Leuconostoc citreum GJ7이 생산하는 박테리오신과 그 유도물질의 특성 규명 및 이의 김치발효에서의 응용

Abstract

A new bacteriocin producing lactic acid bacteria with broad inhibition spectrum, including lactic acid bacteria, other gram-positive and gram-negative bacteria, was isolated from Kimchi. It was identified as Leuconostoc citreum, and accordingly designated as Leuconostoc citreum GJ7. The bacteriocin GJ7 from Leu. citreum GJ7 was stable in the pH range between 2.5∼9.5. Bacteriocin GJ7 was active at wide temperature range from 4℃ to 121℃. It was inactivated by proteinase K, trypsin, α-chymotrypsin and protease treatments, indicating its proteinous nature. Analysis of the purified bacteriocin GJ7 on Tricin-SDS-PAGE suggested a molecular mass of approximately 3,500 daltons. Mixed culture of Leuconostoc citreum GJ7 and an indicator strain, Lactobacillus plantarum KFRI 464, Lactobacillus delbruekii KFRI 347 or Leuconostoc mesenteroides KCTC 1628, increased the production of bacteriocin GJ7. This result suggested an inducing factor(IF) of the bacteriocin GJ7 might be present in the indicator strain. The IF was characterized and purified from Lb. plantarum KFRI 464, which showed the maximum enhancement of bacteriocin GJ7 activity. The IF of bacteriocin GJ7 was very stable at pH 2.5∼9.5 range and 4∼70℃. Moreover pre-heat treatment of IF induced more bacteriocin GJ7. Treatment of IF at 100℃ for 30 min or 121℃ for 15 min was more efficient for bacteriocin GJ7 induction than those of at 4∼70℃ for 24 hr. Treatment of the IF with proteinase K, trypsin, α-chymotrypsin destroyed the inducing activity. The IF was purified by using DEAE-sephacel column and HPLC. The purified IF yielded a single band on a Tricin-SDS-PAGE gel, having a molecular mass of about 6,500 daltons. N-terminal 16 amino acid residues were sequenced and the first amino acid residue was identified as methionine. Artifical peptide with the 16 amino acid residues was synthesized in vitro and the inducing activity of synthetic peptide was analyzed. The synthetic peptide with 16 amino acids showed inducing activity but slightly lower than whole complete IF. This result strongly suggested that bacteriocin GJ7 could be induced by inducing agent, which was originated from sensitive strains such as Lb. plantarum KFRI 464. Molecular weight of inducing agent was 6,500 daltons. Production of bacteriocin GJ7 was enhanced by using bacteriocin GJ7-sensitive strains, Lb. plantarum KFRI 464, and Kimchi was made by using this as starter(named as GJ7 starter Kimchi). Various microorganisms were controlled by starter(Leu. citreum GJ7) in GJ7-starter Kimchi, and Leu. citreum GJ7 was counted to more than 80%, as the most dominant species whereas various microorganisms(bacteria, yeast, fungi) were detected along with lactic acid bacteria in the control(non-starter Kimchi). Tastes of GJ7-starter Kimchi was characterized by soft sweetness, and refreshed, clear and crisp freshness. Control without starter tasted very sour and smelled bad. It was surely recognized that in Kimchi using Leu. citreum GJ7 as starter, controlled microbial growth or fermentations occurred.