C) according to the standard strain (Lactobacillus acid It is shown in comparison with Pilus KCTC 3164).
Figure 9 shows the degree of carbohydrate utilization according to the incubation time (incubation temperature: 37 ° C) of the Lactobacillus species SS-9 strain of the present invention compared with the standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 10 shows the lactic acid production ability of the Lactobacillus species SS-9 strain of the present invention according to the incubation time (incubation temperature: 37 ° C) compared with the standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 11 shows the acid resistance of the Lactobacillus sp. SS-9 strain of the present invention compared with the standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 12 shows the bile resistance of the Lactobacillus sp. SS-9 strain of the present invention compared with that of the standard strain (Lactobacillus acidophilus KCTC 3164).
Figure 13 shows the mucin attachment ability of the Lactobacillus species SS-9 strain of the present invention compared to Salmonella ( S. Typhimurium), B. cereus and the standard strain (Lactobacillus acidophilus KCTC 3164). will be. PBS, phosphate buffered saline (control).
본 발명의 신규 락토바실러스 종(Lactobacillus sp.) SSD-9 균주는 경기도 남양주시 오남읍 오남리 팔현계곡의 쉬땅나무(Sorbaria sorbifolia var. stellipila Maxim.)의 꽃에서 분리되어 국립농업과학원 미생물은행(Korean Agricultural Culture Collection, KACC)에 수탁번호 KACC 92363P로 기탁되어 있다(도 1).The novel Lactobacillus sp. SSD-9 strain of the present invention was isolated from the flowers of Sorbaria sorbifolia var. , KACC) under the accession number KACC 92363P (FIG. 1).
본 발명의 균주는 서열번호 1의 16S rRNA 유전자 서열을 가지며(도 2), 대장균, 살모넬라균, 세레우스균, 황색포도상구균, 칸디다 알비칸스 및 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타낼 수 있고, 우수한 내산성 및 담즙산 내성을 나타낼 수 있고, 뮤신에 대해 우수한 부착능을 나타낼 수 있다.The strain of the present invention has the 16S rRNA gene sequence of SEQ ID NO: 1 (FIG. 2) and exhibits excellent antibacterial activity against Escherichia coli, Salmonella, Cereus, Staphylococcus aureus, Candida albicans and Clavibacter misiganensis. It can exhibit excellent acid resistance and bile acid resistance, and can exhibit excellent adhesion to mucin.
프로바이오틱스 미생물은 섭취된 후 위장을 통과할 때 낮은 pH의 위산과 담즙산에 내성을 갖고 높은 비율로 생존하여 대장으로 이동하고 대장 상피세포에 부착하여 유해 미생물과 경쟁하여 생존하는 등 대사산물의 기능성 뿐만 아니라 장내환경에서의 생존률이 중요한 판단기준이 된다. 본 발명의 균주는 상기와 같이 병원성 세균들에 대한 항균활성을 나타낼 수 있고 낮은 pH와 높은 담즙산에 대한 우수한 생존률을 나타낼 수 있을 뿐만 아니라 장내 부착능도 뛰어나 프로바이오틱스로서의 덕목을 골고루 갖추고 있다. 따라서 본 발명의 균주는 프로바이오틱스로서 유용할 것이며, 우수한 효과를 발휘할 수 있을 것이다.When probiotic microorganisms pass through the stomach after being ingested, they are resistant to gastric acid and bile acids of low pH, survive at a high rate, migrate to the large intestine, adhere to colonic epithelial cells, compete with harmful microorganisms, and survive. The survival rate in the intestinal environment is an important criterion. As described above, the strains of the present invention can exhibit antibacterial activity against pathogenic bacteria, exhibit excellent survival rates against low pH and high bile acids, and have excellent intestinal adhering ability, and are evenly equipped with virtues as probiotics. Therefore, the strains of the present invention will be useful as probiotics and will be able to exert excellent effects.
특히 본 발명의 균주는 매우 우수한(표준 균주인 락토바실러스 애시도필루스(Lactobacillus acidophilus) KCTC 3164에 비해서도 더욱 우수한) 내산성을 나타낼 수 있으며, 매우 우수한(표준 균주인 락토바실러스 애시도필루스 KCTC 3164에 비해서도 더욱 우수한) 뮤신 부착력을 나타낼 수 있다.In particular, the strain of the present invention can exhibit very excellent acid resistance (even better than the standard strain Lactobacillus acidophilus KCTC 3164), and has a very excellent (standard strain Lactobacillus acidophilus KCTC 3164). Even more excellent) can exhibit mucin adhesion.
또한, 본 발명의 균주는 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타낼 수 있어, 식물병 방제를 위해서도 활용할 수 있다. 클라비박터 미시가넨시스는 식물(예를 들어 토마토)에 궤양병을 일으키는 병원균으로, 이의 저해를 통해 관련 식물병을 효과적으로 방제할 수 있다.In addition, the strain of the present invention can exhibit excellent antibacterial activity against Clavibacter misiganensis, and can be utilized for plant disease control. Clavibacter misiganensis is a pathogen that causes canker disease in plants (for example, tomatoes), and through its inhibition, related plant diseases can be effectively controlled.
본 발명의 균주는 유산균, 특히 락토바실러스 속의 유산균을 배양하는데 사용하는 통상의 배양 방법에 따라 배양할 수 있다. 예를 들어, 배지로 MRS 배지를 사용하여 30 내지 40℃의 온도 조건으로 배양할 수 있다. 온도 조건은 보다 바람직하게는 35 내지 40℃, 보다 바람직하게는 36 내지 38℃로 한다. 이는 배양 온도를 달리하여 실험한 결과를 바탕으로 하며, 약 37℃에서 최적의 생장을 나타내는 것으로 확인되었다.The strain of the present invention can be cultured according to a conventional culture method used for culturing lactic acid bacteria, particularly lactic acid bacteria of the genus Lactobacillus. For example, it may be cultured at a temperature of 30 to 40° C. using MRS medium as a medium. The temperature conditions are more preferably 35 to 40°C, more preferably 36 to 38°C. This is based on the results of the experiment by varying the culture temperature, and it was confirmed that the optimum growth was exhibited at about 37 ° C.
본 발명의 프로바이오틱스(probiotics) 조성물은 본 발명의 균주를 포함하는 것을 특징으로 한다. 이때 본 발명의 균주는 균체의 건조물, 예를 들어 동결건조물의 형태일 수 있다. 상기와 같은 균체의 건조물은 균체를 건조시켜 건조물, 예를 들어 동결건조분말의 형태로 제조하는 통상의 처리방법을 사용하여 수득할 수 있다.The probiotics composition of the present invention is characterized by comprising the strain of the present invention. At this time, the strain of the present invention may be in the form of a dried product of the cells, for example, a freeze-dried product. The dry product of the cells as described above can be obtained by using a conventional treatment method for preparing a dry product by drying the cells, for example, in the form of a lyophilized powder.
본 발명의 프로바이오틱스 조성물은 조성물 중 본 발명의 균주를 다양한 함량으로 포함할 수 있다. 예를 들어, 조성물 중 본 발명 균주의 균체 동결건조분말을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다.The probiotics composition of the present invention may include the strain of the present invention in various amounts in the composition. For example, the composition may contain 0.01 to 100% by weight of the cell lyophilized powder of the strain of the present invention, but is not limited thereto.
일 실시형태에서, 본 발명의 프로바이오틱스 조성물에는, 실질적으로, 본 발명의 균주만이 프로바이오틱스로서 포함된다. 다시 말해, 본 발명의 프로바이오틱스 조성물 중 본 발명의 균주만이 유일한 프로바이오틱스로서 포함되고, 다른 프로바이오틱스는 포함되지 않는다. 다른 실시형태에서, 본 발명의 프로바이오틱스 조성물에는 본 발명의 균주 이외에도 다른 프로바이오틱스가 함께 포함된다. 이때 다른 프로바이오틱스에는 다른 속의 프로바이오틱스, 같은 속이지만 다른 균주의 프로바이오틱스가 포함된다.In one embodiment, the probiotic composition of the present invention includes substantially only the strains of the present invention as probiotics. In other words, in the probiotics composition of the present invention, only the strain of the present invention is included as the only probiotic, and other probiotics are not included. In another embodiment, the probiotic composition of the present invention includes other probiotics in addition to the strain of the present invention. At this time, other probiotics include probiotics of different genera, probiotics of the same genus but different strains.
본 발명의 프로바이오틱스 조성물에는 본 발명의 균주 이외에 첨가제, 예를 들어 완충제, 희석제, 부형제 등의 프로바이오틱스 제제에 통상적으로 사용되는 첨가제가 더 포함될 수 있다.In addition to the strains of the present invention, the probiotics composition of the present invention may further include additives commonly used in probiotics preparations, such as buffers, diluents, and excipients.
본 발명의 항균용 조성물은 본 발명의 균주 또는 이의 배양물을 포함하는 것을 특징으로 한다. 이때 본 발명의 균주는 위에서 언급한 바와 같이 균체의 건조물, 예를 들어 동결건조물의 형태일 수 있다.The antimicrobial composition of the present invention is characterized by comprising the strain or culture thereof of the present invention. At this time, as mentioned above, the strain of the present invention may be in the form of a dried product, for example, a lyophilized product.
본 발명 균주의 배양물은 위에서 설명한 바와 같은 방법에 따라 본 발명의 균주를 배양하여 수득할 수 있다. 일 실시형태에서, 본 발명 균주의 배양물은 액체배지에서 배양한 배양물에서 균체를 제거한 배양액이다. 다른 실시형태에서, 본 발명 균주의 배양물은 상기 배양액을 동결건조한 동결건조분말이다. 균체의 제거는 원심분리, 여과 등의 방법을 사용하여 달성할 수 있다. 본 발명 균주의 배양물에는 이 밖에도 배양물을 추출 등의 방법으로 추가 가공한 물질도 포함된다.A culture of the strain of the present invention can be obtained by culturing the strain of the present invention according to the method described above. In one embodiment, the culture of the strain of the present invention is a culture medium in which cells are removed from a culture cultured in a liquid medium. In another embodiment, the culture of the strain of the present invention is a lyophilized powder obtained by lyophilizing the culture medium. Removal of cells can be achieved using methods such as centrifugation and filtration. In addition, the culture of the strain of the present invention includes a material further processed by a method such as extraction of the culture.
본 발명의 항균용 조성물은 조성물 중 본 발명의 균주 또는 이의 배양물을 다양한 함량으로 포함할 수 있다. 예를 들어, 조성물 중 본 발명 균주의 균체 동결건조분말을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다. 또한, 예를 들어, 조성물 중 본 발명 균주 배양액의 동결건조분말을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다.The antimicrobial composition of the present invention may include the strain of the present invention or its culture in various amounts in the composition. For example, the composition may contain 0.01 to 100% by weight of the cell lyophilized powder of the strain of the present invention, but is not limited thereto. In addition, for example, the composition may contain 0.01 to 100% by weight of the lyophilized powder of the culture solution of the strain of the present invention, but is not limited thereto.
일 실시형태에서, 본 발명의 항균용 조성물에는, 실질적으로, 본 발명의 균주 또는 이의 배양물 만이 항균 물질로서(항균의 유효성분으로서) 포함된다. 다시 말해, 본 발명의 항균용 조성물 중 본 발명의 균주 또는 이의 배양물 만이 항균 활성을 나타내는 유일한 물질로서 포함되고, 다른 항균 물질은 포함되지 않는다. 다른 실시형태에서, 본 발명의 항균용 조성물에는 본 발명의 균주 또는 이의 배양물 이외에도 다른 항균 물질이 함께 포함된다. 이때 다른 항균 물질에는 통상적인 항균성 화합물, 항균 활성을 갖는 다른 미생물 또는 이의 배양물이 포함된다.In one embodiment, the antibacterial composition of the present invention, substantially, only the strain or culture thereof of the present invention is included as an antibacterial substance (as an active ingredient of the antibacterial). In other words, among the antibacterial composition of the present invention, only the strain or culture thereof of the present invention is included as the only material exhibiting antibacterial activity, and other antibacterial substances are not included. In another embodiment, the antimicrobial composition of the present invention includes other antimicrobial substances in addition to the strain or culture thereof of the present invention. At this time, other antibacterial substances include conventional antibacterial compounds, other microorganisms having antibacterial activity, or their cultures.
본 발명의 항균용 조성물에는 본 발명의 균주 또는 이의 배양물 이외에 첨가제, 예를 들어 완충제, 희석제, 부형제 등의 항균 제제에 통상적으로 사용되는 첨가제가 더 포함될 수 있다.The antibacterial composition of the present invention may further include additives, for example, buffers, diluents, additives commonly used in antibacterial preparations such as excipients, in addition to the strain or culture thereof of the present invention.
위에서 언급한 바와 같이, 본 발명의 균주는 대장균, 살모넬라균, 세레우스균, 황색포도상구균, 칸디다 알비칸스 및 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타낼 수 있으므로, 본 발명의 항균용 조성물은 상기 미생물들로 이루어진 군으로부터 선택된 하나 이상에 대한 항균용인 것이 바람직하다.As mentioned above, since the strain of the present invention can exhibit excellent antibacterial activity against Escherichia coli, Salmonella, Cereus, Staphylococcus aureus, Candida albicans and Clavibacter misiganensis, the antibacterial composition of the present invention is preferably antibacterial against at least one selected from the group consisting of the above microorganisms.
본 발명의 식품 조성물 또한 본 발명의 균주 또는 이의 배양물을 포함하는 것을 특징으로 한다. 이때 본 발명의 균주 및 이의 배양물에 관한 사항은 위에서 프로바이오틱스 조성물 및 항균용 조성물에서 언급한 사항과 동일할 수 있다.The food composition of the present invention is also characterized by comprising the strain or culture thereof of the present invention. At this time, the details of the strain and its culture of the present invention may be the same as those mentioned in the probiotics composition and antimicrobial composition above.
본 발명의 식품 조성물은 조성물 중 본 발명의 균주 또는 이의 배양물을 다양한 함량으로 포함할 수 있다. 예를 들어, 조성물 중 본 발명 균주의 균체 동결건조분말을 0.001 내지 10중량%로 함유할 수 있으나, 이로 제한되지 않는다. 또한, 예를 들어, 조성물 중 본 발명 균주 배양액의 동결건조분말을 0.001 내지 10중량%로 함유할 수 있으나, 이로 제한되지 않는다.The food composition of the present invention may include the strain or culture thereof of the present invention in various amounts in the composition. For example, the composition may contain 0.001 to 10% by weight of the cell lyophilized powder of the strain of the present invention, but is not limited thereto. In addition, for example, the composition may contain 0.001 to 10% by weight of the freeze-dried powder of the culture solution of the strain of the present invention, but is not limited thereto.
본 발명의 식품 조성물은 다양한 형태, 예를 들어 식육가공품, 어육제품, 두부, 묵, 죽, 라면이나 국수 등의 면류, 간장, 된장, 고추장, 혼합장 등의 조미식품, 소스, 과자, 발효유나 치즈 등의 유가공품, 김치나 장아찌 등의 절임식품, 과실, 채소, 두유, 발효음료 등의 음료수와 같은 형태의 식품 조성물일 수 있으며, 바람직하게는 유가공품 형태의 식품 조성물이다.The food composition of the present invention has various forms, for example, processed meat products, fish products, tofu, jelly, porridge, noodles such as ramen or noodles, seasonings such as soy sauce, soybean paste, gochujang, mixed soy sauce, sauces, confectionery, fermented milk or It may be a food composition in the form of dairy products such as cheese, pickled foods such as kimchi or pickles, fruits, vegetables, soy milk, beverages such as fermented beverages, and preferably a food composition in the form of dairy products.
본 발명의 식물병 방제용 조성물 또한 본 발명의 균주 또는 이의 배양물을 포함하는 것을 특징으로 한다. 이때 본 발명의 균주 및 이의 배양물에 관한 사항은 위에서 프로바이오틱스 조성물 및 항균용 조성물에서 언급한 사항과 동일할 수 있다.The composition for controlling plant diseases of the present invention is also characterized by comprising the strain or culture thereof of the present invention. At this time, the details of the strain and its culture of the present invention may be the same as those mentioned in the probiotics composition and antimicrobial composition above.
본 발명의 식물병 방제용 조성물은 조성물 중 본 발명의 균주 또는 이의 배양물을 다양한 함량으로 포함할 수 있다. 예를 들어, 조성물 중 본 발명 균주의 균체 동결건조분말을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다. 또한, 예를 들어, 조성물 중 본 발명 균주 배양액의 동결건조분말을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다.The composition for controlling plant diseases of the present invention may include the strain or culture thereof of the present invention in various amounts in the composition. For example, the composition may contain 0.01 to 100% by weight of the cell lyophilized powder of the strain of the present invention, but is not limited thereto. In addition, for example, the composition may contain 0.01 to 100% by weight of the lyophilized powder of the culture solution of the strain of the present invention, but is not limited thereto.
일 실시형태에서, 본 발명의 식물병 방제용 조성물에는, 실질적으로, 본 발명의 균주 또는 이의 배양물 만이 식물병 방제 활성 물질로서(식물병 유발 병원체에 대한 항균의 유효성분으로서) 포함된다. 다시 말해, 본 발명의 식물병 방제용 조성물 중 본 발명의 균주 또는 이의 배양물 만이 식물병 방제 효과를 나타내는 유일한 물질로서 포함되고, 다른 식물병 방제 활성 물질은 포함되지 않는다. 다른 실시형태에서, 본 발명의 식물병 방제용 조성물에는 본 발명의 균주 또는 이의 배양물 이외에도 다른 식물병 방제 활성 물질이 함께 포함된다. 이때 다른 식물병 방제 활성 물질에는 통상적인 식물병 방제 활성 화합물, 식물병 방제 활성을 갖는 다른 미생물 또는 이의 배양물이 포함된다.In one embodiment, the composition for controlling plant diseases of the present invention contains substantially only the strain or culture thereof of the present invention as a plant disease controlling active substance (as an antibacterial active ingredient against plant disease-causing pathogens). In other words, among the composition for controlling plant diseases of the present invention, only the strain or its culture of the present invention is included as the only material exhibiting a plant disease controlling effect, and other plant disease controlling active substances are not included. In another embodiment, the composition for controlling plant diseases of the present invention includes other active substances for controlling plant diseases in addition to the strain or culture thereof of the present invention. At this time, other plant disease control active substances include conventional plant disease control active compounds, other microorganisms having plant disease control activity, or their cultures.
본 발명의 식물병 방제용 조성물에는 본 발명의 균주 또는 이의 배양물 이외에 첨가제, 예를 들어 완충제, 희석제, 부형제 등의 식물병 방제용 제제에 통상적으로 사용되는 첨가제가 더 포함될 수 있다.The composition for controlling plant diseases of the present invention may further include additives, for example, buffers, diluents, additives commonly used in preparations for controlling plant diseases, such as excipients, in addition to the strain or culture thereof of the present invention.
위에서 언급한 바와 같이, 본 발명의 균주는 클라비박터 미시가넨시스에 대해 우수한 항균 활성을 나타낼 수 있다. 그리고 이 클라비박터 미시가넨시스는 식물 궤양병, 특히 토마토 궤양병을 주로 유발한다. 따라서 본 발명의 식물병 방제용 조성물은 식물 궤양병 방제용인 것이 바람직하며, 보다 바람직하게는 토마토 궤양병 방제용이다.As mentioned above, the strain of the present invention can exhibit excellent antibacterial activity against Clavibacter misiganensis. And this Clavibacter misiganensis mainly causes plant canker disease, especially tomato canker disease. Therefore, the composition for controlling plant diseases of the present invention is preferably for controlling plant ulcer disease, and more preferably for controlling tomato ulcer disease.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.
[실시예][Example]
실시예 1. 균주 분리Example 1. Strains Isolation
경기도 남양주시 오남읍 오남리 팔현계곡에 서식하는 쉬땅나무의 꽃 한 송이를 균주 분리를 위한 시료로 사용하였다. 시료를 500㎖ 멸균수로 3회 세척하고 물기가 제거된 시료에 20㎖ 멸균수를 첨가하여 막자사발에서 완전히 으깬 후, 원심분리하여 상등액을 취하였다. 상등액은 연속희석법으로 10-1 ~ 10-5으로 희석하였으며, 10-5 희석액 1㎖에서 200㎕씩을 분취하여 MRS 고체배지에 도말하고 37℃에서 24시간 동안 호기 조건에서 배양하였다. 생성된 콜로니를 새로운 MRS 고체배지에 옮겨 희석식 계대법으로 순수배양하여 단일 균주 SSD-9를 확보하였다. 단일 균주로 확보된 SSD-9 균주를 MRS 액체배지에 접종하여 30℃에서 24시간 동안 배양하였다. 배양 후 배양액을 1㎖씩 분취하여 13,500rpm에서 원심분리하였다. 원심분리 후 상등액을 취하여 0.2㎛ 시린지 필터로 여과한 후 -20℃에 보관하면서 다음 실험의 시료로 사용하였다.A flower of the Shitang tree living in Palhyeon Valley, Onam-ri, Onam-eup, Namyangju-si, Gyeonggi-do was used as a sample for strain isolation. The sample was washed three times with 500 ml of sterile water, and 20 ml of sterile water was added to the dried sample, completely mashed in a mortar, and then centrifuged to obtain the supernatant. The supernatant was diluted to 10 -1 to 10 -5 by serial dilution, and 200 μl of each was aliquoted from 1 ml of the 10 -5 dilution, spread on MRS solid medium, and cultured at 37° C. for 24 hours under aerobic conditions. The resulting colony was transferred to a new MRS solid medium and pure culture was performed by dilution-type passaging to secure a single strain SSD-9. The SSD-9 strain obtained as a single strain was inoculated into MRS broth and cultured at 30 ° C. for 24 hours. After culturing, 1 ml of the culture medium was aliquoted and centrifuged at 13,500 rpm. After centrifugation, the supernatant was taken, filtered with a 0.2 μm syringe filter, and then stored at -20 ° C and used as a sample for the next experiment.
실시예 2. 균주의 특성 평가Example 2. Characterization of strains
2-1. 실험 방법2-1. Experiment method
2-1-1. 항균 활성 평가2-1-1. Antibacterial activity evaluation
마이크로티터 플레이트(microtiter plate) 방법을 사용하여 SSD-9 균주의 Escherichia coli ATCC 10536, Salmonella enterica serovar. Typhimurium KCTC 12401, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 6538, Candida albicans KCTC 7122 및 식물병원성 Clavibacter michiganensis에 대한 항균 활성을 측정하였다. Escherichia coli ATCC 10536, Salmonella enterica serovar. Antibacterial activity against Typhimurium KCTC 12401, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 6538, Candida albicans KCTC 7122 and phytopathogenic Clavibacter michiganensis was measured.
항균 활성 분석을 위해 SSD-9 균주를 MRS 액체배지에 30℃, 12 ~ 16시간(overnight, O/N) 전배양하고 배양 상등액을 항균 활성 평가 시료로 사용하였다.For the analysis of antibacterial activity, the SSD-9 strain was pre-cultured in MRS liquid medium at 30 ° C for 12 to 16 hours (overnight, O / N), and the culture supernatant was used as an antibacterial activity evaluation sample.
실험 미생물인 E. coli, S. Typhimurium, B. cereus, S. aureus 및 C. michiganensis는 5㎖ LB 액체배지에 1%로 접종하여 37℃에서 12 ~ 16시간(overnight, O/N) 전배양한 후 106 CFU/㎖로 조정하여 실험에 사용하였고, C. albicans는 5㎖ YM 액체배지에 1%로 접종하여 30℃에서 12 ~ 16시간(overnight, O/N) 전배양한 후 106 CFU/㎖로 조정하여 실험에 사용하였다.Experimental microorganisms E. coli , S. Typhimurium, B. cereus , S. aureus and C. michiganensis were inoculated at 1% in 5 ml LB broth, pre-cultured at 37 ° C for 12 to 16 hours (overnight, O / N), and then adjusted to 10 6 CFU / ml was used in the experiment, and C. albicans was inoculated at 1% in 5 ml YM liquid medium, pre-cultured at 30 ° C for 12 to 16 hours (overnight, O / N), and then adjusted to 10 6 CFU / ml and used in the experiment did
SSD-9 배양 상등액을 농도별로 첨가하고, 106 CFU/㎖로 조정한 각 실험 미생물의 전배양액을 50㎕ 첨가한 후, LB 또는 YM 배지로 200㎕로 조정하였다. 조성된 반응액을 37℃ 또는 30℃에서 24시간 배양한 후 600㎚에서 흡광도를 측정하여 실험 미생물의 성장률을 측정하였다. 이때 SSD-9 배양 상등액을 첨가하지 않은 대조군을 함께 사용하였으며, 대조군의 실험 미생물 성장률을 기준(100%)으로 최저생장저해농도(MIC)를 결정하였다.The SSD-9 culture supernatant was added by concentration, and 50 μl of the pre-culture of each experimental microorganism adjusted to 10 6 CFU/ml was added, and then adjusted to 200 μl with LB or YM medium. After incubating the prepared reaction solution at 37 ° C. or 30 ° C. for 24 hours, the absorbance was measured at 600 nm to measure the growth rate of the experimental microorganisms. At this time, a control group without the addition of SSD-9 culture supernatant was used together, and the minimum growth inhibitory concentration (MIC) was determined based on the experimental microbial growth rate of the control group (100%).
2-1-2. 균주 동정2-1-2. strain identification
SSD-9 균주의 16S rRNA 유전자 염기서열을 분석하고 이 서열을 토대로 NCBI BLAST를 수행하였다.The 16S rRNA gene sequence of the SSD-9 strain was analyzed, and NCBI BLAST was performed based on this sequence.
2-1-3. 성장 특성 분석2-1-3. Analysis of growth characteristics
SSD-9 균주의 성장 특성을 표준 균주(Lactobacillus acidophilus KCTC 3164)와 비교하여 분석하였다. 표준 균주는 한국생명공학연구원 생물자원센터로부터 분양받아 사용하였다.The growth characteristics of the SSD-9 strain were compared and analyzed with the standard strain ( Lactobacillus acidophilus KCTC 3164). Standard strains were purchased from the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology and used.
SSD-9 균주 및 표준 균주를 25, 30 및 37℃에서 5㎖로 24시간 배양하면서, 6시간 간격으로 샘플링하고 600㎚ 흡광도를 측정하여 각 온도에서의 성장력을 비교하였다.The SSD-9 strain and the standard strain were cultured at 25, 30 and 37 ° C. for 24 hours with 5 ml, sampled at 6 hour intervals, and the absorbance at 600 nm was measured to compare growth potential at each temperature.
SSD-9 균주 및 표준 균주를 100㎖ 배지에 1/100로 접종하고, 37℃에서 배양하면서 6시간 간격으로 샘플링하였다. 샘플링된 시료의 600㎚ 흡광도를 측정하여 각 유산균의 성장도를 평가하였다. 각 샘플의 pH는 pH 미터기로 측정하였으며, 탄수화물 함량은 DNS(3,5-dinitrosalicylic acid) 방법으로 측정하였다.The SSD-9 strain and the standard strain were inoculated at 1/100 in 100 ml medium, and sampled at 6 hour intervals while incubating at 37°C. The growth of each lactic acid bacteria was evaluated by measuring the absorbance at 600 nm of the sampled sample. The pH of each sample was measured with a pH meter, and the carbohydrate content was measured by DNS (3,5-dinitrosalicylic acid) method.
또한, 샘플링한 SSD-9 배양 상등액을 사용하여 상기 항목 '2-1-1'에서와 같은 방법으로, 배양 시간에 따른 E. coli 및 S. aureus에 대한 MIC를 측정하였다.In addition, using the sampled SSD-9 culture supernatant, the MIC for E. coli and S. aureus according to the culture time was measured in the same manner as in '2-1-1' above.
유기산 함량은 HPLC 분석법으로 측정하였다. 이때 Aminex 87H 컬럼(300x10㎜, Bio-Rad, USA)을 이용한 Ultimate3000(Thermo Dionex, USA)을 사용하였고, RI(ERC, RefractoMAX520, Japan)로 UV 210㎚에서 검출하였으며, 이동상은 0.01N H2SO4를 사용하여 0.5㎖/분의 유속으로 30분간 수행하였다. 표준품으로 젖산나트륨염(lactic acid sodium salt, Fluka) 및 아세트산(acetic acid, JTB)을 사용하였다. Organic acid content was determined by HPLC analysis. At this time, Ultimate3000 (Thermo Dionex, USA) using an Aminex 87H column (300x10mm, Bio-Rad, USA) was used, and detection was performed at UV 210 nm with RI (ERC, RefractoMAX520, Japan), and the mobile phase was 0.01NH 2 SO 4 was performed for 30 minutes at a flow rate of 0.5 ml/min. As standards, lactic acid sodium salt (Fluka) and acetic acid (JTB) were used.
2-1-4. 내산성 및 내담즙성 분석2-1-4. Acid resistance and bile resistance assay
내산성 및 내담즙성 분석은 이전에 수행된 연구 방법에서 약간 수식한 방법을 사용하여 수행하였다[Ji et al, 2015; Mohd Yusof et al., 2020].Analysis of acid resistance and bile resistance was performed using a slightly modified method from a previously performed research method [Ji et al, 2015; Mohd Yusof et al., 2020].
간략히 서술하면, 내산성 분석의 경우 9㎖ MRS 배지의 pH를 HCl로 pH 2.0 ~ 3.0으로 조정한 후, 12 ~ 16시간 배양된 1㎖의 균주 배양액을 첨가하고 37℃에서 0 ~ 48시간 동안 배양 후 생존 개체수를 조사하였다.Briefly, in the case of acid resistance analysis, after adjusting the pH of 9 ml MRS medium to pH 2.0 ~ 3.0 with HCl, adding 1 ml of strain culture medium cultured for 12 ~ 16 hours, incubating at 37 ° C for 0 ~ 48 hours Survival populations were investigated.
내담즙성 분석은 상기 내산성 분석과 동일한 방법으로 수행하되, pH를 조절하는 대신 0.6 ~ 1.2%가 되도록 oxgall(Oxoid, Bstingstoke, UK)을 첨가하여 수행하였다.The bile resistance assay was performed in the same manner as the acid resistance assay, but instead of adjusting the pH, oxgall (Oxoid, Bstingstoke, UK) was added to a pH of 0.6 to 1.2%.
2-1-5. 뮤신 부착 능력 평가2-1-5. Assessment of mucin attachment ability
돼지 위 type III 뮤신(Sigma-Aldrich, St. Louis, MO, USA)을 10㎎/㎖의 농도로 PBS(phosphate-buffered saline, pH 7.5)에 용해시켰다. 제조된 뮤신 용액을 사용하고, 이전 수행된 연구 방법을 약간 수식한 방법을 사용하였다[Radziwill-Bienkowska et al., 2016]. 간략히 서술하면, 1㎎/㎖ 뮤신을 마이크로티터 플레이트에 처리하여 37℃에서 30분 처리한 후, 4℃에서 16시간 방치하여 부착하였다. PBS 완충액으로 3회 세척한 후 107 CFU/㎖의 시험대상 균주를 첨가하여 실온에서 3시간 또는 37℃에서 24시간 반응하였다. 부착되지 않은 균주는 PBS 완충액으로 3회 세척하여 제거한 후, 100㎕ 0.1% crystal violet을 첨가하여 실온에서 30분 반응한 후, PBS 완충액으로 3회 세척하였다. 100㎕ 30% 초산을 첨가하여 미생물에 부착된 crystal violet을 용출하여 570㎚에서 흡광도를 측정하여 뮤신 결합력을 평가하였다.Porcine gastric type III mucin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in PBS (phosphate-buffered saline, pH 7.5) at a concentration of 10 mg/ml. The prepared mucin solution was used, and a slightly modified method of a previously performed research method was used [Radziwill-Bienkowska et al., 2016]. Briefly, 1 mg / ml mucin was treated on a microtiter plate, treated at 37 ° C for 30 minutes, and then left at 4 ° C for 16 hours to attach. After washing three times with PBS buffer, 10 7 CFU/mL of the strain to be tested was added and reacted at room temperature for 3 hours or at 37° C. for 24 hours. Non-attached strains were removed by washing three times with PBS buffer, followed by adding 100 μl 0.1% crystal violet, reacting at room temperature for 30 minutes, and then washing three times with PBS buffer. 100 μl of 30% acetic acid was added to elute crystal violet attached to microorganisms, and absorbance was measured at 570 nm to evaluate mucin binding ability.
2-2. 실험 결과2-2. Experiment result
2-2-1. 항균 활성 분석 결과2-2-1. Antibacterial activity assay result
E. coli, S. Typhimurium, B. cereus, S. aureus 및 C. albicans에 대한 SSD-9 균주 배양 상등액의 MIC를 측정한 결과, 도 3에서와 같이 SSD-9 균주는 상기 실험 미생물에 대해 우수한 항균 활성을 나타내었다. E. coli , S. As a result of measuring the MIC of the SSD-9 strain culture supernatant for Typhimurium, B. cereus , S. aureus and C. albicans , as shown in FIG. 3, the SSD-9 strain showed excellent antibacterial activity against the experimental microorganisms.
또한, C. michiganensis에 대한 항균 활성에 대해서도 분석한 결과, 도 4에서와 같이 SSD-9 균주 배양 상등액의 첨가에 의해 C. michiganensis의 성장이 유의적으로 저해되며, 이러한 성장 저해는 농도의존적인 것으로 나타났다.In addition, as a result of analyzing the antibacterial activity against C. michiganensis , as shown in FIG. 4, the growth of C. michiganensis was significantly inhibited by the addition of the culture supernatant of the SSD-9 strain, and this growth inhibition was concentration dependent. appear.
2-2-2. 균주 동정 결과2-2-2. Strain identification result
SSD-9 균주의 16S rRNA 유전자 염기서열을 바탕으로 Ezbiocloud를 이용하여 상동성을 검색한 결과, Lactobacillus 속의 균주들과 98.27 ~ 99.86%의 상동성을 보였다(표 1). 이를 토대로 SSD-9 균주는 Lactobacillus 속의 한 종으로 추정되며, 이에 따라 Lactobacillus sp. SSD-9로 명명하였다.As a result of searching for homology using Ezbiocloud based on the 16S rRNA gene sequence of SSD-9 strain, it showed 98.27 ~ 99.86% homology with strains of the genus Lactobacillus (Table 1). Based on this, the SSD-9 strain is estimated to be a species of the genus Lactobacillus , and accordingly Lactobacillus sp. It was named SSD-9.
2-2-3. 성장 특성 분석 결과2-2-3. Analysis of growth characteristics
SSD-9 균주의 생육 최적 온도를 조사하기 위해, 일반적으로 미생물을 배양하는 조건인 25, 30 및 37℃로 배양 온도를 설정하여 평가해 보았다. 이의 결과, SSD-9 균주 및 표준 균주(Lactobacillus acidophilus KCTC 3164) 모두 37℃에서 생육이 가장 우수한 것으로 나타났고, 전반적으로 SSD-9 균주가 표준 균주에 비교하여 각 온도에서 성장이 우수한 것으로 나타났다(도 5).In order to investigate the optimal growth temperature of the SSD-9 strain, the culture temperature was set to 25, 30 and 37 ° C., which are generally conditions for culturing microorganisms, and evaluated. As a result, both the SSD-9 strain and the standard strain ( Lactobacillus acidophilus KCTC 3164) showed the best growth at 37 ° C, and overall, the SSD-9 strain showed excellent growth at each temperature compared to the standard strain (Fig. 5).
생육 최적 온도 조사 결과와 마찬가지로 성장 곡선 관찰 결과에서도 SSD-9 균주는 표준 균주와 비교하여 성장력이 우수한 것으로 나타났다(도 6). 표준 균주는 정지기까지 최대 성장력이 낮은 것으로 보아 내산성이 낮은 것에 기인하여 나타날 수 있는 현상으로 추정되었다.As in the results of the investigation of the optimum growth temperature, the observation of the growth curve showed that the SSD-9 strain had excellent growth potential compared to the standard strain (FIG. 6). The standard strain was estimated to have a low maximum growth potential until the stationary phase, which may be due to low acid resistance.
성장에 따른 pH 변화는 성장 곡선과 유사한 패턴을 보였지만, SSD-9 균주는 pH 3.6까지 감소된 반면, 표준 균주는 pH 4.07까지 감소되는 경향성을 보였다(도 7).The pH change with growth showed a pattern similar to the growth curve, but the SSD-9 strain decreased to pH 3.6, while the standard strain showed a tendency to decrease to pH 4.07 (FIG. 7).
배양 시간별 E. coli 및 S. aureus에 대한 항균력은 SSD-9 균주가 표준 균주에 비교하여 모두 높은 것으로 나타났다(도 8).Antibacterial activity against E. coli and S. aureus by incubation time was found to be higher in all SSD-9 strains compared to the standard strain (FIG. 8).
다른 한편으로, 표준 균주의 성장력 지연에 따라 탄수화물의 이용효율도 감소되는 경향성을 보였다(도 9).On the other hand, the utilization efficiency of carbohydrates tended to decrease according to the growth delay of the standard strain (FIG. 9).
SSD-9 균주는 성장 곡선에 유사하게 젖산 함량이 증가되는 경향성을 보였고, 최종 16g/ℓ의 값을 보였다. 또한, 표준 균주는 성장 곡선과 유사한 양상을 보였고, 최종 9g/ℓ의 값을 보였다. 따라서 SSD-9 균주는 표준 균주에 비교하여 젖산 생산량이 1.8배 높은 것으로 나타났다(도 10).SSD-9 strain showed a tendency to increase the lactic acid content similar to the growth curve, and showed a final value of 16 g / ℓ. In addition, the standard strain showed a similar pattern to the growth curve, and showed a final value of 9 g / ℓ. Therefore, the SSD-9 strain showed 1.8 times higher lactic acid production compared to the standard strain (FIG. 10).
2-2-4. 내산성 및 내담즙성 분석 결과2-2-4. Acid resistance and bile resistance analysis results
48시간 배양에 따른 내산성 분석결과, SSD-9 균주는 0, 12, 18시간에서 pH 2.0에 내산성이 존재하는 것으로 나타났다. 이 결과로 볼 때, 내산성은 대수증식기 말기에서 정지기 초기에 왕성하게 유도되는 것으로 추정된다. SSD-9 균주의 배양 0시간의 pH 2.0에서 내산성이 발생한 것은 전배양 시간이 18시간으로 한정되는 것에 기인된 것으로 추정된다.As a result of acid resistance analysis after 48 hours of cultivation, it was found that the SSD-9 strain had acid resistance at pH 2.0 at 0, 12, and 18 hours. From this result, it is assumed that acid resistance is vigorously induced from the end of the logarithmic growth phase to the early stationary phase. The occurrence of acid resistance at pH 2.0 at 0 hour of culture of the SSD-9 strain is presumed to be due to the limitation of the pre-culture time to 18 hours.
반면 표준 균주는 전배양 시간에 따라 pH 2.0에서 전혀 생존 균수가 존재하지 않는 것으로 나타났다. 따라서 표준 균주는 사람 분변으로 분리된 균주이지만, 내산성이 낮은 것으로 평가되었다. 이러한 결과로 볼 때 SSD-9 균주는 사람 장내에서 분리된 표준 균주 보다 내산성이 매우 우수한 것으로 나타났다(도 11).On the other hand, the standard strain showed no viable bacteria at pH 2.0 according to the pre-incubation time. Therefore, the standard strain is a strain isolated from human feces, but was evaluated to have low acid resistance. From these results, it was found that the SSD-9 strain had much better acid resistance than the standard strain isolated from the human intestine (FIG. 11).
SSD-9 균주의 담즙산 내성은 처리된 모든 oxgall 농도에서 표준 균주와 비슷한 수치를 보였으며, 최종 농도 1.2%에서도 생존률의 변화는 없었다. SSD-9 균주가 장내 담즙의 농도인 0.3%보다 훨씬 높은 1.2% oxgall의 농도에서 높은 생존률을 보인 내담즙성 결과를 토대로, SSD-9 균주는 소장을 통과할 때 담즙산의 영향을 받지 않고 대장으로 이동할 수 있을 것으로 판단된다(도 12).Bile acid tolerance of strain SSD-9 was similar to that of the standard strain at all concentrations of oxgall treated, and there was no change in survival rate even at a final concentration of 1.2%. Based on the results of biliary tolerance in which the SSD-9 strain showed a high survival rate at a concentration of 1.2% oxgall, which is much higher than the concentration of 0.3% in intestinal bile, the SSD-9 strain is unaffected by bile acids when passing through the small intestine and enters the large intestine. It is determined that it can move (FIG. 12).
2-2-5. 뮤신 부착 능력 평가2-2-5. Assessment of mucin attachment ability
뮤신 부착능 평가 결과, SSD-9 균주는 비교 균주로 사용된 S. Typhimurium과 B. cereus 보다 약 6배 정도 부착능이 높았으며, 표준 균주와 비교하여 약 2배 정도 부착능이 우수한 것으로 나타났다(도 13).As a result of mucin adhesion evaluation, SSD-9 strain was used as a comparison strain S. The adhesion capacity was about 6 times higher than that of Typhimurium and B. cereus , and the adhesion capacity was about 2 times higher than that of the standard strain (FIG. 13).
<110> National Institute of Biological Resources <120> Novel Lactobacillus sp. strain isolated from Sorbaria sorbifolia var. stellipila Maxim. <130> ALP21019 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1506 <212> DNA <213> Unknown <220> <223> Lactobacillus sp. SSD-9 <400> 1 ctgctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgaacga actctggtat 60 tgattggtgc ttgcatcatg atttacattt gagtgagtgg cgaactggtg agtaacacgt 120 gggaaacctg cccagaagcg ggggataaca cctggaaaca gatgctaata ccgcataaca 180 acttggaccg catggtccga gcttgaaaga tggcttcggc tatcactttt ggatggtccc 240 gcggcgtatt agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc 300 tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 360 agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgaagaa 420 gggtttcggc tcgtaaaact ctgttgttaa agaagaacat atctgagagt aactgttcag 480 gtattgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 600 agtctgatgt cgaaagcctt cggctcaacc gaagaagtgc atcggaaact gggaaacttg 660 agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag 720 aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg 780 gtagcaaaca ggattagata ccctggtagt ccataccgta aacgatgaat gctaagtgtt 840 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 900 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 1020 agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 1140 ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1200 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 1260 gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 1320 tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgggggga 1380 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1440 gtcggtgggg taacctttta ggaaccagcc gcctaaggtg ggacagatga ttagggtgaa 1500 gtcgta 1506 <110> National Institute of Biological Resources <120> Novel Lactobacillus sp. strain isolated from Sorbaria sorbifolia var. stellipila Maxim. <130> ALP21019 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1506 <212> DNA <213> unknown <220> <223> Lactobacillus sp. SSD-9 <400> 1 ctgctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgaacga actctggtat 60 tgattggtgc ttgcatcatg atttacattt gagtgagtgg cgaactggtg agtaacacgt 120 gggaaacctg cccagaagcg ggggataaca cctggaaaca gatgctaata ccgcataaca 180 acttggaccg catggtccga gcttgaaaga tggcttcggc tatcactttt ggatggtccc 240 gcggcgtatt agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc 300 tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 360 agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgaagaa 420 gggtttcggc tcgtaaaact ctgttgttaa agaagaacat atctgagagt aactgttcag 480 gtattgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 600 agtctgatgt cgaaagcctt cggctcaacc gaagaagtgc atcggaaact gggaaacttg 660 agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag 720 aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg 780 gtagcaaaca ggattagata ccctggtagt ccataccgta aacgatgaat gctaagtgtt 840 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 900 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 1020 agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 1140 ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1200 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 1260 gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 1320 tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgggggga 1380 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1440 gtcggtgggg taacctttta ggaaccagcc gcctaaggtg ggacagatga ttagggtgaa 1500 gtcgta 1506
Claims (7)
상기 식물병은 토마토 궤양병인, 식물병 방제용 조성물.According to claim 6,
The plant disease is a tomato ulcer disease, a composition for controlling plant diseases.
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