RESUMO
Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to 80°C. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.
Assuntos
Bacillus subtilis/metabolismo , Bacteriocinas/metabolismo , Microbiologia de Alimentos , Bacillus subtilis/isolamento & purificação , Bacteriocinas/antagonistas & inibidores , Bacteriocinas/química , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endopeptidase K/metabolismo , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Dados de Sequência Molecular , Peso Molecular , Estabilidade Proteica , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , TemperaturaRESUMO
Bacilli with fibrinolytic activities were isolated from traditionally-prepared Meju and some of these strains showed strong antifungal activities. One isolate, MJ1-4, showed the strongest antifungal activity. MJ1-4 and other isolates were identified as B. amyloliquefaciens strains by recA gene sequencing and RAPD-PCR results. B. amyloliqufaciens MJ1-4 efficiently inhibited an Aspergillus spp.-producing aflatoxin B1 (AFB1) and a Penicillium spp.-producing ochratoxin (OTA) in addition to other fungi. Antifungal activity of B. amyloliquefaciens MJ1-4 culture reached its maximum (40 AU/mg protein) in LB or TSB medium around 48 hr at 37°C. Antifungal activity of the concentrated culture supernatant was not decreased significantly by protease treatments, implying that the antifungal substance might not be a simple peptide or protein. Considering its antifungal and fibrinolytic activities together, B. amyloliquefaciens MJ1-4 can serve as a starter for fermented soyfoods such as Cheonggukjang and Doenjang.
RESUMO
Meju is a traditional Korean fermented soy product used as a key element for soy sauce and doenjang. Bacilli with antimicrobial activity were isolated from meju prepared by traditional methods at Sunchang county, Jeollabukdo, Korea. Six isolates were identified as Bacillus amyloliquefaciens by recA gene sequencing and RAPD-PCR. One isolate, B. amyloliquefaciens MJ5-41, showed the strongest fibrinolytic activity. A 27 kDa active fibrinolytic enzyme, AprE5-41, was purified from the culture supernatant of MJ5-41 grown on LB by chromatographic methods. The optimum pH and temperature for purified AprE5-41 were 7.0 and 45°C, respectively. AprE5-41 quickly degraded Aα and Bß chains but not the gamma-chain of fibrinogen. AprE5-41 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe pnitroanilide, a known substrate for α-chymotrypsin, cathepsin G, and subtilisin BPN'. The structural gene, aprE5-41, was cloned by PCR and successfully expressed in B. subtilis.
Assuntos
Bacillus/enzimologia , Fibrinolisina/isolamento & purificação , Fibrinolisina/metabolismo , Microbiologia de Alimentos , Sequência de Aminoácidos , Bacillus/classificação , Bacillus/genética , Bacillus/isolamento & purificação , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Fibrina/metabolismo , Fibrinolisina/química , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Dados de Sequência Molecular , Peso Molecular , Técnica de Amplificação ao Acaso de DNA Polimórfico , Recombinases Rec A/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , TemperaturaRESUMO
A RAPD-PCR (Randomly Amplified Polymorphic DNA-PCR) method was developed for rapid identification of Bacillus species, especially B. subtilis, B. licheniformis, and B. amyloliquefaciens, the most frequently isolated organisms from fermented soy foods such as Cheonggukjang, a Korean traditional food. A RAPD-PCR using a 10-mer (S-30) produced species specific bands reproducibly. All B. subtilis strains tested produced common bands of 0.5 and 0.88 kb in size. All B. amyloliquefaciens strains generated 1.1 and 1.5 kb bands together with 0.5 kb fragment whereas B. licheniformis strains produced 1.25, 1.70, and 1.9 kb bands with an occasional 0.5 kb band. Using the RAPD-PCR protocol, six bacilli strains isolated from Cheonggukjang were identified to the species level, which was difficult by 16S rRNA gene and recA gene sequencing for some isolates. The 0.5 kb fragment, the major band for B. subtilis strains, was an internal part of a ytcP gene encoding a hypothetical ABC-type transporter. A B. subtilis species specific primer pair was designed based on ytcP sequences and PCR using the primer pair produced a 0.46 kb fragment only from B. subtilis strains.
