CRISPR/Cas systems and anti-repeat sequences of Lactobacillus curvatus, Lactobacillus graminis, Lactobacillus fuchuensis, and Lactobacillus sakei genomes

Ilıkkan Özge Kahraman 한국미생물학회 2021 미생물학회지 Vol.57 No.1

CRISPR/Cas system (Clustered regularly interspaced short palindromic repeats–CRISPR associated genes) helps the bacteria or the archaea to gain immunity against phage or plasmid invasions. Lactobacillus curvatus has been known to be phylogenetically related to Lactobacillus fuchuensis, Lactobacillus graminis, and Lactobacillus sakei. In this study, this relationship has been investigated in terms of CRISPR/Cas systems, Cas1, Cas2 amino acid sequences, protospacers, and anti-repeat sequences. A total of 54 genomes (34 whole-genome and 20 draft genome) were investigated, 25 genomes had CRISPR/Cas system, namely, 23 type II-A, 2 II-C, and 2 I-E. The pairwise identity of anti-repeat sequences was 77.4%. Phylogeny analysis and alignment of Cas1 and Cas2 amino acid sequences were carried out to evaluate the relationship among CRISPR subtypes and four species. The phylogenetic tree tended to split into branches corresponding to the CRISPR/Cas subtype II-A, II-C, and I-E. According to protospacer analysis, common invaders of strains were Pediococcus damnosus, Pediococcus claussenii, Lactobacillus brevis, Lactobacillus backii, Lactobacillus plantarum, and Lactobacillus sakei. The secondary structure of seven different repeat sequences was depicted. Recently, although detection of CRISPR/Cas systems has been highly attractive for genotyping, this intention has also been used for endogenous genome editing to improve the industrial benefits of lactic acid bacteria. This detailed study will provide a perspective on these approaches.

김치에서 분리한 Lactobacillus sakei proBio65의 면역 조절 특성

임정희 ( Jeong Heui Lim ),서병주 ( Byoung Joo Seo ),김정은 ( Jung Eun Kim ),채창석 ( Chang Suk Chae ),임신혁 ( Sin Hyeog Im ),한윤수 ( Youn Soo Hahn ),박용하 ( Yong Ha Park ) 한국미생물생명공학회(구 한국산업미생물학회) 2011 한국미생물·생명공학회지 Vol.39 No.3

김치로부터 새로운 프로바이오틱균주인 Lactobacillus sakei proBio65를 분리하고 명명하였다. 형질전환 생쥐 (Foxp3-GFP KI 생쥐)를 이용하여 L. sakei proBio65의 면역조절 메커니즘 규명 및 면역 조절능을 확인하고 in vivo 적용 질환제어 응용 가능성을 평가하였다. 조절 T 세포의 master 전사조절인자로 알려진 Foxp3+를 선정하고, L. sakei가 Foxp3+전사조절인자를 증가시키는지 확인하기 위해 확립된 세포기반 screening system을 이용하였다. 항 염증성 사이토카인 전사조질인자의 증가에 이어 Foxp3+ 전자조절 인자발현의 상당한 증가를 확인하였다. L. sakei proBio65는 염증성면역 장애의 조절에 치료적으로 유용할 것이다. We isolated and identified a novel probiotic strain, Lactobacillus sakei proBio65 from Kimchi. To determine whether L. sakei proBio65 has an immunomodulatory effect, we investigated cells via an in vitro screening system which co-cultured freshly isolated mesenteric lymphocyte with probiotics. A significant increase of Foxp3+ transcription regulatory factor expression was observed, followed by an increase in anti-inflammatory cytokines transcription regulatory factor. L. sakei proBio65 exhibited high levels of the IL-10/IL-12 production ratio and enhanced Foxp3 expression in vitro. L. sakei proBio65 may thus be therapeutically useful for the modulation of inflammatory immune disorders.

동치미로부터 분리한 Lactobacillus sakei J4의 항균활성 및 생균제로서 특성

박중현,오덕환,정하열 한국산업식품공학회 2012 산업 식품공학 Vol.16 No.2

본 연구는 probiotics 균주 선발을 위해 동치미로부터 식중독균에 대해 높은 항균활성을 보이는 유산균을 분리동정하여 배양조건을 확립하고, 위산, 담즙산, 췌장액에 대한 저항성 실험을 비교, 분석하여 probiotics 균주로 사용 가능성을 탐색하고자 하였다. 2% CaCO3를 첨가한 MRS agar를 이용하여 김치 무리로부터 식중독균의 저해능이 우수한 8 균주를 분리하였다. 식중독균에 대해 항균활성실험 결과 J4가 B. cereus는 19.85±0.53 mm, L. monocytogenes는 18.37±0.38 mm, S. aureus는 22.49±0.37 mm, E. coli는 17.90±0.64 mm, S. enterica는 16.28±0.53 mm, V. parahaemolyticus는 24.59±0.29 mm의 생육저해환을 형성하여 가장 높은 항균력을 나타내었다. J4 균주의 16S rDNA 염기서열을 분석한 결과 L. sakei와 99% 상동성을 나타내어 L. sakei J4라 명명하였다. L. sakei J4의 최적배양조건은 pH 8.5, 30, 정치배양시 가장 활발하게 생육이 진행됨을 확인하였다. L. sakei J4는 인공위액에서의 생존율 24.3±3.0%, 담즙산에서의 생존율 25.8±0.4%, 췌장액에서의 생존율 101.2±4.5%로 다른 균주보다 상대적으로 생존율이 높았으며, 향후 alginate를 이용한 캡슐화, chitosan을 이용한 재코팅 등의 연구방법을 정립하여 인공위액에 대한 저항성을 높인다면 프로바이오틱스 균주로서 조건을 충족시킬 수 있으리라 사료된다. Various lactic acid bacteria were isolated from Korean Dongchimi (whole radish Kimchi with added water) in order to study their antimicrobial activity, optimal culture conditions and probiotics. Among the eight isolated strains, J4 showed the highest inhibitory effect on pathogens (Bacillus cereus KCTC 3624, Listeria monocytogenes KCTC 13064, Staphylococcus aureus subsp. aureus KCTC 3881, Escherichia coli KCTC 2441, Salmonella enterica subsp. enterica KCTC 2514, Vibrio parahaemolyticus KCTC 2729). It was identified as Lactobacillus sakei based on its morphological and 16S rRNA gene sequence, and designated as L. sakei J4. The optimal pH of the medium, culture temperature and shaking speed for the antimicrobial substance (s) production were pH 8.5, 30oC and 0 rpm, respectively. L. sakei J4 had 24.3% of survival after 2 hr incubation in the artificial gastric juice, 25.8% of survival after 24 h incubation in the presence of 0.3% oxgall and 101.2% of survival after 24 hr incubation in the 0.5% pancreatic juice. These results suggest that Lactobacillus sakei J4 may be commercially used for the probiotic culture.

김치로부터 Lactobacillus sakei 생육저해 Leuconostoc 및 Weissella 속 균주의 분리

이광희 ( Kwang Hee Lee ),이종훈 ( Jong Hoon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2011 한국미생물·생명공학회지 Vol.39 No.2

Kimchi is a group of traditional fermented vegetable foods in Korea and known to be the product of a natural mixed-fermentation process carried out principally by lactic acid bacteria (LAB). According to microbial results based on conventional identification, Leuconostoc mesenteroides and Lactobacillus plantarum were considered to be responsible for the good taste and over-ripening of kimchi, respectively. However, with the application of phylogenetic identification, based on 16S ribosomal RNA gene similarities, a variety of Leuconostoc and Lactobacillus species not detected in the previous studies have been isolated, together with a species in the genus Weissella. Additionally, Lactobacillus sakei has been accepted as the most populous LAB in over-ripened kimchi. In this study, Leuconostoc and Weissella species inhibiting the growth of Lb. sakei were isolated from kimchi for future applications to do with kimchi fermentation. From 25 kimchi samples, 378 strains in the genera Leuconostoc and Weissella were isolated and 68 strains identified as Lc. mesenteroides, Lc. citreum, Lc. lactis, W. cibaria, W. confusa, and W. paramesenteroides exhibited growth inhibition against Lb. sakei. Most of the strains also had antagonistic activities against Lb. brevis, Lb. curvatus, Lb. paraplantarum, Lb. pentosus, and Lb. plantarum. Their antagonistic activities against Lb. sakei were more remarkable at lower temperatures of incubation.

단보 : Lactobacillus sakei 생육저해활성 보유 Leuconostoc mesenteroides가 생산하는 Bacteriocin의 특성

이광희 ( Kwang Hee Lee ),이종훈 ( Jong Hoon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2011 한국미생물·생명공학회지 Vol.39 No.4

Lactobacillus sakei is known to be the most populous lactic acid bacteria in over-ripened kimchi. Twenty three strains of Leuconostoc species inhibiting the growth of Lb. sakei were isolated from kimchi and amongst these the Leuconostoc mesenteroides strain CK0122 exhibited the highest antagonistic activity against Lb. sakei. The culture supernatant of the strain CK0122 was fractionated by a molecular weight cutter and lyophilized. The fraction with a molecular weight of less than 3,000 Da showed antagonistic activity against Lb. sakei. The antibacterial activity of the active fraction was sensitive to proteinase K treatment, confirming its proteinaceous nature (bacteriochi). The crude bacteriocin was active in the pH range of 4 to 7 and extremely stable after 15 min of heat treatment at 121℃. The crude bacteriocin inhibited the growth of Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Alcaligenes xylosoxydans, Flavobacterium sp., and Salmonella typhimurium.

비단백질성 항균물질을 생산하는 김치발효용 내산성 Hetero 발효형 유산균주 선발

김혜림 ( Hye Rim Kim ),이종훈 ( Jong Hoon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.1

선행연구에서 김치발효 후기의 우점종으로 알려진 Lactobacillus sakei의 생육을 저해하는 Leuconostoc 속 23균주와 Weissella 속 45 균주를 김치로부터 분리, 동정하였다. 발효후기까지 생존할 수 있는 김치발효용 hetero 발효형 종균 선발을 위하여 이들 균주에 대한 내산성을 평가한 결과, Lc. mesenteroides CK0128, W. cibaria CK0633, W. cibaria KK0797 균주가 acetic acid와 lactic acid 혼합용액을 이용하여 pH를 4.3으로 조정한 MRS broth에서 상대적으로 높은 생존율을 보였고, 다량의 세포 외 다당류를 생산하였다. 세균주가 생산하는 항균물질의 분자량은 3,000 Da 이하로 추정되며 Staphylococcus aureus와 Lb. sakei에 대한 생육저해를 나타내었다. 분획한 3,000 Da 이하의 조항균물질 모두가 121oC, 15분의 열처리에도 항균활성을 유지함으로써 항균물질의 열에 대한 높은 안정성이 확인되었다. pH의 감소에 따른 항균활성의 증가가 pH 5 이하의 산성조건에서 확인되어, 이들 항균물질은 pH 5 이하의 산성조건에서 활성을 갖는 것으로 추정된다. α-amylase, lipase, pepsin, proteinase K 처리가 항균활성에 아무런 영향을 미치지 않는 것으로 보아 이들 균주가 생산하는 항균물질은 탄수화물, 지질을 포함하지 않으며, 비단백질성 물질로 추정된다. 또한, 선발균주가 생산하는 비단백질성 항균물질은 식중독균의 생육을 효과적으로 저해하였다. Twenty-three strains of Leuconostoc species and 45 strains of Weissella species inhibiting the growth of Lactobacillus sakei, one of the most populous lactic acid bacteria in over-ripened kimchi, were isolated from kimchi in our previous study. Among these hetero-fermentative 68 strains, Leuconostoc mesenteroides CK0128, Weissella cibaria CK0633, and W. cibaria KK0797 exhibited a relatively high survival rate in MRS medium, which was adjusted to pH 4.3 using an acid mixture consisting of acetic and lactic acids, and produced a large amount of exopolysaccharides. The culture supernatants of 3 strains were fractionated by a molecular weight cutter and lyophilized. The fractions with a molecular weight smaller than 3,000 Da showed antagonistic activity against Staphylococcus aureus and Lb. sakei. The anti-bacterial substances were very stable to heat treatments (121oC, 15 min) and active at acidic conditions below pH 5. α-Amylase, lipase, and proteolytic enzymes (proteinase K and pepsin) did not affect their activities. These non-proteinaceous anti-bacterial substances inhibited the growth of several food pathogens.

<i>Lactobacillus sakei</i> BK19 enriched diet enhances the immunity status and disease resistance to streptococcosis infection in kelp grouper, <i>Epinephelus bruneus</i>

Harikrishnan, Ramasamy,Balasundaram, Chellam,Heo, Moon-Soo Elsevier 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.29 No.6

<P><B>Abstract</B></P><P>The effect of <I>Lactobacillus sakei</I> BK19 (10<SUP>8</SUP> cells g<SUP>−1</SUP>) supplemented diet fed to kelp grouper, <I>Epinephelus bruneus</I> against streptococcosis caused by <I>Streptococcus iniae</I> and <I>Streptococcus parauberis</I> with reference to the innate immune response and disease resistance was evaluated at 1, 2, and 4 weeks. Maximum reduction in mortalities was observed in kelper feeding the probiotic diet for two weeks after challenged with the pathogens when compared to the infected group fed with basal diet; similarly the cellular and humoral immune responses such as head kidney macrophage phagocytic and peroxidase activities, serum lysozyme activity, and total protein levels increased significantly. The results reveal that, in streptococcosis infected kelp grouper feeding <I>L. sakei</I> BK19 enriched diet affords a higher level of disease protection due to stimulation of immune system.</P>

Extracellular vesicles from Lactobacillus sakei prevent RANKL-induced osteoclast differentiation

Joo-Hee Choi,Kyung-Ku Kang,Soo-Eun Sung,Si-joon Lee,Min-Soo Seo 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

The postbiotics are the complex mixture of metabolites secreted by probiotics and relatively not yet been clearly defined. Recent studies suggest that postbiotics may elicit health benefits because they have efficacy equivalents to that of probiotic bacteria. However, the inhibitory effect and molecular mechanisms of L. sakei-derived extracellular vesicles (LEVs) on osteoclast differentiation have yet to be clearly elucidated. Thus, we investigated the effects of LEVs on RANKL-induced osteoclast differentiation. LEVs were isolated from Lactobacillus sakei-cultured supernatant. Characterization of EVs included typical evaluation of size analysis and concentration of EVs by nanoparticle tracking analysis (NTA) and morphology analysis by transmission electron microscopy (TEM). Osteoclastogenesis was determined by TRAP staining, F-actin ring formation, and bone resorption assay. Expression of osteoclastogenesis-related genes and activation of NF-kB and MAPKs were determined by RT-PCR and Western blotting, respectively. The isolated LEVs had an average size of 153.2 nm and morphological assessments showed a spherical shape with a lipid bilayer structure. LEVs inhibited the RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by LEVs treatment of RANKL-treated BMDMs. In addition, LEVs decreased the RANKL-induced activation of the MAPKs/NF-κB axis at the early stage and the expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. PRMT1 levels, new biomarkers for osteoclastogenesis, were decreased by LEVs treatment. These findings suggest that LEVs inhibited RANKL-induced osteoclast differentiation via the downregulation of molecular mechanisms and could be a preventive candidate for destructive bone diseases.

Anti-colitis effect of <i>Lactobacillus sakei</i> K040706 via suppression of inflammatory responses in the dextran sulfate sodium-induced colitis mice model

Seo, Seunghwan,Shin, Ji-Sun,Lee, Woo-Seok,Rhee, Young Kyoung,Cho, Chang-Won,Hong, Hee-Do,Lee, Kyung-Tae Elsevier 2017 Journal of Functional Foods Vol.29 No.-

<P><B>Abstract</B></P> <P>Lactobacillus strains are used in the functional food industry as promising probiotics with purported beneficial effects. We recently reported on the immunostimulatory effects of <I>Lactobacillus sakei</I> K040706 (K040706) in macrophages and in a cyclophosphamide-induced immunosuppression model. And this study assessed the anti-inflammatory effects and molecular mechanisms involving K040706 in a mouse model of colitis with dextran sodium sulfate (DSS). K040706 significantly attenuated the clinical signs and histological characteristics in DSS-induced colitic mice, which are well-correlated with the restoration of tight junction protein expression and the significant reduction of immune cell infiltration and expression of ICAM-1 and VCAM-1. Moreover, K040706 reduced the abnormal expression of pro-inflammatory mediators, such as iNOS, TNF-α, IL-1β, and IL-6, and the activation of NF-κB, STAT3 and TLR4. K040706 also improved the composition of intestinal microbiota. These results suggest K040706 has shown significant anti-inflammatory effects in a DSS-induced colitis model and has the potential to treat IBD.</P> <P><B>Highlights</B></P> <P> <UL> <LI> K040706 attenuated DSS-induced colitis severity and epithelial injury. </LI> <LI> K040706 suppressed the DSS-induced expression of pro-inflammatory mediators. </LI> <LI> K040706 downregulated the DSS-induced activation of TLR4/NF-κB/STAT3 signaling. </LI> <LI> K040706 improved the intestinal microbiota composition. </LI> <LI> K040706 has potential as a treatment option for inflammatory bowel disease. </LI> </UL> </P>

김치 미생물 조성을 바탕으로 한 김치 스타터의 선정 및 효과

진효상(Hyo Sang Jin),김종범(Jong Bum Kim),윤영주(Yeong Ju Yun),이경자(Kyung Ja Lee) 한국식품영양과학회 2008 한국식품영양과학회지 Vol.37 No.5

대다수 숙성 김치의 미생물 조성이 L. mesenteroides와 L. sakei로 구성되며, 이러한 혼합조성을 가진 김치가 맛이 있다는 점을 바탕으로 김치 맛을 가장 좋게 하며 김치 미생물을 조절하는 능력을 가진 두 균주 L. mesenteroides K2M5와 L. sakei K5M3을 각각 김치스타터로 선정하였다. 선정된 두 가지 균주들은 베타 용혈현상을 보이지 않았고, 암모니아나 인돌을 생성하지 않았으며, 젤라틴을 용해하지 않았고, phenylalanine deaminase, β-glucuronidase, β-glucosidase, 7α-dehydroxylase, nitroreductase 등의 효소활성에서 음성을 보여 산업적으로 사용하여도 안전한 것으로 나타났다. 선정된 두 가지 균주를 첨가하여 제조한 김치는 비첨가 김치에 비해 실험실 평가와 다수의 일반인을 상대로 한 비교평가에서 맛이 더 우수하였고, 미생물 조성은 오직 첨가한 균주로만 구성되어 선정한 균주들은 김치 맛을 좋게 하고 미생물 조성을 일정하게 조절하는 능력이 있는 스타터로 사용될 수 있음을 보여주었다. 스타터를 첨가한 김치는 대조김치에 비해 젖산 등의 유기산 함량이 특이하게 높았으나 산도, 염도, 당 함량 등에서는 큰 차이를 나타내지않았다. Based on information about the major microbial composition of kimchi and its relation to the taste, Leuconostoc mesenteroides K2M5 and Lactobacillus sakei K5M3 were selected as kimchi starter candidates. These two strains were found to be safe for industrial use because they showed neither harmful characteristics like β-hemolysis, ammonia and indole formation, and gelatin liquefaction, nor enzymatic activities like phenylalanine deaminase, β-glucuronidase, β-glucosidase, 7α-dehydroxylase and nitroreductase. Starter kimchi made with these strains were better in taste than the conventional kimchi when they are evaluated both by laboratory personnel and the public. Also microbial analysis of starter kimchi showed only starter bacteria after they were fermented to have the optimum acidity. Starter kimchi prepared with these two strains were not much different in physicochemical properties to the conventional kimchi except in that the starter kimchi were much higher in volatile organic acid content such as lactic acid. These results suggest that kimchi quality can be controlled to have consistent properties, both in taste and microbial composition, by using bacterial starters.