Purification and characterization of multi-functional peptide produced by Bacillus strain isolated from Korean food

Abstract

국내 식품에서 분리한 바실러스 약 100여종을 대상으로 항균, 항산화, 항염 펩타이드를 생산하는 균주를 스크리닝 하였다. 후보 미생물 중 MP40 발효물이 항균 및 항산화 효과가 있음을 확인하였다. 선별한 MP40 균주는 16S rRNA 염기서열분석법을 통하여 신규한 균주임을 확인하고 Bacillus sp. MP40이라 명명하였다. 본 균주의 최적 배양 조건은 탄소원 sorbitol (1 % w/v), 질소원 peptone (1 % w/v), tryptone (0.5 % w/v), soytone (0.5% w/v), 무기염류 calcium chloride (0.01 %), 37℃, 12시간임을 확인하였다. 최적 배양 후 황산 암모늄 침전법과 Sepharose CL-6B, Sephadex G50컬럼 크로마토 크래피를 이용하여 분리정제 후 펩타이드의 분자량이 1000Da 정도임을 확인하였다. MP40 항균펩타이드는 그람 음성균인 E.coli와 다약제 내성균인 반코마이신 저항성 장알균 (VRE)에 좋은 항균 활성을 나타내었다. 특히, 장알균(VRE)에 대한 항균 활성이 뛰어 났으며 VRE의 항생제에 대한 최소 저해 농도값은 10-20 μg/ml 이었다. MP40 펩타이드는 pH 와 온도 안정성 시험을 통해pH 2.0-10.0, 20~100 ℃까지 항균 활성이 유지되었으며 단백질 분해효소인lipase, proteinase K, α-chymotrypsin, tyrpsin에 안정함을 확인하였다. 다양한 chemical에 대한 안정성을 확인한 결과 다양한 유기용매, EDTA, TCA, 계면활성제 등에 활성이 저해되지 않고 유지되었다. MP40 항균 펩타이드의 항산화 활성은 DPPH 라디칼 소거능, 환원력, 총 페놀 함량 측정, 아질산 소거능 시험을 통해 분석하였다. 이 펩타이드의 DPPH 소거능과 환원력, 아질산 소거능은 대조군인 아스코르브산과 같은 농도에서 비교하였을 때 뛰어난 수준의 항산화 활성을 나타내었으며, 총 페놀 함량은 같은 농도에서 갈산보다 높은 수준의 페놀을 함유함을 확인하였다. Murine macrophage인 Raw264.7 대식세포는 LPS에 의해 자극되면 일산화 탄소의 생성이 증가하는데 MP40 항균 펩타이드를 10~200 ug/ml의 농도로 처리하였을 때 이의 생산을 억제하는 효과를 보였으며 이 농도에서의 세포독성도 나타내지 않았다. 본 연구를 통해 국내 식품에서 분리한Bacillus sp. MP40 균주가 생산하는 펩타이드가 항생제 내성 세균에 항균 효과를 가지며, 높은 항산화 활성 및 NO 생성을 억제하는 것을 확인하였다.|Bacillus sp. MP40 was isolated from several hundreds of Bacillus strains preserved in our laboratory isolated from Korean traditional foods. Fermentation broth of MP40 showed antimicrobial, antioxidant activities. Therefore we selected this strain for the study. Based on the 16s rRNA sequencing, MP40 was found related to the Bacillus amyliquefaciens. Therefore the strain was classified as Bacillus sp. MP40. Antimicrobial peptide production from the strain was optimized by using several nutritional parameters. Sorbitol, peptone, tryptone, soytone, CaCl2 were found the best carbon, nitrogen and metal ion sources. After fermentation of MP40 strain under optimized condition for 12 h, broth was harvested. The antimicrobial peptide was precipitated using ammonium sulfate. MP40 AMP (ca. 1000Da) was purified to homogeneity by using sequential chromatographic steps. MP40 AMP performed antimicrobial activity against Gram negative bacteria and MDR bacteria such as Escherichia coli, vancomycin-resistant enterococci (VRE). Minimal inhibitory concentrations of MP40 AMP against VRE were in the range of 10–20 μg/ml. MP40 AMP was found to be stable at pH 2.0–10.0 and up to 100 ℃. The AMP activity was resistant to the proteolytic action of lipase, proteinase K and α-chymotrypsin, trypsin. The effect of several chemicals on the AMP was evaluated. The AMP was resistant to various organic solvents and detergents. The antioxidative activities of the AMP were determined by using 2,2-diphenyl-a-picrylhydrazyl (DPPH), reducing antioxidant power, total phenol contents, nitrite scavenging activity methods. The MP40 AMP has good antiradical potency against the DPPH. The reducing power of MP40 AMP was increased in a concentration dependent manner. The DPPH scavenging, nitrite scavenging activity and reducing power of MP40 AMP was more effective than ascorbic acid (control). The total phenol contents of MP40 AMP were increased concentration dependent. Total phenol contents of peptide was significantly higher than gallic acid in same concentration Bacillus sp. MP40 AMP was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. In conclusion, antimicrobial peptide producing MP40 strain was purified from a new Bacillus strain isolated from Korean foods. This AMP has antimicrobial, high antioxidant activities and inhibited nitric oxide production.