김치에서 분리한 Lactococcus sp. J-105가 생산하는 Bacteriocin의 특성

곽규숙,구재관,배경미,전홍기 한국생명과학회 1999 생명과학회지 Vol.9 No.1

우리 고유의 전통 발효 음식인 김치로부터 bacteriocin 생성능이 우수한 Lactococcus sp. J-105균주를 분리하였다. Lactococcus sp. J-105의 생육 및 bacteriocin 생성에 대한 최적 조건을 검토한 결과 bacteriocin 생성은 대수 증식기 말기에서 정지기 초기에 가장 많이 생성되었다. Lactococcus sp. J-105의 생육 및 bacteriocin 생성 최적 탄소원 및 질소원은 각각 maltose와 polypeptone이었다. 균의 생육 및 bacteriocin 생성 최적 온도는 $25^{\circ}C$였으며 생육 최적 pH는 pH 8 부근이었다. bacteriocin 생성 또한 pH 8에서 최고를 나타내었다. 한편 NaCl은 오히려 균체 증식과 bac-tericoin 생성을 저해하였다. Lactococcus sp. J-105가 생산하는 bacteriocin의 pH 및 열에 대한 안정성을 검토한 결과 pH 2~4, $121^{\circ}C$ 15분에서 각각 안정하였다. 또한 J-105의 항균 spectrum을 검토한 결과 Lactobacillus sp. Leuconostoc s 그리고 Bacillus subtilis 등의 Gram 양성균에 대해서 항균활성 나타내었으며 Gram 음성균인 Acetobacter acid에 대해서도 항균 활성을 나타내었으나 E. coli에 대해서는 항균 활성을 나타내지 않았다. 본 bacteriocin은 trypsin, protease와 같은 peptide 분해효소에 대해서는 안정한 반면, pepsin에 의해서는 활성이 상실되는 특이한 성질을 나타내었다 A bacteriocin-producing strain, J-105, was isolated from Kimchi and identified as Lactococcus sp. The optimum conditions for the bacteriocin production from the isolated microorganism were evaluated. For maximum yield of bacteriocin from Lactotoccus sp. J-105, the cell should be harvested at the early stationary phase and temperature, pH and NaCl concentration should be $25^{\circ}C$, pH 8.0 and without the addition of NaCl, respectively. Maltose should be used as a carbon source and organic nitrogen such as polypeptone should be used as a nitrogen source for the best yield. The bacteriocin from isolate was inhibitory against Acetobacter aceti, Bacillus subtilis and several strains of lactic acid bacteria. The bacteriocin of J-105 was sensitive to pepsin, but stable for heat treatment. It was stable even at autoclaving temperature for 15 min.

항균성 미강 단백질 필름 개발을 위한 Bacteriocin 생성균주의 선별 및 특성

김은정,김경미,한혜경,김영호,권기성,배동호 한국응용생명화학회 2003 Applied Biological Chemistry (Appl Biol Chem) Vol.46 No.4

경제성과 항균력을 갖춘 미강단백질 필름을 개발하기 위한 연구의 일환으로 미강배지에서 bacteriocin을 생성할 수 있는 균주를 선별하고 그 특성을 조사하였다. 그 결과, Pseudomonas aeruginosa 9027에 대한 항균효과를 지닌 Pseudomonas aeruginosa 21025를 선별하였으며, 이 균이 생성한 bacteriocin은 쌀에 존재하는 미생물 및 토양미생물에 대하여 넓은 항균활성을 갖는 것으로 나타났다. 지금까지이용되고 있는 미강단백질 추출 조건에 대한 적합성을 알아보기 위해 bacteriocin의 특성을 조사하였다. Pseudomonas putida 21025에서 생성되는 bacteriocin은 50℃이사에서는 한 시간 후 활성이 저하되었으며, 헥산과 에탄올을 제외한 메탄올, 톨루엔, 클로로포름, 아세톤, 메틸 클로로포름등의 유기용매에서 활성이 현저하게 저하되었다. 그러나 이 bacteriocin은 pH 6.0-9.0에서 2시간 동안 안정하였고, 50℃이하에서 안정하였으며, 에탄올에 대하여 3시간동안 안정한 특성을 가지고 잇는 것으로 나타났다. 또한 부분정제한 bacteriocin의 분자량은 약 21.6kDa로 비교적 작은 분자량을 나타내었다. This study was conducted to select the bacteriocin-producing microorganism cultivated in the rice bran culture and to characterize the produced bacteriocin for the further purpose of economical and anti-bacterial rice bran protein film. Pseudomonas putida 21025 was cultivated from rice bran and identified as a producer of a bacteriocin which showed bacteriocin activity against Pseudomonas aeruginosa 9027. Bacteriocin produced by Pseudomonas putida 21025 showed a broad spectrum of activity against spoilage and soil bacteria. The activity of the bacteriocin produced by Pseudomonas putida 21025 decreased after 1 hr of staying at the temperature of 50℃ and with the presence of some organic solvents, except hexane and ethanol. However, the bacteriocin activity was stable throughout the pH ranges of 6-9 for 2 hrs, at the temperature lower than 50℃, and with the presence of ethanol for 3 hrs. The bacteriocin was partially purified by 50% ammonium sulfate precipitationn followed by subsequent dialysis. Direct detection of the partially purified bacteriocin on SDS-PAGE suggested that it had an apparent molecular mass of about 21.6 kDa.

Bacillus속 세균 유래 박테리오신의 특성과 응용

이지영,강대욱 한국생명과학회 2023 생명과학회지 Vol.33 No.1

Bacteriocins are antimicrobial peptides synthesized on ribosomes, produced by bacteria, that inhibit the growth of similar or closely related bacterial strains. Since the discovery of nisin, many bacteriocins with unique structures and various modes of antibacterial activity have been described, and genes encoding production, secretion, and immunity have been reported. Nisin is one of the bacteriocins applied in cheese, liquid eggs, sauces and canned foods. Many of the bacteriocins of the genus Bacillus belong to lantibiotics, which are modified peptides after translation. Other genus Bacillus also produce many non-lantibiotic bacteriocins. Bacteriocins of the genus Bacillus are sometimes becoming more important because of their broader antibacterial spectrum. Bacteriocins are considered attractive compounds in the food and pharmaceutical industries to prevent food spoilage and growth of pathogenic bacteria. Bacteriocins can be used as biological preservatives in a variety of ways in the food system. Biopreservation refers to extending shelf life and improving safety of foods using microorganisms and/or their metabolites. The demand for new antimicrobial compounds has generated great interest in new technologies that can improve food microbiological safety. Applications of bacteriocins are expanding from food to human health. Today, many researchers are shifting their interest in bacteriocins from food preservation to the treatment of bacteria that cause infections and antibiotic-resistant diseases. This exciting new era in bacteriocin research will undoubtedly lead to new inventions and new applications. In this review, we summarize the various properties and applications of bacteriocins produced by the genus Bacillus.

Lactococcus sp. CU216이 생산하는 박테리오신을 함유한 pH Sensitive Liposome의 응용

박성수,김명희,한경식,오세종 한국축산식품학회 2004 한국축산식품학회지 Vol.24 No.1

The objective of this study was to control Kimchi fermentation using pH sensitive bacteriocin entrapping liposome(bacteriocin-liposome). The liposomes were prepared by the reverse-phase evaporation method from a mixture of DPPC(dipalmitoyl phosphatidylcholine, DPPE(dipalmitoyl phosphatidylethanolamine), DOPC(dioleoyl phosphatidylcholine) and cholesterol in a molar ration of 4:2:1:4. The bacteriocin-liposome was disruptured at pH 4 of buffer and was stable at alkaline pHs(6 and 7). Irrespective of the addition of the bacteriocin-liposomes, the pH of every Kimchi sample decreased to 5 during 5 days storage at 5$^{\circ}C$. Kimchi samples treated with bacteriocin-liposomes maintained pH 4 or higher, while Kimchi samples not treated with bacteriocin-liposomes exhibited pH 3.58 or lower. In general, the pH of Kimchi samples stored at 20$^{\circ}C$ decreased faster, compared to that of Kimchi samples stored at 5$^{\circ}C$. The pH of Kimchi samples treated with the bacteriocin-liposomes was 3.9 during 90 days storage, while that of the samples not treated with the bacteriocin-liposomes was 3.68 and 3.32 during 30 days and 90 days storages, respectively. Lactic acid bacteria in Kimchi treated with the bacteriocin-liposome grew relatively slow at 5$^{\circ}C$. The viable cell number of lactic acid bacteria increased up to 4${\times}$10$\^$7/ cells/ml and then decreased to 8${\times}$10$\^$6/ cells/ml during 90 days storage at 5$^{\circ}C$. 발효식품의 후산발효를 조절하기 위하여 pH sensitive liposome의 이용 가능을 검토하였다. Lactococcm sp. CU216이 생산하는 박테리오신은 L.. acidophilus를 제외하고는 대부분의 유산균주들에 생육억제 효과가 있는 것으로 확인되었으며, 이를 Octyl-Sepharose column으로 분획하여 정제하였다. 정제된 박테리오신을 dipalmitoyl phosphocholine, dipalmitoyl phosphoethanolamine, dioleoyl phosphocholine 및 콜레스테롤를 각각 4:2:1:4(㏖ ratio)의 비율로 섞은 liposome을 제조하여 최종적으로 pH sensitive bacteriocin-liposome을 제조하였다. 이렇게 제조된 liposome은 pH 4부근에서 대부분 유리되는 것으로 확인되었으나 pH 6이상에서는 일어나지 않았다. 또한 pH sensitive bacteriocin-liposome 한국의 전통 발효식품인 김치에 적용하여 저장시 pH의 저하를 억제시키는 것으로 확인되었다. 본 실험 결과, pH sensitive bacteriocin-liposome은 발효식품의 후산발효 억제에 적용될 수 있으며 추후에 요구르트를 포함한 다른 발효식품에 대한 추가적인 실험이 필요한 것으로 생각되었다.

Bacillus subtilis Bacteriocin Bac 14B with a Broad Inhibitory Spectrum: Purification, Amino Acid Sequence Analysis, and Physicochemical Characterization

Ines Hammami,Bassem Jaouadi,Abir Ben Bacha,Ahmed Rebai,Samir Bejar,Xavier Nesme,Ali Rhouma 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.1

Bacillus subtilis strain 14B was used to produce a novel antimicrobial peptide (bacteriocin) called Bac 14B. Pure bacteriocin was obtained after heat and acidic treatments (80°C and pH 4), precipitation by ammonium sulfate, and chromatography on Sephadex G-50 and Mono Q Sepharose columns. Based on MALDI-TOF mass spectrometry analysis, purified Bac 14B is a monomer protein with a molecular mass of 20110.13 Da. N-terminal sequencing allowed for the straightforward identification of its first 12 residues, which were of a pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound. Bac 14B was stable for 1 h at temperatures up to 80°C and pH of 4 ~ 8. It also proved sensitive to various proteases, which demonstrated its protein nature. Bac 14B displayed a bacteriolytical mode of action and a broad range of inhibitory spectra toward Gram-positive and -negative pathogens. Interestingly,based on conventional agronomic seed vigor parameters,the application of Bac 14B (500 activity units/mL)to various crops revealed that this bacteriocin was a potent exogenous enhancer of growth that stimulated the seedling vigor of tomatoes and muskmelons. Compared to those of the control, the germination percentage, shoot weight,shoot height, and root length were all significantly enhanced in Bac 14B-treated plant seeds. Bac 14B also exhibited effective disinfectant properties against a wide range of seedborne diseases and significant effects on the control of damping off diseases, particularly at the pregermination stage. It also proved to be effective against root rot diseases caused by Alternaria solani and other bacterial seedborne pathogens such as wilt diseases. The findings indicate that Bac 14B is the first B. subtilisproduced bacteriocin ever reported to exhibit such promising biological properties. Bacillus subtilis strain 14B was used to produce a novel antimicrobial peptide (bacteriocin) called Bac 14B. Pure bacteriocin was obtained after heat and acidic treatments (80°C and pH 4), precipitation by ammonium sulfate, and chromatography on Sephadex G-50 and Mono Q Sepharose columns. Based on MALDI-TOF mass spectrometry analysis, purified Bac 14B is a monomer protein with a molecular mass of 20110.13 Da. N-terminal sequencing allowed for the straightforward identification of its first 12 residues, which were of a pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound. Bac 14B was stable for 1 h at temperatures up to 80°C and pH of 4 ~ 8. It also proved sensitive to various proteases, which demonstrated its protein nature. Bac 14B displayed a bacteriolytical mode of action and a broad range of inhibitory spectra toward Gram-positive and -negative pathogens. Interestingly,based on conventional agronomic seed vigor parameters,the application of Bac 14B (500 activity units/mL)to various crops revealed that this bacteriocin was a potent exogenous enhancer of growth that stimulated the seedling vigor of tomatoes and muskmelons. Compared to those of the control, the germination percentage, shoot weight,shoot height, and root length were all significantly enhanced in Bac 14B-treated plant seeds. Bac 14B also exhibited effective disinfectant properties against a wide range of seedborne diseases and significant effects on the control of damping off diseases, particularly at the pregermination stage. It also proved to be effective against root rot diseases caused by Alternaria solani and other bacterial seedborne pathogens such as wilt diseases. The findings indicate that Bac 14B is the first B. subtilisproduced bacteriocin ever reported to exhibit such promising biological properties.

Antimicrobial Activity of a Bacteriocin Produced by Enterococcus faecalis KT11 against Some Pathogens and Antibiotic-Resistant Bacteria

Hilal Seval Abanoz,Buket Kunduhoglu 한국축산식품학회 2018 한국축산식품학회지 Vol.38 No.5

In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Karg₁ Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gramnegative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, α-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at 121℃ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.

Brevicin 174Aγ-like novel bacteriocin produced by Lactobacillus brevis JP0063 isolated from kimchi

박재용 한국미생물학회 2023 미생물학회지 Vol.59 No.4

To isolate bacteriocin-producing lactic acid bacteria (LAB) that inhibit Lactobacillus plantarum, which can induce kimchi spoilage, a strain of L. plantarum was first isolated from kimchi and used as an indicator. The isolate was identified as L. plantarum through 16S rRNA sequencing and recA multiplex PCR analysis and was named L. plantarum JP0059. An LAB strain that inhibits JP0059 was isolated from kimchi, identified as Lactobacillus brevis through 16S rRNA sequencing, and named L. brevis JP0063. Lactobacillus brevis JP0063 showed maximum production of bacteriocin in the exponential and stationary phases. This bacteriocin primarily inhibited homolactic fermentable Lactobacillus, but not Leuconostc. The bacteriocins were partially purified from the culture supernatant using a cation-exchange column. SDS-PAGE confirmed that the partially purified bacteriocin had a molecular weight of approximately 5 kDa and was relatively stable under pH and heat treatment. However, the bacteriocin activity disappeared after proteinase K treatment. Bacteriocins produced by JP0063 inhibited JP0059 through bactericidal action. The N-terminal amino acid sequence of the bacteriocin was analyzed as KKKKKVACTWGNAAAA, which was highly homologous to that of brevicin 925A-γ produced by L. brevis 925A. Primers were designed based on the brevicin 925A-γ neighboring genes, and PCR amplification and sequencing was performed; two bacteriocin genes were found. The amino acid sequences deduced from the bacteriocin genes were compared with those of brevicin 925A. In the breB gene, a single amino acid in the signal peptide varied, whereas in breC gene, three amino acids in the bacteriocin structural gene varied.

Characterization of a Bacteriocin Produced by Enterococcus sp.T7 Isolated from Humans

Moon, Gi Seong,Jeong, Jong Jin,Ji, Geun Eog,Kim, Jong Sang,Kim, Jeong Hwan 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.4

A bacteriocin-producing organism, Enterococcus sp. T7, was isolated from human fecal samples. Bacteriocin T7, named tentatively as the bacteriocin, was produced by Enterococcus sp. T7 and it inhibited some strains of Lactobacillus, Staphylococcus, Enterococcus, and Streptococcus, but not all the lactococci and gram-negative bacteria tested. Bacteriocin T7 inhibited the growth of Listeria monocvtogenes Scott A, but the degree of inhibition was less than those for other sensitive gram-positive bacteria. Bacteriocin T7 in MRS broth started to produce at the middle of the exponential growth phase and the inhibitory activity reached its maximum level during the stationary growth phase. Bacteriocin T7 was stable against heat treatments, pH variations (pH2- 10), and exposure to organic solvents. The molecular weight of bacteriocin T7 was estimated to be 6,500 Da by SDS-PAGE. All these facts, including physico-chemical stabilities, small molecular size, and inhibition of Listeria monocytogenes, indicate that bacteriocin T7 is likely to be a member of the class Ila bacteriocins.

In vitro Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated from Nem Chua, a Traditional Vietnamese Fermented Pork

Pilasombut, Komkhae,Rumjuankiat, Kittaporn,Ngamyeesoon, Nualphan,Duy, Le Nguyen Doan Korean Society for Food Science of Animal Resource 2015 한국축산식품학회지 Vol.35 No.4

The aim of this study was to screen and In vitro characterize the properties of bacteriocin produced by lactic acid bacteria isolated from Vietnamese fermented pork (Nem chua). One hundred and fifty LAB were isolated from ten samples of Nem chua and screened for bacteriocin-producing lactic acid bacteria. Antimicrobial activity of bacteriocin was carried out by spot on lawn method against both gram positive and gram negative bacteria. One isolate, assigned as KL-1, produced bacteriocin and showed inhibitory activity against Lactobacillus sakei, Leuconostoc mesenteroides and Enterococcus faecalis. To characterize the bacteriocin-producing strain, optimum temperature, incubation period for maximum bacteriocin production and identification of bacteriocin-producing strain were determined. It was found that the optimum cultivation temperature of the strain to produce the maximum bacteriocin activity (12,800 AU/mL) was obtained at 30℃. Meanwhile, bacteriocin production at 6,400 AU/mL was found when culturing the strain at 37℃ and 42℃. The isolate KL-1 was identified as L. plantarum. Antimicrobial activity of cell-free supernatant was completely inhibited by proteolytic enzyme of trypsin, alpha-chymotrypsin and proteinase K. Bacteriocin activity was stable at high temperature up to 100℃ for 10 min and at 4℃ storage for 2 d. However, the longer heating at 100℃ and 4℃ storage, its activity was reduced.

In vitro Characterization of Bacteriocin Produced by Lactic Acid Bacteria Isolated from Nem Chua, a Traditional Vietnamese Fermented Pork

Komkhae Pilasombut,Kittaporn Rumjuankiat,Nualphan Ngamyeesoon,Le Nguyen Doan Duy 한국축산식품학회 2015 한국축산식품학회지 Vol.35 No.4

The aim of this study was to screen and In vitro characterize the properties of bacteriocin produced by lactic acid bacteria isolated from Vietnamese fermented pork (Nem chua). One hundred and fifty LAB were isolated from ten samples of Nem chua and screened for bacteriocin-producing lactic acid bacteria. Antimicrobial activity of bacteriocin was carried out by spot on lawn method against both gram positive and gram negative bacteria. One isolate, assigned as KL-1, produced bacteriocin and showed inhibitory activity against Lactobacillus sakei, Leuconostoc mesenteroides and Enterococcus faecalis. To characterize the bacteriocin-producing strain, optimum temperature, incubation period for maximum bacteriocin production and identification of bacteriocin-producing strain were determined. It was found that the optimum cultivation temperature of the strain to produce the maximum bacteriocin activity (12,800 AU/mL) was obtained at 30°C. Meanwhile, bacteriocin production at 6,400 AU/mL was found when culturing the strain at 37°C and 42°C. The isolate KL-1 was identified as L. plantarum. Antimicrobial activity of cell-free supernatant was completely inhibited by proteolytic enzyme of trypsin, alpha-chymotrypsin and proteinase K. Bacteriocin activity was stable at high temperature up to 100°C for 10 min and at 4°C storage for 2 d. However, the longer heating at 100°C and 4°C storage, its activity was reduced.