• Title/Summary/Keyword: subtilis

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Development of a Molecular Selection Marker for Bacillus licheniformis K12 (Bacillus licheniformis K12 균주 분자 선발 마커 개발)

  • Young Jin Kim;Sam Woong Kim;Tae Wok Lee;Won-Jae Chi;Woo Young Bang;Ki Hwan Moon;Tae Wan Kim;Kyu Ho Bang;Sang Wan Gal
    • Journal of Life Science
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    • v.33 no.10
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    • pp.808-819
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    • 2023
  • This study was conducted to develop a selection marker for the identification of the Bacillus licheniformis K12 strain in microbial communities. The strain not only demonstrates good growth at moderate temperatures but also contains enzymes that catalyze the decomposition of various polymer materials, such as proteases, amylases, cellulases, lipases, and xylanases. To identify molecular markers appropriate for use in a microbial community, a search was conducted to identify variable gene regions that show considerable genetic mutations, such as recombinase, integration, and transposase sites, as well as phase-related genes. As a result, five areas were identified that have potential as selection markers. The candidate markers were two recombinase sites (BLK1 and BLK2), two integration sites (BLK3 and BLK4), and one phase-related site (BLK5). A PCR analysis performed with different Bacillus species (e.g., B. licheniformis, Bacillus velezensis, Bacillus subtilis, and Bacillus cereus) confirmed that PCR products appeared at specific locations in B. licheniformis: BLK1 in recombinase, BLK2 in recombinase family protein, and BLK3 and BLK4 as site-specific integrations. In addition, BLK1 and BLK3 were identified as good candidate markers via a PCR analysis performed on subspecies of standard B. licheniformis strains. Therefore, the findings suggest that BLK1 can be used as a selection marker for B. licheniformis species and subspecies in the microbiome.

Effects of Microbial Fermentation on the Antioxidant Activities of Protaetia brevitarsis Larvae (미생물 발효가 흰점박이꽃무지(Protaetia brevitarsis) 유충의 항산화 활성에 미치는 영향)

  • Han Bi Kim;Hye Soo Kim;Soo Jeong Cho
    • Journal of Life Science
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    • v.33 no.12
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    • pp.1052-1061
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    • 2023
  • This study was carried out to evaluate the effect of fermentation by B. subtilis (BPLE), L. brevis (LPLE), S. cerevisiae (SPLE) and C. militaris (CPLE) on the antioxidant activity of Protaetia brevitarsis larvae fed with mushroom substrates (king oyster mushroom). The total polyphenol content of Protaetia brevitarsis larvae (PLE), BPLE, LPLE, SPLE and CPLE were 58.07±0.67, 83.33±0.98, 79.21±1.32, 61.02±0.87 and 57.90±1.02 mg GAEs/extract g, respectively. The flavonoid contents of the PLE, BPLE, LPLE, SPLE and CPLE were 17.35±1.57, 19.49±0.95, 16.90±1.57, 18.12±0.95 and 16.99±0.95 mg QEs/extract g, respectively. The DPPH radical scavenging activity showed no significant difference between the PLE, BPLE, LPLE, SPLE and CPLE at a concentration of 0.2 mg/ml. However, at a concentration of 0.4 mg/ml or more, the DPPH radical scavenging activity of the BPLE and LPLE was higher than that of the PLE. The reducing power of the BPLE and LPLE was also higher than that of the PLE, and more than twice as high at a concentration of 0.8 mg/ml or more. The ORAC value of the BPLE (79.77±0.82 uM TEs/extract g) was higher than that of the PLE (61.34±0.97 uM TEs/extract g). A WST-1 assay of the RAW 264.7 cells indicated that the PLE, BPLE, LPLE, SPLE and CPLE showed no cytotoxicity.

Metagenomic Analysis of Jang Using Next-generation Sequencing: A ComparativeMicrobial Study of Korean Traditional Fermented Soybean Foods (차세대 염기서열 분석을 활용한 장류의 메타지놈 분석 : 한국 전통 콩 발효식품에 대한 미생물 비교 연구)

  • Ranhee Lee;Gwangsu Ha;Ho Jin Jeong;Do-Youn Jeong;Hee-Jong Yang
    • Journal of Life Science
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    • v.34 no.4
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    • pp.254-263
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    • 2024
  • Korean jang is a food made using fermented soybeans, and the typical products include gochujang (GO), doenjang (DO), cheonggukjang (CH), and ganjang (GA). In this study, 16S rRNA metagenome analysis was performed on a total of 200 types of GO, DO, CH, and GA using next-generation sequencing to analyze the microbial community of fermented soybean foods and compare taxonomic (biomarker) differences. Alpha diversity analysis showed that in the CHAO index, the species richness index tended to be significantly higher compared to the DO and GA groups (p<0.001). The results of the microbial distribution analysis of the GO, DO, CH, and GA products showed that at the order level, Bacillales was the most abundant in the GO, DO, and CH groups, but Lactobacillales was most abundant in the GA group. Linear discriminant analysis effect (LEfSe) analysis was used to identify biomarkers at the family and species levels. Leuconostocaceae, Thermoactinomycetaceae, Bacillaceae, and Enterococcaceae appeared as biomarkers at the family level, and Bacillus subtilis, Kroppenstedtia sanguinis, Bacillus licheniformis, and Tetragenococcus halophilus appeared at the species level. Permutational multivariate analysis of variance (PERMANOVA) analysis showed that there was a significant difference in the microbial community structure of the GO, DO, CH, and GA groups (p=0.001), and the microbial community structure of the GA group showed the greatest difference. This study clarified the correlation between the characteristics of Korean fermented foods and microbial community distribution, enhancing knowledge of microorganisms participating in the fermentation process. These results could be leveraged to improve the quality of fermented soybean foods.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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A Study on the Manufacturing of Soysauce by the Combined use of Aspergillus sojae and Bacillus subtilis (단백질분해세균(蛋白質分解細菌)을 병용(倂用)한 간장제조(製造)에 관(關)한 연구(硏究))

  • Kim, Ze-Uook;Cho, Sung-Hwan
    • Applied Biological Chemistry
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    • v.18 no.1
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    • pp.1-9
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    • 1975
  • In order to study on the preparation of improved soysauce, Koji(soybean and wheat mash fermented by the strain Aspergillus sojae), Koji-Natto(mixed with the various proportion of Koji to Natto; 8 : 2, 6 : 4, 4 : 6, 2 : 8) and Natto (prepared using the strain: Bacillus subtilis) were prepared as soysauce materials. Sensory evaluation for the soysauces made from above-mentioned soysauce materials and the contents of total nitrogen, amino nitrogen, ammonia nitrogen, reducing sugar, total acid and enzyme activity were determined. The results were summarized as follows: 1. Protease activity of Natto was over twice higher than that of Koji and amylase activity of Koji was three times higher than that of Natto. Koji-Natto showed successive increase of amylase activity and successive decrease of protease activity as Koji proportion was increased to Natto. 2. The contents of total nitrogen and amino nitrogen in all the soysauce mashes displayed linear increases or no changes of these contents as aging. The contents of total nitrogen and amino nitrogen in the mahes were higher as Natto proportion increase to Koji during the aging period. 3. In Natto soysauce mash and Koji-Natto soysauce mashes the contents of ammonia nitrogen were gradually increased until $1{\sim}2$ months and rapidly reduced after that period, while Koji soysauce mashes were continuatively increased. 4. In all the soysauce mash the reducing sugar increased preeminently until the two months and after then there was a rapid decrease as aging the contents of reducing sugar in the mash were higher as Natto proportion increased to Koji during aging period. 5. The total acid of Koji and Koji-Natto (8 : 2) soysauce mash showed gradual increase as aging, while contents of total acid in Natto and other Koji-Natto soysauce mashes increased preeminently until 50${\sim}$70 days aging, after then decreased. 6. The results of sensory evaluation for three-month-fermented soysauces showed the most excellent in Koji-Natto (6 : 4) and successive order in the soysauces consisting of Koji and Natto proportion: 8 : 2, 10 : 0, 4 : 6, 2 : 8, 0 : 10.

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Fermentation Characteristics of Juice Pomace Feed by Horse Feces Microbes (말분변 미생물을 이용한 주스박 사료의 발효 특성)

  • Hwang, Won-Uk;Kim, Gyeom-Heon;Niu, Kai-Min;Lim, Joung-Ho;Woo, Jae-Hoon;Chae, Hyun-Seok;Park, Nam-Geon;Kim, Soo-Ki
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.37 no.4
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    • pp.290-300
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    • 2017
  • In vitro fermentation was conducted to figure out alternative fiber sources for horse feed. For the development of value-added products as a horse feed resource, the pomaces from apple, carrot, grape, and citrus were fermented under solid-state conditions in the presence of 60% soybean meal with 40% of each fruit pomace at 60% of moisture content. Lactobacillus plantarum SK3873, Lactobacillus plantarum SK3893, Weissella cibaria SK3880, and Bacillus subtilis SK3889 were isolated from the fermented fruit pomace by inoculation of horse feces. For the growth of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum, they were inoculated in 3-step order at 0, 12, and 24 h, respectively. The fruit pomace was fermented for 48 h at $35^{\circ}C$. The pH of the apple, carrot, grape, citrus and all mixed pomaces decreased from 5.45~6.25 to 4.40~4.77. Microbial growth was maintained at $10^8{\sim}10^9cfu/g$. After 12 and 24 h incubation, dry matter of carrot pomace were highest at 54.84 and 56.66%, respectively (P<0.05) and that of grape pomace was lower than others during fermentation (P<0.05). Dry matter was generally reduced by about 20%. NDF decreased gradually or maintained after 24 h, indicating the fiber degradation. Ash content tended to decrease during fermentation. After 48 hours fermentation, Bacillus, yeast and Lactobacillus showed an excellent growth by using juice by-products. These results suggest that fermented juice pomace has a potential as horse feedstuff with probiotics to maintain beneficial microflora in horse gut.

Antioxidant and Antibacterial Activities of Glycyrrhiza uralensis Fisher (Jecheon, Korea) Extracts Obtained by various Extract Conditions (한국 제천 감초(Glycyrrhiza uralensis Fisher)의 추출 조건별 추출물의 항산화 및 항균 활성 평가)

  • Ha, Ji Hoon;Jeong, Yoon Ju;Seong, Joon Seob;Kim, Kyoung Mi;Kim, A Young;Fu, Min Min;Suh, Ji Young;Lee, Nan Hee;Park, Jino;Park, Soo Nam
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.41 no.4
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    • pp.361-373
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    • 2015
  • This study was carried out to evaluate the antioxidant and antibacterial activities of Glycyrriza uralensis Fisher (Jecheon, Korea) extracts obtained by various extraction conditions (85% ethanol, heating temperatures and times), and to establish the optimal extraction condition of G. uralensis for the application as cosmetic ingredients. The extracts obtained under different conditions were concentrated and made in the powdered (sample-1) and were the crude extract solutions without concentration (sample-2). The antioxidant effects were determined by free radical scavenging activity ($FSC_{50}$), ROS scavenging activity ($OSC_{50}$), and cellular protective effects. Antibacterial activity was determined by minimum inhibitory concentration (MIC) on human skin flora. DPPH free radical scavenging activity of sample-1 ($100{\mu}g/mL$) was 10% higher in group extracted for 6 h than 12 h, but sample-2 didn't show any significant differences. The extraction yield extracted with same temperature for 12 h was 2.6 times higher than 6 h, but total flavonoid content was 1.1 times higher. These results indicated that total flavonoid content hardly increased with increasing extraction time. Free radical scavenging activity, ROS scavenging activity and cellular protective effects were not dependent on the yield of extraction, but total flavonoid content of extraction. Antibacterial activity on three skin flora (S. aureus, B. subtilis, P. acnes)of sample-1 in different extraction conditions were evaluated on same concentration, and the group extracted at 25 and $40^{\circ}C$ showed 16 times higher than methyl paraben ($2,500{\mu}g/mL$). In conclusion, 85% ethanol extracts of G. uralensis extracted at $40^{\circ}C$ for 6 h showed the highest antioxidant and antibacterial activity. These results indicate that the extraction condition is important to be optimized by comprehensive evaluation of extraction yield with various conditions, yield of active component, and activity test with concentrations, and activity of 100% extract, for manufacturing process of products.

Biochemical Composition of Marine Microalgae and Their Potential Antimicrobial Activity

  • Kim Se-Kwon;Jeon You-Jin;Kim Won-Suk;Back Ho-Cheol;Park Pyo-Jam;Byun Hee-Guk;Bai Sungchul C.
    • Fisheries and Aquatic Sciences
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    • v.4 no.2
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    • pp.75-83
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    • 2001
  • This study is to investigate biochemical compositions of two species of marine microalgae, Chlorella ellipsoidea of Chlorophyta and Tetraselmis suecica of Prasinophyta, and to assess their potential antimicrobial activities. Crude protein, lipid and carbohydrate for C. ellipsoidea were $43.15\%$, $12.63\%$ and $13.09\%$, respectively, and those for T. suecica were $44.95\%$, $4.80\%$ and $24.05\%$, respectively. The major amino acids of the two micro algae were aspartic acid, glutamic acid, glycine, alanine, valine, leucine, lysine and proline, and no significant difference between the amino acid compositions of both micro algae was observed. The major sugars for both microalgae were glucose, galactose and mannose, and glucose contents showed the highest level, $58.70\%$ for C. ellipsoidea and$57.86\%$ for T. suecica. The major mineral contents of both micro algae for 100g were Ca (3,114mg in C. ellipoidea and 3,389mg in T. suecica) and followed by Na (2,881mg), K (548mg) and Mg (545mg) for C. ellipsoidea and Na (1,832 mg), Mg (1,510mg) and K (548mg) for T. suecica. In the content of ATP-related compound, hypoxanthine in C. ellipsoidea and IMP in T. suecica were absolutely dominant compounds. The highest content of fatty acid in C. ellipsoidea was 20:4, $27.15\%$ and that in T. suecica was 18:3 (w-6), $18.10\%$. In case of physiologically important polyunsaturated fatty acids like eicosapentaenoic acid (20: 5) and docosahexaenoic acid (22: 6), both microalgae possessed just trace amounts but was rich in arachidonic acid (20: 4). Vitamin content in both microalgae was significantly high in choline and inositol. In antimicrobial activity by water- and fat-soluble fraction of the micro algae, hexane extract in the fat-soluble fraction of C. elliposidea inhibited the growth of Bacillus subtilis by $96\%$ bactericidal activity and tetrachlorocarbon extract of T. suecica indicated relatively excellent antimicrobial activity $(81\%\;bactericidal\;activity)$ against Escherichia coli. Hot water extract among water-soluble fraction of both micro algae almost suppressed the growth of Staphylococcus aureus by $96\%$ bactericidal activity.

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Effect of Starter Cultures on the Fermentative (발효 균주에 따른 청국장의 발효특성)

  • Baek, Lag-Min;Park, La-Young;Park, Kuem-Soon;Lee, Shin-Ho
    • Korean Journal of Food Science and Technology
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    • v.40 no.4
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    • pp.400-405
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    • 2008
  • The effects of different starter cultures on the fermentative characteristics of cheonggukjang were examined by using three Bacillus strains. The strains included Bacillus subtilis KCTC 1021 as a control, Bacillus sp. Kn-10 (Kn-10) isolated from a commercial cheonggukjang, and Bacillus sp. B-59 (B-59) isolated from rice straw. There were no significant differences in pH or viable cells among the different cheonggukjang samples during fennentation for 72 hr at $40^{\circ}C$. However, the sample prepared with B-59 had higher slime content and protease activity than the controls and Kn-10 samples. DPPH free radical scavenging activity was higher in the B-59 sample and lower in the control and Kn-10 samples when compared to steamed soybeans after fermentation for 72 hr at $40^{\circ}C$. The total amino acid contents the cheonggukjangs were 34869.98 mg% (B-59), 34481.89 mg% (control), and 31791.09 mg% (Kn-10). Glutamic acid and lysine contents were higher in the B-59 sample than in the control. Finally, the cheonggukjang fermented using the B-59 strain had improved sensory qualities such as color, taste, texture, and overall acceptability compared to the control and Kn-10 samples.

Studies on Relationships between Marine Bacteria and Phytoplankton in Suyeong Bay -1. On Relationshops of Dominant Species between Marine Bacteria and Phytoplankton- (해양미생물과 식물플랑크톤의 상호관계 -1. 수영만의 해양세균과 식물플랑크톤 우점종 간의 상호관계-)

  • Kim, Dong-Won;Lee, Won-Jae
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.26 no.5
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    • pp.446-457
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    • 1993
  • Monthly distribution of marine bacteria and phytoplankton in Suyeong Bay were investigated with laboratory experiment of dominant algal species and bacterial flora. During the periods of study, the highest density of phytoplankton and bacteria occurred in May with the number of $3.3{\times}10^6cells/l$ and $1.93{\times}10^8cells/ml$, respectively. 10 genera of bacteria and 22 genera of phytoplankton were isolated and identified. In May when phytoplankton bloom occurred, dominant species of bacteria were Acinetobacter calcoaceticus($29.1\%$) and Bacillus subtilis($22.9\%$), and dominant species of phytoplankton were Chaetoceros spp.($62.8\%$) and Skeletonema spp. ($19.4\%$). Pseudomonas spp., which was the most abundant bacterial species during the study periods, were rapidly decreased in May. In laboratory studies of culturing bacteria and phytoplankton isloated in May, the growth of Pseudomonas vesicularis seems to be influenced by the concentrations of excretion matter of Chaetoceros spp. To examine the result colsely, the problem of pure isolation for phytoplankton must be solved and more experimental process have to be conducted.

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